Bioavailability and Recent Advances in the Bioactivity of Flavonoid and Stilbene Compounds

*Chin Giaw Lim and Mattheos A. G. Koffas

Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, New York 14260.

ABSTRACT:

Polyphenols such as flavonoids and stilbenes are abundant in our daily diet, and their roles in the protection and prevention of

various diseases are substantial. However, the bioavailability varies among polyphenols and the actual compounds acting on the

designated tissues are often not the native molecules consumed in the diet. Hereby, we review the bioavailabilty of the main

classes of stilbenes and flavonoids, namely flavanones, flavones, isoflavones, flavanols, flavonols and anthocyanins, emphasizing

on their absorption, distribution, metabolism and excretion (ADME). The bioavailability summary can be useful for future

experimental design, especially the emphasis on the bioactivity on targeted tissues and organs. In addition, we review the

bioefficacy of the polyphenols, emphasizing recent advances on health benefits both in vivo and in vitro. Other issues of importance, such as structure, food source and synthesis methods, are also considered.

KEYWORDS: Polyphenols, flavonoids, resveratrol, bioavailability, absorption, distribution, metabolism, excretion, bioactivity

INTRODUCTION

Flavonoids are plant secondary metabolites found in the plant

kingdom and the largest phenolic group in nature. They are common

in our daily diet such as fruits, vegetables, herbs, red wine and tea. A

major part of their function in plant is related to their interactions

with extreme environments. For example, flavonoids accumulated in

epidermal cells by UV induction can act as a UV protectant by

absorbing the radiation. Upon wounding or infection by pathogens,

flavonoids also act as toxin and fungal pathogens as well as

antimicrobial compounds. The high concentration and wide variety of

flavonoids found in seed coats is attributed to the antifungal and

antimicrobial properties of flavonoids to protect the seeds and

indirectly facilitate plant reproduction[1]. Microbial is not necessary

harmful to plants, especially in the case with leguminous plant, when

they are in their nutrient-limited condition or more specific nitrate-

deficient state. The plants induce flavonoids accumulation to attract

nitrogen-fixing bacteria, Rhizobium[2], which convert nitrogen from

the atmosphere into plant usable ammonium in the nodule. In return,

the plant provides microaerobic environment and offers organic acids

necessary for the bacteria as carbon sources[3]. This form of

relationship between the plant and the bacteria is known as legume-

Rhizobium symbiosis. Other proposed functions of flavonoids in

plants include reproductive tissues protection, pollinator attraction,

seed dispersal, coloration, feed deterrent and enzyme inhibition.

Flavonoids are also involved in photosynthesis, morphogenesis and

sex determination[1, 2, 4]. From all the benefits flavonoids possess,

even though these compounds are not essential for cell survival, they

serve a key role in giving the organism an evolutionary advantage to

survive and reproduce[5], both in terms of physiological function and

biochemical properties.

Stilbenes do not belong to the flavonoids but they share high

resemblance to flavonoids in both functions in plant and chemical

structure. Stilbenes are synthesized naturally by distinct plants and

they are synthesized in response to infection by pathogens

(phytoalexins) and ultraviolet light exposure, and are also involved in

bacterial root nodulation and coloration[6, 7].

Chemical Structure

Flavonoids share a common three-ring structure but different

subclasses, including chalcones, flavanones, flavones, flavonols, and

anthocyanidins, differ mainly at the middle heterocyle ring (C-ring)

where two benzene rings are linked. In the case of isoflavones, in

addition to the difference in the C ring, there is also a position shift

on the phenyl ring B. All structures are shown in Fig.(1) and detailed

in the Table (1) below.

Polyphenol Structure Description (A- and B-ring at position 2)

Flavanones Linked by a tetrahydropyrone Isoflavones Linked by a pyrone, but ring B is substituted at position 3 Flavones Linked by a pyrone Flavonols Linked by a tetrahydropyrone hydroxylated at position 3 Flavanols Linked by a pyrone hydroxylated at position 3 Anthocyanins Linked by a pyrilium hydroxylated at position 3 Stilbenes Linked by a methylene bridge

Table 1| Structure description of individual flavonoids and stilbenes

In addition to the structure of flavonoids discussed, subsequent

substituent can occur with the attachment of organic acids, sugar

group and hydroxyl group. On the A ring, typical flavonoids are

hydroxylated at the 5 and 7 positions but modification in the A ring

can exist, for instance, isoflavone hydroxylated at both 5,7 positions

is known as genistein while the other counterpart with only one

hydroxyl group at position 7 is named daidzein. On the B ring,

hydroxyl and methoxyl groups substitution usually occur on the 3‟, 4‟

or 5‟ position. At last, C ring substitution is the uncommon one to

flavonoids except for catechins and anthocyanidins, in which both the

Category Source Polyphenol (content in mg/kg or mg/L)

Fruits Orange Flavanones (400-600) Flavonols (0-50) Lemon Flavanones (150-250) Flavonols (0-28) Flavones (0-15)

Lime Flavanones (450) Flavonols (5)

Blackcurrant Anthocyanins (1300-4000) Flavonols (30-130) Flavanols (10)

Strawberry Anthocyanins (150-750) Flavanols (40) Flavonols (10-20)

Olives Flavones (80-200) Flavonols (60-120)

Avocado Flavanols (0.2-5)

Plum Anthocyanins (20-250) Flavanols (50-100) Flavonols (10-15)

Black Grape Anthocyanins (300-7500) Stilbenes (1.5-10)

Flavanols (30-175) Flavonols (5-40)

Vegetables Soybean Isoflavones (200-900) Flavonols (5-35)

Celery Flavones (20-140)

Broccoli Flavonols (50-100)

Peanut Stilbenes (0.4-5.1)

Onions Flavonols (300-1500)

Kale Flavonols (300-600)

Tomato Flavonols (3-20) Flavanones (6-15)

Cabbage Anthocyanins (250) Flavonols (5)

Beverages Green Tea Flavanols (100-800) Flavonols (40)

Black Tea Flavanols (60-500) Flavonols (10-60)

Beer Flavonols (1-10) Flavanols (0.5-10)

Cider Flavanols (40)

Soy milk Flavanols (30-175)

Red Wine Anthocyanins (200-350) Stilbenes (5-18)

Flavanols (80-300) Flavonols (2-30)

Miscellaneous Foods

Chocolate Flavanols (35-600)

Honey Flavonols (1-60) Flavones (3-50)

Fruit Jams Flavonols (1-380) Flavanols (10-100)

Rosemary Flavones (10)

Parsley Flavones (200-2400) Flavonols (5-10)

structure lack oxygen grouyp at 4-position and the presence of

hydroxyl group at 3-position on the C ring. For anthocyanidin,

different sugar like glucose, arabinose and galactose can be attached

to this 3-position while for flavanols, gallic acid can attach to the

same position forming gallic acid esters.

Stilbene chemical structure consists of 2 aromatic rings (ring A

and B) joined by a methylene bridge[7]. One of the most well known

compounds in the stilbene family is trans-resveratrol, which is

hydroxylated at 3, 5and 4‟ position. Substituent can occur both on the

A and B ring. For substituent on the A ring, piceid is one typical

example. It is the glycosidic form of resveratrol with substituent on

the 5 position on the A ring. On the other hand, compounds with

varied substituent on the B ring are pinosylvin with no substituent

and piceatannol with hydroxyl groups at position 3‟ and 4‟.

Presence in food

Flavonoids and stilbenes are presented in an abundant amount in our

daily diet. Table (2) presents the polyphenols distributions in a few

common foods.

Flavanones

Flavanones main dietary sources are restricted to citrus products such

as orange (50-60mg/100g), grapefruit (30-55mg/100g), lemon (17-

26mg /100g) and lime (17-47mg/100g)[8-10]. The main aglycones of

flavanones are naringenin and hesperetin, but they are more

commonly found in glycosides[11].

Isoflavones

In human diet, isoflavones are primarily found in leguminous plant.

Soy bean is a major source of isoflavones, comprise of genistein,

daidzein and their glucosides, genistin, daidzin. In both soybean and

soybean related products like miso, soymilk and soy flour,

isoflavones exist mostly in glucosides rather than the aglycone[12].

In miso, the concentration of genistein is higher than other soy

product due to the microbes in the fermentation process which help

convert glucosides to the unconjugated form.

Flavones

Flavones are present in human diet as glycosides[13]. Major flavones

in plant are luteolin and apigenin. In plant, parsley and celery are two

major sources of flavones (240-1850mg/kg and 20-950mg/kg

respectively)[10, 11].

Flavonols

Flavonols are widely distributed in human diet and similar to

flavones, flavonols usually occur in diet as glycosides[13]. The major

aglycones of flavonols are kaempferol and quercetin. Flavonols

found in vegetables, fruits and beverages are generally low except for

kale (300-450mg/kg), onion (300-1300mg/kg), broccoli, leeks, beans,

apple, red wine and tea[11, 13].

Flavanols

Unlike most of flavonoids, the presence of flavanol glucosides are

rare[13]. The most abundant flavanols in plant are catechins,

gallocatechins and catechin gallates. Catechins can be found in a

variety of fruits, red wine, tea and chocolate. Apricot is reported to

have the highest amount of flavanols among fruits (67- 250 mg/kg),

while in beverages, flavonols content can reach up to 800 mg/L in

green tea and 300mg/L in red wine[11].

Anthocyanins

Due to the fact that anthocyanins are responsible for most of the red,

blue and purple colors of different parts of plant, there is no surprise

that anthocyanin are abundant in berries, grapes, grains and red wine.

The richest source of anthocyanins is in berries, in which their

content can top 10mg per 100g of edible portion of fruits[10]. Red

wine also contain anthocyanin but in a minute amount. The reason

the glycoside forms, anthocyanins are presented in this review instead

of the aglycones, anthocyanidin is because the aglycones are highly

unstable and rarely found in the plant. In recent years, anthocyanins

have drawn huge interest in their function as natural food colorants,

much preferred than synthetic dyes. As a result, small amount of

anthocyanins could also be consumed through healthy food colorant.

Table 2| Flavonoids and stilbene distribution in common foods[8-11].

Stilbenes

One of the most recognized stilbene, resveratrol is found in tiny

quantities in human diet and its primary dietary sources are peanuts

(0.02-1.92mg/kg), cranberries and grapes(0.16-3.54 mg/kg)[14].

Therefore, there is no surprise that resveratrol is also found in wine

(0.1-14.3mg/L)[14]. Red wine possesses a higher concentration of

resveratrol compared to white because red wine is fermented together

with the grape skin. This revealed that resveratrol is accumulated

mostly in the skin and is consistent with the „red-better-than white‟

hypothesis[15]. Generally, stilbene does not exist as single compound

but as a mixture of multiple isomers or metabolites[7]. Besides trans-

resveratrol, its cis isomer, resveratrol glycosides: trans- and cis-piceid

also exist.

Biosynthesis in Nature

Individual pathways for the biosynthesis of flavonoids and stilbene

are shown in Fig. (1). In plant, hydroxycinnamates-CoA ester,

particularly p-coumaroyl-CoA serves as precursor for the

biosynthesis of various plant secondary products, such as stilbenes

and flavonoids. The ester is synthesized from amino acids building

blocks, phenylalanine or tyrosine. First, the enzyme phenylalanine

ammonia lyase (PAL) or tyrosine ammonia lyase (TAL) converts the

amino acids into phenylpropanoic acids followed by another pivotal

enzyme 4-coumaroyl-CoA ligase (4CL) that converts the

phenylpropanoic acids to their CoA-esters.

Stilbenes and Chalcones

To synthesize both stilbenes and chalcones, the CoA-esters are

extended by three rounds of stepwise decarboxylative condensation

with malonyl-CoA to form a tetraketide intermediate. This is where

two closely related enzymes, stilbene synthase (STS) and chalcone

synthase (CHS) take place. Austin et al. published an excellent article

that clarifies the intramolecular condensation mechanisms of the two

highly similar sequence enzymes, CHS and STS which lead to the

fabrication of two different products[16]. CHS mediates the claisen

cyclization of the tetraketide intermediate (carbon 6 to carbon 1) and

offloading naringenin chalcone, which is the first flavonoid

compound formed. On the other hand, stilbene synthase (STS) is

responsible for the aldol-type cyclization (carbon 2 to carbon 7) and

decarboxylation to form trans-3, 5, 4‟-trihydroxystilbene or

resveratrol[17].

Flavanones

Flavanones are the direct precursor of different subgroups in the

flavonoids pathway. After the naringenin chalcones are formed by

4CL and CHS enzymes, the chalcones are stereospecifically

isomerized into (2S)-flavanones by chalcone isomerase (CHI).

Combining 4CL, CHS and CHI, they are known as the “upper

pathway”.

Isoflavones

One main feature that differentiates isoflavone from other flavonoids

is the aryl group location on the C ring (position 3 instead of position

2). Isoflavone synthase (IFS) is responsible for the oxidative attack

and the ring migration of the flavanone precursor[18]. IFS, a type II

P450 enzyme requires bounding to the intracellular membrane and

association with another membrane bound protein CPR that provides

an electron to IFS via flavin mononucletide (FMN) and flavin

adenine dinucleotide FAD. As a result, 2-hydroxyisoflavanones are

formed with 3-hydroxyflavanones as the byproduct. Subsequently,

dehydration occurs spontaneously to form the final product,

isoflavone. This 1,2-elimination of a water can be facilitated by a

third enzyme, namely2-hydroxyisoflavanone dehydratase (HID)[19].

Figure 1| Flavonoids and stilbene

biosynthetic pathway. Enzyme abbreviations

in text are as follows: PAL: phenylalanine ammonia-lyase; C4H: cinnamate 4-hydroxylase;

TAL: tyrosine ammonia-lyase; 4CL: 4-

coumaroyl:coenzyme A ligase; STS: stilbene synthase; CHS: chalcone synthase; CHI:

chalcone isomerase; IFS: isoflavones synthase;

FSI: flavone synthase; FHT: flavanone 3β-hydroxylase; FLS: flavonol synthase; DFR:

dihydroflavonol 4-reductase; LAR: leucoanthocyanidin reductase; ANS:

anthocyanidin synthase; 3GT: UDPG-flavonoid

3-O-glucosyl transferase.

Flavones

Flavones are another subgroup of flavonoids that utilize flavanones

as the precursors. In plant, the flavones biosynthesis is governed by

two distinct enzymes: flavone synthase I (FSI), which is found only

in plants belong to the Apiceae family, and flavone synthase II

(FSII), a membrane-bound protein ubiquitous in plants[18].

Flavonols

Two vital enzymes are required to produce flavonols. First,

flavanones are converted to dihydroflavonols (DHF) by flavanone

3-hydroxylase (F3H or FHT). DHFs are then used as the substrate

for the second enzyme, flavonol synthase (FLS) to synthesize

flavonols. Both the enzymes are 2-oxoglutarate-dependent enzymes.

Flavanols and Anthocyanins

The further down the molecules in the flavonoid biosynthesis

pathway, the more tedious it is to synthesize. This is the case for

anthocyanins in which five enzymes are required prior to the

formation of flavanone (FHT, DFR, LAR, ANS and 3GT). Both

anthocyanin and flavanol are grouped into the same section because

flavanol can be one of the intermediates for anthocyanin biosynthesis.

Similar to the first step towards producing flavonols, the biosynthesis

of anthocyanidins utilize FHT to form DHKs. DHKs are used as

substrates for dihydroflavonol 4-reductase (DFR) to produce

leucoanthocyanidins. Subsequently, catechins are formed using

leucoanthocyanidin reductase (LAR), in which it removes the

hydroxyl group at position 4 from leucoanthocyanidins. Next,

anthocyanidin synthase (ANS) takes place and this enzyme catalyzes

the reduction of either catechins or leucoanthocyanidins to form

anthocyanidins. At last, anthocyanidins are converted to anthocyanins

with UDPG-flavonoid 3-O-glucosyl transferase (3GT).

Biosynthesis Methods

In the past few decades, different attempts have been made to

synthesize flavonoids and stilbenes with the purposes of utilizing

them as valuable nutraceuticals and colorants. Chemical synthesis or

to be more precise organic synthesis is one of the more common

approaches for polyphenols biosynthesis and often, it takes multiple

steps to synthesize the product of interest. However, this approach

has a few limitations: first, being the starting materials for some of

the reactions are cost prohibitive for large scale synthesis and second,

in some instances, products are formed as racemic mixtures, which

are highly unfavorable for purification. By-products of each synthesis

steps can also be problematic and they have to be removed to prevent

contamination of the final products. On the other hand, the main

benefit of this method is it offers diverse structure modification to

bioactive polyphenols. By using the polyphenols as a lead, this

approach offers huge potentials in fabricating more desirable

molecules, potentially with a better potency and more desirable traits,

for instance higher binding affinity, less toxicity, altered lipophilicity

and higher oral bioavailability. Biddle et al. recently reported the

discovery of a bifunctional quinine-derived thiourea catalyst that

effectively regulates the chemical outcome and produces either a left-

handed molecule or a right-handed molecule, not a one-to-one

mixture of both. This catalyst was used in flavanones and

chromanones synthesis and high yields were reported with excellent

enantioselectivity, in addition to the advantages of further

modification capability[20]. Apart from chemical synthesis, different

polyphenols biosynthesis approaches with each offers advantages and

disadvantages are available, for example plant extract, plant cell

cultures, and recombinant microororganisms.

Plant extracts

Figure 2| Recombinant bacteria and

anthocyanidins colored compounds. (A) Schematic representation of gene of interest

isolated from the origin, expressing it in a plasmid and transforming into bacteria host cell. (B)

Biosynthesis of diverse chemistry of natural and

unnatural anthocyanidins utilizing recombinant microorganism.

Plant kingdom is the origin of many potent drugs, and plant extract

remains one of the most abundant and classic way to produce natural

compounds. The disadvantage of using plant extract is the hefty

amount of plant tissues required to produce a significant amount of

individual flavonoid. This method can be very costly and time

consuming. In addition, the “browning effect” of flavonoids extract

remains one of the biggest obstacles in the field[18].

Plant cell cultures

Plant cell cultures can be one good alternative to plant extracts as

there is no heavy depletion of environmental components; instead,

plant cells are mass produced in the bioreactor. The major challenge

of this production method is with the plant cells. They tend to form

cells aggregates mainly due to the affinity of the cells not to separate

after cell division[21]. This issue leads to a fraction of the cells

within the aggregates not uniformly exposed to lightning. In the

nature of plant cells, a few enzymes along the flavonoid and stilbene

biosynthetic pathway are induced primarily by UV[22, 23], and it had

been shown that the intensity of lightning improved the overall

flavonoids productivity[24]. Without uniform lightning, there are

limited induction and subsequently low protein expression within the

cells, which will directly affect the output of the bioactive

compounds. Until this issue is solved, this method remains

economically non-feasible.

Recombinant microorganisms

Microbial production of polyphenols is one of the more popular

methods which can involve both bacteria and yeast. Fig. (2A)

demonstrates the essential steps of embedding a gene of interest

isolated from plant or animal cells, cloning the gene into a functional

plasmid, transforming the whole plasmid into the microorganisms

and subsequently subjecting the microorganisms for fermentation.

Microbial production possesses a few distinctive advantages. For one,

microbial does not form aggregates and has a significant shorter

doubling time compared to the plant counterpart. Secondly, plant

biosynthetic pathways cloned have to be under the respective

microbial promoters, which indeed exclude the dependent of UV

induction exist in plants. By using this method the end products

generated are not only limited to natural polyphenols, but also a

diverse library of high-valued unnatural compounds. Fig. (2B) shows

the potential of microbial to create diverse natural and unnatural

dihydroflavonol through combinatorial mutasynthesis and

subsequently generate a plethora of different colored anthocyanidin

[Chemler et al. unpublished].

The only drawback using microbial for natural compound

productions is the complication involved in embedding the

biosynthetic pathways and to functionally express individual pathway

in the microorganism. Escherichia coli strains have been engineered

to produce natural compounds primarily due to the ease of gene

manipulation and their rapid cell division. One major disadvantage of

using the bacteria platform is the absence of intracellular membrane

or endoplasmic reticulum that lead to the non-fuctionality of many

key enzymes, for example the membrane bound cytochrome P450

family, which in some cases requires the association of another

membrane bound protein or redox partner, namely cytochrome P450

reductases (CPRs). Effendi et al. successfully designed an artificial

P450 system that is functionally expressed in E. coli by mimicking

the architecture of the bacterial P450BM-3[25, 26]. Other than the

slight lengthy doubling time, yeast or Saccharomyces cerevasiae can

be advantageous over bacteria especially of its capability to support

the challenge of P450 enzymes with the existence of its endoplasmic

reticulum. It also contains endogenous CPR that can facilitate

electron transfer to the cytochrome P450 enzymes.

Both flavonoids and stilbenes have been shown to possess a

range of beneficial effects across distant species and disease models.

Oxidation in the body is detrimental to the health and it has been

proved to be responsible for a few major degenerative diseases,

including arteriosclerosis, cancer and heart disease. Polyphenols are

known to prevent many of the oxidative processes by acting as potent

metal chelators, free radical scavengers and chain-breaking

antioxidant[4]. The number of flavonoids antioxidant activities

related publications have increased exponentially in these past two

decades covering activities in vitro, ex vivo and also in vivo[27].

Since the polyphenols‟ antioxidant properties have been extensively

studied, only the ones with higher antioxidant activity will be

discussed in this review.

Flavonoids and stilbenes associated health benefits are apparent

but there are still a lot of inquiries on how these small molecules

proceed in the body once ingested, followed by promoting their vital

bioactivities. This can be addressed by the study of absorption,

distribution, metabolism and excretion (ADME), which is crucial in

defining the pharmacokinetics and pharmacodynamics of

polyphenols, most importantly addressing the fate of the glycosides

and aglycones. Reviews by Walle et al., Zhang et al. and Prasain et

al. provide detailed explanations on the possible transport

mechanisms and metabolisms of polyphenols in the body. The

absorption patents between glycosides and the aglycones are

distinctively different mainly due to the variation in size and polarity.

Polyphenol aglycones are more permeable across human intestinal,

therefore more readily absorbed while the glycosides are hydrolyzed

into their aglycones prior to absorption. Hydrolysis of these

glycosides could be accomplished by β-glucosidase in the small

intestinal epithelium of human or lactase phlorizin hydrolase (LPH)

bound to the intestinal lumen[28]. LPH was initially known only to

hydrolyze lactose. Besides being metabolized, the glycosides can

also remain intact and directly be absorbed using the sodium-

dependent glucose transporter 1 (SGLT-1)[28, 29], with absorption

efficiency greatly determined by the sugar moiety of individual

molecules[28]. These absorbed glycosides could possibly be

transported back into lumen by the multidrug resistance protein 2

(MRP2). Although the majority of the flavonoids absorbed are

aglycones followed by a minute amount of glycosides, both groups

are found in very low concentrations in the plasma. Compounds that

predominate the blood circulation are primarily phase II metabolites

such as glucuronides, sulfo and methyl conjugates as a result of

glucuronidation, sulfonation and methylation[30]. Glucuronidation

reaction is mediated by UDP-glucuronosyltransferases (UGTs),

sulfonation is mediated by sulfotransferases (SULTs) while

methylation is regulated by catechol-O-methyl transferases

(COMTs)[29, 30]. Walle et al. also suggested a few additional

metabolic pathways including bacteria metabolism that happened

mostly in the colon and metabolites oxidation by reactive oxygen

species (ROS)[29]. Flavonoids and their metabolites that reach the

colon may be further metabolized and degraded by bacterial enzymes

into phenolics, carboxylic acids and carbon dioxide. On the other

hand, oxidized flavonoids were reported to be able to covalently bind

to DNA and protein in human intestinal and hepatic cells[31].

This review focuses on investigating the bioavailability of

individual flavonoids/stilbene, the different approaches attempted to

improve polyphenols‟ bioavailability as well as their current

advances in biological activities. This review includes in vitro

studies, in vivo studies in both animals and humans as well as a few

clinical trials. This study seeks to offer a greater understanding of the

bioavailability of different polyphenols and their metabolites, their

role in preventing and curing diseases, and ultimately help improve

human well-being.

BIOAVAILABILITY AND BIOEFFICACY

FLAVANONES

Flavanones are among the most prominent phenolics due to their

role as the direct precursors of all other flavonoids. The most

representative flavanones are naringenin, hesperetin as well as their

glycosides. Flavanones are most abundant in citrus fruit and juice

compared to other food sources[32], with hesperidin and narirutin,

the 7-O-rutinoside version of the aglycone hesperetin and naringenin

respectively, as the major flavanone glucosides in oranges. Several

studies have looked into the in vivo bioavailibililty of flavanone after

consumption of citrus fruit or juice and cooked tomato paste[33-35].

In the plasma and urine, flavanones and their metabolites were

determined by measuring their concentration throughout the course of

the studies.

Overall, flavanones are poorly bioavailable and their reported

peak plasma concentrations are in the low µM range[32, 33, 36].

Manach et al. conducted a human study in which 0.5 or 1 L of

commercial orange juice (444mg/L of hesperidin and 96.4 mg/L

narirutin) was ingested. The peak plasma concentration of aglycones

hesperetin and naringenin were 0.46µM and 1.28µM,

respectively[33]. In another human study, 150 subjects whose age

ranged from 18 to 80-year-old were given either orange fruit (150g)

or juice (300g). The reported mean peak plasma concentration of

flavanone was in the range of 0.05-0.1µM. This particular study

reported a few important observations: first, there were no significant

bioavailability variation in flavanones ingested from fruit or juice and

second, there was a huge inter-individual flavanone excretion

variation but it was independent of age, sex, body mass index, or use

of contraceptive pill[32]. The major reason behind the low

bioavailability and absorption of flavanone glycosides in human is

the individual sugar moieties[37]. Hesperidin with a rutinoside is one

of the glycosides with low bioavailability. By treating the consumed

orange juice with hesperidinase enzyme to remove the rhamnose

group yielding hesperetin-7-glucoside, Nielsen et al. were able to

improve the bioavailability of hesperetin by 4-fold compared to

volunteers who consumed orange juice with natural hesperidin alone,

and by 1.5-fold compared to those who consumed modified orange

juice with 3 times more hesperidin. The researcher proposed that the

improved bioavailability was caused by the change in the absorption

site from the colon to the small intestine[38].

In order to eliminate doubts that other active compounds present

in orange that decrease the overall flavanone bioavailability, some

studies used pure flavanones to scrutinize flavanone bioavailability.

Six healthy volunteers were orally administered with 135 mg of

hesperetin or naringenin under fasting conditions. Both the flavanone

aglycones were rapidly absorbed and detected in the blood samples

20 minutes after dosing. Their mean peak plasma concentrations were

about 2.3µM and 7.3µM respectively; however, their urinary

excretions were merely 3.26 percent and 5.81 percent the

administered dose, respectively[39]. These results confirmed the low

bioavailability of flavanone aglycones.

The flavanone metabolites found in the circulating plasma,

urinary excretion were mainly glucuronides and

sulfoglucuronides[40]. Manach et al. reported four major metabolites:

4‟- and 7-O-monoglucuronides of naringenin and the 3‟- and 7-O-

monoglucuronides of hesperetin in addition to two hesperetin

diglucuronides and a hesperetin sulfo-glucuronide in plasma and

urine[33]. Phenolic acids were also reported as flavanone metabolites

resulted from intestinal microflora degradation[33, 41]. Kanaze et al.

attributed the low cumulative flavanone urinary recovery to possible

cleavage of flavanone aglycones on the C ring[39].

Even though flavanone aglycone and glycosides possess very

low bioavailability, numerous in vivo animal and human as well as in

vitro studies have shown that flavanones exhibit an array of health

benefits including antioxidant, anti-inflammatory, anti-tumor and

anti-carcinogenic activities. There have been reports in the past two

decades suggesting the antioxidant activities of flavanone in vitro[42,

43]. On the contrary, Andrade et al. reported no antioxidant or pro-

oxidant effects in the rat model when the rats were fed with 3

increasing concentrations of naringenin (30, 60 and 120mg/kg or

diet)[44].

Several in vivo animal studies looked into the function of

flavanones as anti-carcinogenic and anti-cancer agents. Oral

administration of hesperidin on azoxymethane (AOM)-induced colon

carcinogenesis in rats, significantly decreased the incidence and

multiplicity of neoplasms in the rats‟ large intestines, reduced the 5'-

bromodeoxyuridine-labeling index and argyrophilic nuclear organizer

region's number in crypt cells, colonic mucosal ornithine

decarboxylase activity, and polyamine levels in the blood, suggesting

hesperidin possible chemoprevention effect against colon

carcinogenesis[45]. They later reported a similar chemopreventive

effect of a commercial Satsuma mandarin (Citrus unshiu Marc.)

juice, which is rich in hesperidin (3.58 percent) and β-cryptoxanthin

(0.67 percent)[46]. Later studies were conducted to investigate

whether citrus juices could modify carcinogenesis in other organs. A

modified Satsuma mandarin juice with higher hesperidin (100mg)

and β-cryptoxanthin (3.9mg) was reported to possess a

chemopreventive ability against 4-(methylnitrosamino)-1-(3-pyridyl)-

1-butanone-induced mouse lung tumorigenesis[47]. Apart from the

glycosides, flavanone aglycones themselves were also reported

similar anti-carcinogenic effect. In 1,2-dimethylhydrazine-induced

colon carcinogenesis rats, hesperetin reduced the formation of

preneoplastic lesions and modulated the xenobiotic-metabolizing

enzymes in rats[48], whereas in N-methyl-N‟-nitro-N-

nitrosoguanidine-induced gastric carcinogenesis rats, naringenin

significantly up-regulated the redox state in rats to decrease the risk

of cancer[49]. Liquiritigenin, a naringenin analog lacking the 5-

hydroxyl group also showed potent cytotoxic effect against human

cancer cells in vitro via reactive oxygen species production and

subsequently lead to cell apoptosis[50, 51].

Besides the evidence on the anticarcinogenic and anti-tumor

activities in animals, flavanones have been shown to protect against

various inflammatory injuries. Vafeiadou et al. studied the role of

flavanones as neuroprotective agents. In vitro assays performed to

assess flavonoids‟ role in glial cells suggested that naringenin had

one of the strongest activity among flavonoids tested against

attenuating LPS/IFN-γ-induced TNF-α production and inhibiting

LPS/IFN-γ-induced iNOS expression, p38 mitogen-activated protein

kinase phosphorylation and nitric oxide production. Moreover, it also

protected inflammatory-induced neuronal death in a primary

neuronal-glial co-culture system[52]. In periodontitis, an

inflammatory disease of tooth supporting tissues, naringenin could

act as a potent inhibitor of the pro-inflammatory cytokine response

induced by lipopolysaccharide in both macrophages and in human

whole blood model[53]. Both naringin and the aglycones were

evaluated for their anti-inflammatory effect on acute model of

induced colitis in mice, a disease affecting the tissue that lines the

gastrointestinal system. Results revealed both the flavanones were

capable of reducing edema of the gut and the levels of nitrate/nitrites

through inhibition of inducible nitric oxide synthetase, and

minimized lipid peroxidation by oxygen radicals scavenging; hence

suggesting anti-inflammatory potentials[54]. Another flavanone

derivative, isopedicin also showed potent anti-inflammatory function

in inhibiting superoxide anion production in formyl-l-methionyl-l-

leucyl-l-phenylalanine (FMLP)-activated human neutrophils[55].

Hypocholesterolemic and cardioprotective activities of

flavanones also attracted a few animal studies and human clinical

trials. Supplementation of naringenin in 1 percent high-cholesterol

diet-fed rats effectively improved the plasma or hepatic lipid profiles

and antioxidant capacities compared to the control group[56].

Naringin also showed cardioprotective role in ISO-induced

myocardial infarction in rats that were pretreated with 10 to 40mg/kg

for a period of 56 days[57]. In contrast, one study contradicted the

benefits declared. 50mg/kg of naringenin ingested in both control and

ethanol-drinking rats for 2 weeks only exerted minor influence on

lipid metabolism. Likewise, isolated adipocytes incubated with

naringenin revealed similar lack of effect[58]. In a human clinical

trial, orange juice fed three times daily to thirteen healthy subjects for

three weeks successfully decreased the low density lipoprotein to

high density lipoprotein cholesterol (LDL/HDL) ratios but

cholesterol concentrations in the plasma did not vary

significantly[59].

In anti-diabetic related area, Ghanim et al. employed a different

approach using orange juice as possible glucose substitution. Glucose

intake could induce oxidative stress and inflammation but these

effects were absent following fructose, orange juice and water

ingestion. In vitro proved that reactive oxygen species in

mononuclear cells was suppressed by naringenin and hesperetin in

orange juice but not by fructose or ascorbic acid[60]. Naringenin was

also shown to facilitate extra-pancreatic action and to inhibit

carbohydrate absorption from intestine, thereby promoting anti-

diabetic effect on diabetic rat models[61].

ISOFLAVONES

Leguminous plants such as soya are major source of isoflavone.

Isoflavones glycosides predominate in soy compared to the

aglycones, but once it is fermented, the soya product primarily

contains the aglycones.

When it comes to the bioavailability of isoflavones (aglycones

and glycosides), the results are highly contradictory. There are

reports suggested that isoflavone aglycones are more rapidly

absorbed than their glucosides[62-64]; whereas Setchell et al.

reported higher bioavailability of glucosides form of isoflavones[65].

More interestingly, Richelle et al. and Zubik et al. did not observe any

absorption difference when either aglycones or glucosides were

administered[66, 67]. Discrepancy among the reports could be

attributed to the used of soybean extract (mixture of isoflavones)

compared to pure compounds in some other studies. Setchell credited

the inconsistent results to the importance of multiple time points

sampling during the elimination phase and after attaining steady-state

rather than sampling merely 2 time points in the elimination

phase[65].

In the plasma, concentration of genistein is consistently higher

than daidzein[64, 65]. Similar to most other flavonoids, isoflavones

glycosides were not found in human plasma after ingesting either 50

mg of isoflavone glycosides (daidzin or genistin) or 250mL of

soymilk[68]. Isoflavone aglycones could be detected when pure

daidzein and genistein were administered[65]. In addition, data of

individual conjugates of daidzein and genistein have also been

reported. Glucuronides predominates in human plasma after

metabolism. Clarke et al. determined the intact isoflavone conjugates

in human urine and the average pattern was found to be 54 percent 7-

glucuronide, 25 percent 4′-glucuronide, 13 percent monosulfates, 7

percent free daidzein, 0.9 percent sulfoglucuronides, 0.4 percent

diglucuronide, and less than 0.1 percent of disulfate[69].Cassidy et al.

investigated the effect of age, gender, and the food matrix on the

bioavailability of isoflavones. Firstly, they showed gender differences

in peak concentrations, in which women attained higher levels of

daidzein in the plasma. Consumption of predominantly isoflavone

aglycones (tempeh) attained a higher serum peak and area than the

administration of corresponding isoflavones glucosides (textured

vegetable protein). At last, consuming isoflavone through liquid

matrix reached a higher absorption rate and earlier peak than solid

soy food[64]. Another study looked into the role of ethnical

difference in isoflavones pharmacokinetic and bioavailability. On

acute intake of soya cheese, Asians absorbed daidzein and genistein

better than Caucasians, while on chronic ingestions, the concentration

of isoflavones in the plasma increased in Caucasians but not in

Asians[70].

Isoflavone family attracted a lot of in vivo studies and they

remain one of the most investigated polyphenols mainly due to their

estrogen-like structures[71]. They belong to the phytoestrogen family

and have been demonstrated to have a plethora of pharmacological

benefits in illnesses such as cardiovascular disease, breast and

prostate cancer, post-menopausal syndrome and osteoporosis. In

cardiovascular-related area, genistein was shown to improve

endothelium function in postmenopausal women after one year of

therapy with dose of 54 mg/day, and the result was comparable to a

similar extent as the estrogen/progestin treatment[72]. A review by

Siow et al. concluded that isoflavones are able to activate

intracellular signaling pathways, leading to increased NO

bioavailability and improving antioxidant gene expression via key

transcription factors NFκB and Nrf2, and hence protecting ones from

cardiovascular-related diseases[73].

Breast cancer is the most common and second leading cause of

cancer deaths among women in the United States. One possible

reason Asian women are less prone to breast cancer compared to

American women could be that traditional Asian diet is rich in soy

products such as miso, tofu and soymilk. This is supported by the fact

that Asian population has a higher mean daily soy intake of 10 to 50g

compared to the intake of 1 to 3g in the USA[74]. In a rodent study,

Lamartiniere et al. looked into the mammary glands of immature rats

after genistein treatment. The expression of epidermal growth factor

was up-regulated shortly after treatment, suggesting that early

genistein action promotes cell differentiation that might result in a

less active epidermal growth factor signaling pathway in adulthood,

and eventually lead to the suppression of the development of

mammary or breast cancer[75]. In a clinically-relevant breast tumor

animal model, bioactive genistein was shown to enhance Tamoxifen

(an estrogen antagonist that interferes with the activity of estrogens in

most estrogen sensitive tissues) activity in a synergistic manner via

induction of tumor cell apoptosis and inhibition of tumor cell

proliferation[76]. In addition, isoflavones have also shown positive

effects in decreasing the likelihood of prostate cancer. Dietary

genistein dose of up to 250mg/kg was shown to decrease human

prostate cancer metastasis in a dose-dependent manner through

mediated inhibition of prostate cancer cell detachment[77]. Another

cancer related case-control study was conducted in Japan on

colorectal adenoma or colon cancer. The researchers observed a

ceiling effect associated with higher isoflavone intake on colon

cancer and showed an inverse association between isoflavone intake

and the risk of colorectal adenoma[78].

In obese Zucker rat (OZR), high isoflavones soy protein

consumption resulted in an improved lipid metabolism and activated

peroxisome-proliferator activated receptors (PPARs), which

subsequently led to an anti-diabetic effect[79]. Besides the potent

effects of various illnesses, isoflavones could also decrease the risk of

osteoporosis[80, 81], improve cognitive performance[82, 83] and

reduce LDL cholesterol and LDL/HDL ratio[84, 85].

Despite all the aforementioned positive reports, there are also

studies that presented contradictory results. Taku et al. reported no

improvement of LDL cholesterol levels in normocholesterolemic

menopausal women ingested with 70 mg of extracted soy

isoflavones/day for 1–3 months[86]. Kreijkamp-Kaspers et al. found

no apparent correlation between dietary isoflavone intake and

cognition functions such as memory, processing capacity and speed,

and executive function[87]. Furthermore, no beneficial effects of

isoflavones were detected on vascular function in older

postmenopausal women[88]. In a review, Cassidy et al. concluded

that the evidence of isoflavone potentials on cancer, diabetes and

cognitive function are inconclusive due to the lack of appropriately

designed studies and inconsistent methodological approaches[89].

FLAVONES

Flavones present in diet primarily as 7-O-glycosides and C-

glycosides[13]. Chrysin, apigenin and luteolin are the most popular

flavones with 0, 1 and 2 hydroxyl substitutions on the B ring

respectively. To date, only a handful of studies have reported the

absorption and bioavailability of flavone family. Shimoi et al. looked

into the bioavailability of luteolin in both rats and humans[90].

Plasma of rats orally administered with luteolin contained free

luteolin, glucuronide, sulfate and methyl conjugates. For the rats

administered with luteolin 7-O-β-glucoside, the glucoside was not

detected in the plasma but luteolin glucuronides were identified. The

author suggested that flavone glucoside was hardly absorbed by

itself; it was hydrolyzed to luteolin by the microbacteria presented on

the surface of the intestinal mucosa and later glucuronidated.

Pharmacokinetic studies revealed the rats‟ luteolin plasma

concentration was about 15µM, 30 minutes after 50µmol/kg was

administered. In the same study, two volunteers were involved in

which they ingested 50mg of luteolin. In the serum of both

volunteers, free luteolin and its monoglucuronide were detected[90].

Similar study with human subjects was conducted, in which two

different artichoke leaf extracts with varied luteolin glucoside

concentrations (equivalent to 14.4mg and 35.2mg of luteolin

respectively) were administered to 14 healthy volunteers. The

reported peak luteolin plasma concentration was 206nM (extract 1)

and 546nM (extract 2) after 0.36 and 0.46 hours respectively[91]. In

another human study, 11 healthy subjects were administered a single

oral bolus of 2g/kg blanched parsley (65.8µmol equivalent of

apigenin) to scrutinize the bioavailability of apigenin. The peak

apigenin plasma concentration in urine was reported to be 127 nM

after about 7.2 hours, and the average apigenin content for the first

24hour was 144nmol or 0.22 percent of the ingested dose[92]. Walle

et al. looked into the bioavailability of chrysin, after 7 subjects

ingested two 200mg capsules of chrysin. The highest chrysin peak

plasma concentration among the volunteers was 16mg/mL or 64nM

and surprisingly, the chrysin sulphate concentratrions were found to

be 30-fold higher than the aglycone concentration; whereas in urine,

free chrysin and its glucuronide accounted for 0.2–3.1mg and 2–

26mg, respectively and only trace amounts of chrysin sulphate were

found[93]. In their earlier in vitro study in human intestinal Caco-2

and hepatic Hep G2 cells, the only detected metabolites were flavone

conjugates[94, 95].

In order to overcome the low bioavailability bottleneck that

flavones possess, Walle et al. proposed methyl-capping all free

hydroxyl groups of flavones. In rats, the oral bioavailability of

methylated flavone was tested by comparing chrysin and its

methylated version 5,7-dimethoxyflavones. When both compound

were co-administered, only the methylated flavone was detected in

the plasma with a peak concentration of 2.3 µM at 1 hour, and it was

found to be accumulated highly in tissue, especially in the liver[96].

In conclusion, methylated flavone could resulted in higher

bioavailability and improved metabolic stability compared to the

aglycones[97-99]. This approach might be a way to overcome the

bottleneck of flavonoids being such low bioavailable polyphenols.

Similar to the bioavailability studies, the most investigated

flavones are the aglycones luteolin, chrysin, apigenin, and their

derivatives. Based on numerous in vitro studies and animal

experiments, flavones exhibit a wide range of biological and

pharmacological benefits. Michels et al. used H4IIE rat hepatoma

cells as a model system for studying luteolin capability on inducing

oxidative stress and apoptosis. It was reported that luteolin is

relatively toxic and it triggered cell apoptosis through the

mitochondria pathway. At a concentration of 250µM, luteolin was

capable of activating and at the same time increasing the activity of

different caspases(used in cell apoptosis)[100]. Besides the capability

of inducing oxidative stress, surprisingly flavone apigenin was found

to have a protective role against oxidative stress and DNA damage in

N-nitrosodiethylamine (NDEA)-induced hepatocellular

carcinogenesis in Wistar albino rats[101]. Apigenin was also

demonstrated to successfully manage the evasiveness of tumor cells

by preventing them from penetrating healthy tissues[102]. In human

pancreatic cancer cells MiaPaCa2, 20µM of luteolin successfully

blocked the epidermal growth factor receptor (EGFR) tyrosine kinase

activity, inhibited the growth of tumor cells and induced

apoptosis[103]. In human prostate cancer DU145 and PC-3 cells,

apigenin was reported to significantly inhibit insulin like growth

factor (IGF)-I-stimulated cell proliferation, dephophorylate Akt and

induced cell death, both in vitro and in vivo[104, 105]. Flavones also

showed potent effects on many other cancer diseases, for example

leukemia[106], breast cancer[107] and etc.

In cardiovascular-related diseases, Jin et al. investigated

apigenin potential on endothelium-dependent vasorelaxation in

isolated rat aortic rings exposed to superoxide anion. 0.5 to 72.0 μM

of apigenin were used and resulted in a concentration-dependent

relaxation in aortas and also provided protection against the

dysfunction caused by the superoxide anion[108]. Similarly, Ma et al.

verified superoxide anion capability to impair acetylcholine-induced

relaxation and hyperpolarization of smooth muscle cells in resistance

arteries, and both luteolin and apigenin protected resistance arteries

from injury, which further verified flavonols‟ vasoprotective

activity[109]. Another article looked into the effect of flavones as

angiotensin converting enzyme (ACE) inhibitors, which are

commonly used to treat cardiovascular diseases. Among the flavones

tested, apigenin, luteolin and luteolin-7-O-β-glucopyranoside

appeared to be potential inhibitor candidates with IC50 values of 280,

290 and 280µM respectively, as compared to the therapeutic drug

captopril (IC50 0.02 µM). Yamagata revealed apigenin function as a

arteriosclerotic vascular disorder protectant by regulating the

activation of NF-κB[110]. Some studies also underlined the anti-

inflammatory effect of flavones. Chrysin concentrations of 1, 5 and

10mg/kg were administered orally to dextran sodium sulfate (DSS)-

induced colitis in mice and flavone was able to attenuate symptoms

and colitis DAI scores of DSS-induced colitis, and inhibited NF-κB

activation in TNF-α-stimulated intestinal epithelial cells[111]. These

results suggested chrysin as a potential therapeutic agent for intestinal

inflammation. Also in mouse model of LPS-induced acute pulmonary

inflammation, two less common flavones, fisetin (3,3',4',7-

tetrahydroxyflavone) and tricetin (3',4',5,5',7-pentahydroxyflavone)

were compared to an anti-inflammatory glucocorticoid

dexamethasone as potential therapeutic agents against pulmonary

inflammation. Fisetin was shown to have the highest potential and it

significantly reduced lung myeloperoxidase-levels and gene-

expression of different inflammatory mediators[112]. Other studies

also examined flavone therapeutic effects on anti-aging, antibacterial,

neuprotective, etc.

Lack of medicinal effects of flavones in some studies was also

reported. Human leukemia cells HL60 and their MDR-1 resistant

subline HL60/VCR were treated with both luteolin and apigenin, but

neither of them regulated the cell apoptotic program[113]. Janssen et

al. reported that at a relatively high concentration of 2500 µmol/L in

vitro, apigenin inhibited collagen- and ADP-induced platelet

aggregation; whereas at lower concentration, it showed no effect. The

hypothesis was again tested in vivo and no significant effects were

found on platelet aggregation, thromboxane B2 production, factor

VII, or other hemostatic variables[114].

In more recent years, methylated flavones have attracted

voluminous studies mainly due to their improved bioavailability

compared to the flavone aglycones. The downside of using the

methylated compounds is associated with their antioxidant potentials.

The major determinants for radical-scavenging ability are based on

the presence of catechol group in the B ring, 2,3-double bond

conjugated with the 4-oxo group in the C ring[115]. O-methylation of

the hydroxyl groups particularly on the B-ring greatly reduces the

overall antioxidant capability of the molecules. Michels et al.

demonstrated that the anti-oxidative activity of luteolin was lowered

by methylation (5,3‟-dimethylflavone and 5,7,3‟,4‟-

tetramethylflavone) in H4IIE rat hepatoma cells, and hence led to the

decline of radical-scavenging activity and to the reduction of luteolin

aglycone‟s inducing-apoptotic potential[116]. Walle et al. who

suggested the methyl-capping approach confuted the fact that even

though the methylated molecules lack antioxidant properties, their

effects as modulators of protein and lipid kinase signaling remain and

in some cases even more potent[97, 117]. For instance, 30µM of

nobiletin (3',4',5,6,7,8-hexamethoxyflavone) was capable of

attenuating the apoptosis induced by H2O2 exposure in human

neuroblastoma SH-SY5Y cells[118]. Methylated flavone also showed

potent inhibitory activity on carcinogen bioactivating enzymes

CYP1A1 and CYP1B1 at low concentration[119, 120]. On cancer

chemoprevention in vitro, methylated flavones were ten times more

potent inhibitors of cell proliferation than the unmethylated flavones

on humanoral SCC-9 cancer cells[96]. Likewise, Cai et al. revealed

the effectiveness of the methoxy group in promoting gastrointestinal

cancer chemopreventive efficacy both in vitro and in vivo[121]. The

issue of using methyl-capping to significantly improve the

bioavailability of not just flavones but also flavonoids and stilbene

requires further investigation as its possible consequences: lower

antioxidant activity might jeopardize a handful of polyphenols vital

bioactivities.

FLAVONOLS

Flavonols occur as glycosides in diet. Onion, kale and apple are

a few major sources of flavonols and they are best represented by

quercetin, kaempferol and myricetin. It has long been known that

flavonol aglycones‟ bioavailability is low. A number of studies have

looked into the bioavailability of quercetin, mostly by using onion as

the source. Quercetin glycosides were generally more efficiently

absorbed than quercetin aglycones, except rutin (quercetin 3-O-

rutinoside), which was reported to be less rapidly absorbed mainly

due to its sugar moeity[122], and these results were confirmed by

Graefe et al.‟s study. A four way crossover study was conducted, in

which 12 subjects were administered with two isolated quercetin

glucosides (quercetin-4‟-O-glucoside and quercetin-3-O-rutinoside)

and two plant extracts (onion and buckwheat tea). Quercetin

glucuronides were found in human plasma but not the aglycones and

the peak plasma concentrations were 7.0, 1.1, 7.6 and 2.1µM

respectively. It was concluded that sugar moiety, glycosylation

position and the dietary sources contribute to the overall flavonols‟

bioavailability[123]. Flavonol bioavailability studies up to date

concluded that flavonols are less bioavailable than their glucosides;

however, this conclusion have defied the typical assumption of

aglycone should be more bioavailable than its glucosides due to the

requirement of either preliminary hydrolysis to aglycones or active

transport for absorption. Wiczkowski et al. attributed the low

bioavailability of the aglycones to their low solubility in the digestive

tract, and they suggested dispersing flavonol quercetin from shallot

into the food matrix. As a result, a higher quercetin bioavailability

than its glucosides was reported[124].

Similar to most flavonoids, flavonol glucuronides and sulfates

occurred predominantly in the circulating plasma. Major metabolites

of quercetin detected in human plasma 1.5 hours after consumption

of onions were quercetin-3-glucuronide, 3'-methylquercetin-3-

glucuronide and quercetin-3'-sulfate[125]. Isorhamnetin (3‟-

methylquercetin) contributed to 20 percent of the absorbed quercetin,

one third was sulfated and the remaining were quercetin glucuronides

or mixed conjugates[40]. Walle et al. also found oxidized quercetin

as a result of ROS oxidation and these quercetin were reported to

covalent bound to DNA and cellular protein in hepatic cells and

human intestinal[31].

There are also some studies that looked into amending the

bioavailability of flavonols, intending to maintain a higher flavonol

plasma concentration for greater distribution and subsequently

greater bioactivity. In a review, Manach et al. reported the baseline

quercetin plasma concentration after overnight fasting and promising

improvement on the baseline after prolonged supplement of flavonols

was reported[126]. A rat study was conducted to further elucidate the

phenomenon. Male CD rats were fed with two distinct administration

procedures: intragastric administration (single 50mg/kg dose) and

free access administration (diet with 1 percent quercetin for 4 weeks).

The total quercetin concentration was higher in the free access group

(7µM) compared to intragastric group (2.5µM), which supported the

improved flavonol baseline hypothesis. The methylated to non-

methylated metabolites ratio was also accounted in which the free

access group had a much higher ratio of 3.79 compared to 0.39 for

the intragastric group[127]. On the other hand, experiments by

Bieger et al. in pig reported no greater flavonol accumulation in any

tissue or plasma between orally administered a single dose quercetin

aglycone (25mg/kg) and twice a day orally administered at the same

dosage for 4 weeks[128]. These results indicated possible

bioavailability variation among organisms of interest and this issue

requires further elucidation. Dietary quercetin combined with pectin

(a heteropolysaccharide used as a thickening agent) could

significantly enhance the intestinal absorption of quercetin. Male

mice fed with pectin-rutin(PR) diet or cellulose-rutin(CR) diet for 14

days resulted in varied plasma concentration of quercetin (PR 2.5µM

versus CR 0.89 µM) and of isorhamnetin (PR 6.66 µM versus CR

0.64 µM)[129]. Similarly, the effect of apple pectin (AP) on

quercetin and rutin bioavailability was investigated. Suprisingly, for

rats with quercetin administration, the maximum plasma

concentration of quercetin metabolites was significantly higher in

AP-fed rats. In contrast, this phenomena was not seen in AP-fed rats

administered with rutin. In short, the apple pectin did not affect rutin

bioavailability[130]. The researchers attributed the disagreement

between the 2 studies to the manner in which rutin was fed.

Flavonols have prominent antioxidant properties which are

among the best among flavonoids, particularly with quercetin. This

compound fulfilled all requirements as a potent free radical

scavenger, with 3‟ and 4‟ hydroxyls on the B ring (catechol group),

2,3-double bond conjugated with the 4-oxo group and 3-hydroxyl on

the heterocyclic C ring[115]. In a recent study, rats with nicotine-

induced prooxidant and antioxidant imbalance in circulation were

given quercetin by intragastric intubations for 22 weeks. Quercetin

modulated the lipid peroxidation and antioxidant status of nicotine-

induced rat, decreased DNA damage and showed protective effect

against nicotine toxicity to the extent of N-acetylcysteine (NAC), a

popular antioxidant[131]. In plant, it has long been demonstrated that

adaptation of plants to high light stress involves accumulation of

protective pigments namely, flavonols which absorb solar radiation in

broad spectral ranges extending from UV to the green and, in some

cases, to the red regions of the spectrum. The build-up of such

substances in specific cell and tissue structures reduces the fraction of

radiation absorbed by potent photosensitizers, and thereby diminishes

light-induced damage. As an important defence mechanism against

the deleterious effects of solar radiation, long-term adaptation of

higher plants involves synthesis of relatively stable flavonols capable

of serving as light screens and/or internal traps. Similar

photoprotection effect was also demonstrated on mice skin by

Campos et al., in which flavonols was applied on dorsal skin of

hairless mice irradiated by UVA/B for 15 minutes[132]. The

association of flavonols to the vitamin was shown to further improve

the antioxidant activity in vitro and protect the mice skin from UV

damage by reducing the number of sunburn cells. Similar outcome

was seen when topical application of 0.2mg/cm2 standardized black

tea extract in SKH-1 hairless mice prior to UVB exposure resulted in

40 percent reduced incidence, 64 percent reduced severity of

erythema and 50 percent reduction in skinfold thickness by day 6

when compared to nontreated UVB-exposed mice[133]. In addition to antioxidant and photoprotective potentials,

flavonols also possess anti-inflammatory effect. In a very recent

study, kaempferol, quercetin and other flavonoids were found to

block tumor necrosis factor (TNF)-α induced interleukin-8 promoter

activation and gene expression in HEK 293 cells. Interestingly,

kaempferol was the only flavonoid compound that did not affect the

cell viability during pretreatment[134]. Similarly, quercetin effect as

an anti-inflammatory compound on neuroinflammation was

investigated and it showed potent effect on lipopolysaccharide (LPS)-

induced mRNA levels of two proinflammatory genes, interleukin 1-α

and tumor necrosis factor-α, in which both were greatly reduced.

Moreover, the inflammation-mediated apoptotic death of neuronal

cells was also inhibited[135]. During inflammation, nitric oxide (NO)

formation is enhanced and Hamalainen et al. revealed that both the

flavonols: kaempferol and quercetin repressed inducible nitric oxide

synthase (iNOS) protein and mRNA level as well as NO production

by first inhibiting the activation of nuclear factor-kappaB (NF-κB) as

well as signal transducer and activator of transcription 1 (STAT-1),

both transcription factor of iNOS[136].

Quercetin is a potent molecule for treating or preventing cancer

due to its anti-mutagenic, anti-proliferative, antioxidative activities

and its role in cellular receptor interactions and modification of signal

transduction[137]. Ginkgo biloba extract was proven to induce

apoptosis by activation of caspase-3 in oral cavity cancer cells[138]

and the individual molecules responsible for the anti cancer potential

were the flavonols, kaempferol and quercetin. Both at concentration

of 40µM were able to induce apoptosis on oral cancer cell lines SCC-

1483, SCC-25 and SCC-QLL1, and showed cleavage of poly (ADP-

ribose) polymerase[139]. Quercetin also inhibited cell proliferation

and induced apoptosis in HepG2 cells in dose- and time-dependent

manner through activation of caspases and down-regulation of two

anti-apoptosis gene, survivin and Bcl-2[140]. Shan et al.

demonstrated quercetin‟s antitumor potential in SW480 colon cancer

cells by inhibiting expression of cycline D1 and survivin through

Wnt/beta-catenin signaling pathway[141]. Reports on quercetin anti-

cancer potentials in vivo have been published. Administration of a

diet containing 4.5g/kg of quercetin was capable of suppressing the

formation of early preneoplastic lesions in azoxymethane (AOM)-

induced rat colon carcinogenesis, together with inhibition of cell

proliferation and induction of apoptosis[142]. Similar studies were

done by comparing the anti-cancer properties of rutin to quercetin but

results are strongly contradictory. Compared to the control (no test

reagent) in AOM-induced rat colon cancer model, quercetin and rutin

decreased the number of aberrant crypt foci, a histological tumor

marker by 4 and 1.2 fold respectively[143]. In contrast, with dextran

sulfate sodium (DSS)-induced colitis in mice, diet containing 0.1

percent of rutin but not quercetin attenuated DSS-induced body

weight loss and shortening of the colorectum, improved colitis

histological scores and significantly blunted colonic mucosal IL-1β

levels[144]. The author attributed the discrepancy again to the model

of study and pointed out that there are differences among mouse, rat

and human gut with respect to the mechanisms each organism utilize

or exclude luminal flavonoids. Quercetin also showed promising

effect on breast cancer[107, 145] and prostate cancer[146, 147].

Apart from inducing apoptosis in flavonols‟ anticancer activity,

they can also reverse apoptosis when cells are under oxidative

stress[137]. Chow et al. showed that quercetin prevented H2O2-

induced apoptosis in macrophages through its antioxidant potential

and heme oxygenase 1 expression; but similar effects were not seen

in both rutin and quercitrin (quercetin 3-O-rhamose)[148]. Quercetin

also facilitated anti-apoptosis in its anti-neurodegenerative potential,

in which it regulated the mRNA levels and protein expression of pro-

apoptotic (Bax) and anti-apoptotic Bcl-2 genes and as a result, the

apoptotic neuronal cell death was diminished[149]. Similar effect of

quercetin was observed in neuronal cultures treated with amyloid

beta (Aβ), an Alzheimer disease protein[150, 151]. Apart from

inhibiting apoptotic cell death, quercetin also attenuated the peptide-

induced cytotoxicity, protein oxidation and lipid peroxidation.

Interestingly, this promising Alzheimer disease treating approach was

only attained at lower quercetin doses (5µM); higher doses (20 µM or

40 µM) were not only non-neuroprotective but toxic effects are

observed[152].

In cardiovascular-related diseases, flavonol has also been shown

to be a powerful cardioprotective agent. Daunorubicin (DNR) was

commonly used for cancer treatment, but its use was limited because

of its association with the development of cardiac toxicity. Mojzisova

et al. revealed quercetin at concentrations between 10µM and 100

µM were able to protect H9c2 cardiomyocyte cells against DNR-

induced damage[153]. As mentioned earlier, flavonol metabolites

predominate in plasma while the aglycone exists in a tiny amount.

Lodi et al. assessed quercetin and its glucuronidated, methylated and

sulfated metabolites vasorelaxant effects, and their roles on NO

bioavailability and endothelial function in rat aorta. It was found that

the free quercetin and its metabolites offered protection against

endothelial dysfunction. The only downside was these conjugates

lack direct vasorelaxant effects, unlike the quercetin aglycone[154].

Flavonols‟ other reported health benefits involved life-

prolonging property in Caenorhabditis elegans[155], anti-

leishmanicidal activity[156], anti-viral activity[157] etc. A recent

review by Bischoff et al. provides a comprehensive assessment of the

biological effects of quercetin[158].

Despite promising results related to the effects of flavonol on

human health, possible adverse outcomes or lack of effects are also

matters of debate. Back in 1997, Hertog et al. investigated the

hypothesis that high flavonols intake resulted in low rate of ischemic

heart disease (IHD). This was done in 1900 Welsh man aged range

from 45 to 59 years old but surprisingly, no correlation was

found[159]. Similarly, effects of the dietary antioxidant

supplementation (quercetin and vitamin C) at 900mg/day on the

blood inflammatory biomarkers and on the severity of rheumatoid

arthritis in patients were assessed after treatment. Again, no

significant differences of pro-inflammatory cytokines such as C-

reactive protein (CRP) in the plasma after 4 weeks of

supplementation were observed. Moreover, the scores of disease

severity measurement were not significantly decreased[160].

Kääriäinen et al. investigated quercetin antioxidant and cytotoxicity

in vitro and neurotoxicity in vivo. Quercetin showed significant

antioxidant effect against 6-hydroxydopamine(6-OHDA)-induced

oxygen radical formation in catecholaminergic SH-SY5Y

neuroblastoma cells. At low concentration in the range of 10 to

100μM, quercetin protective effect was seen by a reduction of

caspase-3 like activity, but at the highest concentration tested (100

μM), enhanced toxicity occurred reversing the protective effect. In

vivo results showed no consistent neuroprotective effect of quercetin

concentration of up to 200mg/kg in 6-OHDA rat lesion models of

Parkinson's disease[161]. In a later study, it was revealed that

quercetin‟s protective in vitro was time-dependent, in which initially,

quercetin protected against 6-OHDA-induced cell death, but after

prolonged treatment, the protective effect began to diminish[162].

The author attributed the discrepancy to the fact that articles reporting

quercetin anti-neuroprotective property only analyzed results in the

early protective stage, missing the toxicity effect that might have

followed. In a phase I clinical trial, cancer patients who were given

intravenous quercetin at escalating doses reported renal toxicity,

chest pain, nephrotoxicity, nausea[163].

FLAVANOLS

One unique characteristic of flavanols that distinguish them

among other flavonoids is instead of being present in glycosides, they

can occur in diet as aglycones. Flavanols are abundant in tea,

chocolate, fruits and red wine[13, 126], and the most representative

molecules of flavanols are (-)-epicatechin and (+)-catechin epimers,

gallocatechins, and catechin gallates, the gallic acid esters of

catechins. Bioavailability and pharmacokinetic studies of flavanols

have been investigated mainly after the administration of tea, cocoa

or red wine[126]. In some studies, pure flavanol compounds were

used. In tea, the most abundant flavanols are (-)-epicatechin gallate

(ECg), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate

(EGCg)[13, 164, 165]. In red wine, relatively high levels of (-)-

epicatechin and (+)-catechin epimers were found[13], while in cocoa

(-)-epicatechin and (-)-catechin, instead of the more common (+)-

enantiomer, were reported[166].

In a human pharmacokinetic study, the peak plasma

concentrations (Cmax) of EGCg, EGC, and EC were found to be 0.17,

0.73 and 0.43µM, respectively at Tmax 1.3 to 1.6 hours after a single

dose of 20mg green tea solids/kg was administered[165]. The results

elucidated the relatively low bioavailability of all three catechin

found in green tea. However, Nakagawa et al. report a much higher

EGCg Cmax of 4.3 and 4.4µM, 1.5 hours after ingesting 375mg and

525mg EGCg equivalent of green tea capsules respectively[167].

Flavanol bioavailability was also tested in cocoa beverage and red

wine. After 5 healthy adults were given 0.375 g cocoa/kg as a

beverage, the Cmax for (-)-epicatechin, and (+)-catechin were 5.92 µM

and 0.16 µM, respectively after 2 hours. Interestingly, epicatechin

was the predominant plasma flavanol even though the measured ratio

during cocoa preparation of epicatechin to catechin was 1:1[168].

Possible explanation for this observation can be that the cleavage of

dimers or other procyanidin oligomers that form epicatechin

monomers were then absorbed[168, 169]. In another study that

involved the consumption of black chocolate, much lower Cmax of

0.383 and 0.7µM were reported after the administration on

epicatechin equivalent dose to 82 and 164mg in chocolate. In wine,

the bioavailability of catechin is extremely low compared to other

flavanol food sources. An intake of 120mL of red wine (35mg

catechin) resulted in a catechin Cmax of 91nM[170].

Investigations on flavanols using pure individual tea catechins

also revealed low bioavalability of each flavanol. It was reported that

no EGC or EC in the plasma after administration of pure EGCg and

the reported EGCg Cmax was 0.96µM[164]. Van Amelsvoort et al.

reported similar Cmax of 1.3µM, but traces of EGC (5.1 percent of

AUC) was detected in the plasma, while after ingesting 663mg of

ECg, its Cmax was found to be 3.1µM with traces of the

epicatechin(3.3 percent of AUC). Interestingly, after the consumption

of 459mg of EGC (the only tea polyphenols without gallic acid

substituent), a separate peak was found in addition to EGC, which

was confirmed as the O-methylated EGC. EGC Cmax was 5.0 µM

whereas the Cmax for the methylated EGC was reported to be 1.9 µM.

In urine, no traces of EGCg or ECg was found but 13.6 percent of

EGC was excreted (9.8 percent aglycone and 3.8 percent

methylated)[171].

In plasma, flavanols can appear as aglycones or as glucuronited,

sulfated or methylated conjugates[13]. For (+)-catechin, Donovan et

al. reported the formation of 3'-O-methylcatechin and their sulfate,

glucuronide, sulfo-glucuronide conjugates after consuming 35mg of

catechin in 120mL of red wine[172]. On the other hand, for (-)-

epicatechin, three metabolites namely (-)-epicatechin-3′-O-

glucuronide, 4′-O-methyl-(-)-epicatechin-3′-O-glucuronide, and 4′-O-

methyl-(-)-epicatechin-5 or 7-O-glucuronid were purified from

human urine[173]. Lu et al. investigated extensively the enzymology

of the human cytosol catechol-O-methyltransferase (COMT)-

catalyzed methylation of EGCg and EGC. This enzyme catalyzed the

methylation of EGC to form 4‟-O-methylEGC and at a lower

concentration, 3‟-O-methyl-EGC. For EGCG, it was first metabolized

to 4”-O-methyl-EGCG, then to 4‟-4”-di-O-methyl-EGCG. The

possible glucuronidation sites of EGCG was reported at 7 position on

the A ring, 3‟ position on the B ring and 3”,4” position on the gallate

ester D ring. Besides, the structure-inhibition activity was also

proposed. It was found that glucuronidated EGCG on the B- or the D-

ring greatly inhibited methylation on the same ring but

glucuronidation on the A ring of both EGC and EGCG did not affect

methylation[174]. Apart from the flavanol conjugates, phenolic acids

were also reported after the consumption of flavanol-rich food.

Colonic microflora has been reported to degrade flavanols to simple

phenolic acids or ring-fission metabolites, valerolactones. 6 phenolic

acids were found in the urine after chocolate intake: m-

hydroxyphenylpropionic acid, ferulic acid, 3,4-

dihydroxyphenylacetic acid, m-hydroxyphenylacetic acid, vanillic

acid, and m-hydroxybenzoic acid[175] while 2 ring-fission

metabolites (-)-5-(3',4',5'-trihydroxyphenyl)-γ-valerolactone and (-)-

5-(3',4'-dihydroxyphenyl)-valerolactone were also identified[165].

Higher and repeated dosing were shown to improve flavanol‟s

bioavailability[164, 176]. Compared to a single grape seed

polyphenolic extract (GSPE) dose, repeated daily exposure to GSPE

in mouse models was found to significantly improve the

bioavailability of catechin and epicatechin by 253 and 282 percent

respectively[176]. The effects of alcohol and milk on the

bioavailability of flavanols were also scrutinized. No difference was

seen in the proportion of individual flavanol metabolites after the

consumption of red wine and de-alcoholized red wine, but ethanol

was able to reduce the elimination half-life of catechin in plasma by

facilitating elimination in urine[172]. Results on the effect of milk on

plasma flavanol are mixed. Serafini et al. reported the absorption of (-

)epicatechin into the bloodstream after ingestion of chocolate was

significantly less when chocolate was accompanied by milk (-

46.4percent) or chocolate itself containing milk(-69.1percent). The

author hypothesized a formation of secondary bonds between the

flavanols and milk proteins that reduced the absorption of

flavanols[177]. Other studies clearly oppose the reported effects.

Roura et al. demonstrated that milk did not affect the bioavailability

of flavanols[178]. Likewise, Mullen et al. revealed negligible effects

on the plasma pharmacokinetics of catechin metabolites when cocoa

beverage was made with water or milk. However, milk had a major

effect on the flavanol metabolite concentrations in urine, with

significant reductions in the amounts excreted of the 4 flavanol

metabolites[179]. The author credited such discrepancies to the

amount of flavanols available in cocoas. With high flavanol

concentrations, milk that reduces absorption have minimal effect

compared to typical commercial cocoas with lower flavanol content

in which milk is capable to affect the absorption.

Besides flavonol, flavanols also possess prominent antioxidant

properties, with both ECg and EGCg being the best of breed,

comparable to or even greater than that of quercetin. The presence of

pyrogallol group in the B ring greatly increases the radical

scavenging activity and this pyrogallol ring is known to outperform

the typical catechol ring. In addition, galloylation of the 3 position

further enhances the radical scavenging capacity. Catechin, which

lacks all these features, had a 50 percent lower Trolox Equivalent

Antioxidant Capacity (TEAC) than both EGCg and ECg[115]. Tea

flourishing with flavonoids, especially flavanols, is not surprising to

possess potent beneficial antioxidant effects. In a recent study on

experimentally induced cerebral hypoperfusion rat models, 400mg/kg

of green tea polyphenols were found to scavenge oxygen free

radicals, enhance antioxidant potential, decrease lipid peroxide

production and reduce oxidative DNA damage in rats. In addition,

the green tea treated rats had better spatial learning and memory

compared to the control rats[180]. Similarly, on cholesterol-fed rats,

green tea polyphenol reduced the susceptibility of LDL to oxidation,

decreased malondialdehyde (oxidative stress marker), improved

antioxidative activity of serum and increased HDL cholesterol

level[181]. However, an in vivo human study showed that single or

double dosage of tea polyphenol extract did not improve the

antioxidant activity measured by ferric reducing antioxidant power

(FRAP) assay. No antioxidant activity increment was detected even

after 7 days of continuous consumption of tea catechins but

significant decrease in the activity was observed 7 days after the

withdrawal from the intake[182]. Cocoa, another food source that

contains abundant flavanols was also shown to exert strong

antioxidant effects. After heat exposure, flavanols in rats fed with

cocoa polyphenolic extract inhibited the generation of free radicals

by activated leukocytes and protected the rat from subsequent

cognitive impairment[183]. Consumption of cocoa also contributed to

the inhibition of lipid peroxidation in human[184] and attenuated in

vitro LDL oxidation[185].

Besides boosting individuals‟ antioxidant defense system,

flavanol-rich green tea and cocoa can affect cardiovascular health and

function, by modulating inflammation, platelet aggregation, nitric

oxide availability and lipid profile that directly affects blood

pressure[186, 187]. In a human study, one week of dark chocolate

consumption elucidated flavanols‟ cardioprotective properties, in

which they improved lipid profile (lowered LDL by 6 percent,

increased HDL by 9 percent) and decreased platelet reactivity[188].

Similarly, a 5-week treatment with green tea extract, or equivalent of

270 mg of EGCg on 14 healthy women resulted in 37.4 percent

reduction in the oxidized LDL concentration[189]. The role of platlet

in the development and manifestation of atherosclerosis, myocardial

infarction and stroke is well documented[186]. Compared to the

placebo group, Murphy et al. reported significantly decreased platelet

function, P-selectin expression, and ADP- as well as collagen-

induced aggregation after healthy volunteers were administered with

234 mg cocoa flavanols and procyanidins for 28 days[190]. When

human umbilical-vein endothelial cells were treated with pure EC,

ECg, EGC and EGCg, only EGCg showed inhibition properties on

endothelial exocytosis in a dose-dependent manner. Besides, EGCg

increased Akt phosphorylation, eNOS phosphorylation, and nitric

oxide (NO) production, which help attenuated vascular

inflammation[191].

Many experimental animal studies and in vitro cell culture

studies have also tested the antimutagenic, ant-carcinogenic and

anticancer potential of flavanols. Kavanagh et al. revealed the effect

of green tea on breast cancer both in vivo and in vitro. Green tea

ingested by 7,12-dimethylbenz(a)anthracene (DMBA)-induced

mammary cancer in rats significantly improved the mean latency of

first tumor, reduced the tumor burden and decreased the number of

invasive tumors. In vitro experiment with the treatment on EGCg on

breast cancer cell lines further confirmed that it inhibited cell

proliferation[192]. Similar in vitro studies on the effect of flavanols

on mammary cancer elucidated two diverse mechanisms of EGCg

and EGC independently inhibited heregulin-beta1 (HRG)-induced

migration/invasion of MCF-7 human breast carcinoma cells and

prevented the metastasis of the cancer cells. EGCg inhibited the

migration/invasion through downregulation of

ErbB2/ErbB3/PI3K/Akt signaling, while EGC functioned through the

disruption of the HRG-stimulated activation of ErbB2/ErbB3 but not

Akt[193]. Aqueos black tea extract reversed testosterone-induced

oxidative stress in Wistar rats that if untreated might result in

development of prostate cancer[194]. In vitro studies also showed

that at 0.2 percent, cocoa polyphenols extracts had a growth

inhibition effect on two prostate cancer cell lines (nonmetastatic

22Rv1 cells and metastatic DU145 cells) but not on normal prostate

cells. The anti-proliferative activity was significantly greater than β-

sitosterol, the most common phytosterol used which only showed

minor growth inhibition. Interestingly, there was no synergistic effect

when both cocoa extract and β-sitosterol were combined for

treatment[195]. Lee et al. presented a detailed review article on the

possible effects of tea and coffee on cancer, particularly prostate

cancer[196]. Green tea, cocoa and pure flavanols have also been

reported to treat other cancer diseases, for instance oral and

gastrointestinal cancer[197-199], lung cancer[200], pancreatic

cancer[201] and liver cancer[202, 203].

Flavanol consumption has also been associated with life

prolonging benefits. Saul et al. found that catechin treatment caused a

marked reduction in Caenorhabditis elegans‟ body length, but an

increase in lifespan, which supported the “Disposable Soma” theory.

Further investigation revealed the importance of catechin in assisting

modulation of stress response and repair system that resulted in a

prolonged lifespan[204]. This result was replicated by Abbas et al.

with the use of main tea polyphenols, EGCg[205]. Flavanols are

also considered to be beneficial for providing ultraviolet protection,

especially with EGCG[206]. Some studies also suggest that

flavanols‟ neuroprotective and anti-neurodegenerative activities[207,

208]. In addition, flavanols administration led to improved body

composition and possible anti-obesity potential[209, 210]. Finally,

flavanols are also associated with anti-diabetic effect, such as

improved glucose tolerance and insulin sensitivity[211, 212].

There are also studies that reported unfavorable or lack of

beneficial effects associated with flavanols. Ried et al. reported no

blood pressure lowering effect of flavonol-rich dark chocolate, after

50g or 750 mg polyphenols equivalent of consumption for 8

weeks[213]. Moreoever, neuroprotective effect of catechin was not

observed in nigrostriatal dopaminergic neurons in Parkinson‟s

disease rat model, unlike other flavonoids, such as flavones,

procyanidins and isoflavones that could attenuate the 6-

hydroxydopamine-induced dopaminergic loss[214]. A cohort study

conducted by Kikuchi et al. revealed no association of green tea

consumption and prostate cancer incidences among 19561 Japanese

men, who on average consumed more tea than the Westerners[215].

Some adverse effects after ingestion of flavanols were reported.

Bonkovsly et al. and Stevens et al. both demonstrated hepatotoxicity

following the consumption of supplements containing tea extracts:

green tea extract from Camellia sinensis and Hydroxycut, a

concoction of plant extracts respectively[216, 217].

ANTHOCYANINS

Studies investigating the bioavailability of anthocyanins that

looked into the pharmacokinetics, absorption, metabolic fate and

excretion have increased exponentially over the past decades in both

human and animal models. In plants, both cyanidin aglycone and its

glycosides, with hydroxyl on the 3-position on the C ring of the

aglycone being occupied by different sugar groups, exist in leaves,

fruits, vegetables and flowers. Anthocyanins pH dependent

colorations from red, purple to blue have attracted interest from food

industries due to their promising properties as natural food colorants.

They can be used to replaced artificial food color that was reported to

increase hyperactivity in children[218]. In order to study the

bioavailability of anthocyanins, animal or volunteers are generally

administered with anthocyanin-abundant berries (elderberry,

blackcurrant, blackberries, chokeberry, etc), juices, wines, extracts or

pulps. After a certain period, plasma and urinary excretion are

collected for analysis. In general, the peak plasma concentrations

(Cmax) of anthocyanins were extremely low, varying from the mid to

low nM range[219]. The highest reported anthocyanin Cmax at 96nM

was attained 2.8 hours after volunteers were administered with 7.1g

of encapsulated chokeberry extract containing 721.4mg of cyanidin 3-

O-glycosides[220]. A merely 5.17nM and 2.53nM Cmax were

reported after consumption of acai pulp and clarified acai juice,

respectively[221]. Similarly, the anthocyanin urinary excretions were

relatively low. Elimination of plasma total anthocyanins followed

first-order kinetics, with rate of urinary excretion reaching maximum

3-4 hours after post-administration and then decreased

exponentially[222, 223]. Kay et al. reported the total urinary

excretion of metabolites and parent compounds of merely 0.15

percent of the initial dose over the course of 24 hours[220]. An even

lower total anthocyanin urinary excretion of 0.05 percent of ingested

dose was reported after the consumption of 12g of vitis vinifera grape

peel extract (equivalent anthocyanin dose of 183.9mg)[224].

Unlike flavonoids, in which glucuronidated, sulfated or

methylated conjugates are generally uncovered in plasma and urine

with little or no native forms found, both intact anthocyanin

glycosides and the metabolized derivatives have been identified in

both plasma and urine[126, 219]. Various glycosides (glucoside,

galactoside, rutinoside, sambubioside) glycosylated at the 3-position

and anthocyanin derivatives on the B ring (delphinidin, malvidin, etc)

could be absorbed directly into the plasma. Miyazawa et al. showed

that in both humans and rats, cyanidin-3-O-glycoside and cyanidin-

3,5-diglycoside were absorbed from the digestive tract into the blood

circulation system in structurally intact forms[225]. In addition,

cyanidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3-

sambubioside, cyanidin-3-sambubioside-5-glucoside have also been

detected unaltered in plasma, urine or both[226-228]. In a recent

human study, following 12g of grape extract oral ingestion, 3-

monoglucosides of delphinidin, cyanidin, petunidin, peonidin and

malvidin, were detected in their intact forms in both plasma and

urine, except for cyanidin 3-glucosides, which could not be identified

in the plasma alone[224]. No further explanation was given on the

undetected glucosides.

In addition to the intact anthocyanin glycosides, their

metabolites have also been profoundly investigated. Tian et al.

showed that the methylation patterns of anthocyanin glycosides were

primarily affected by the structures of the glycoside moieties

(monoglycosides, diglycosides or triglycosides). Methylation

occurred on the 3‟-hydroxy on the B-ring of anthocyanin

monoglycoside and diglycoside, whereas for anthocyanin

triglycosides like cyanidin 3-xylosylrutinoside, methylation could

occur at either 3‟- or 4‟-hydroxyl[219, 229]. Both glucuronide and

sulfate conjugates of cyanidin have been studied. In most of the

studies, mono-glucoronidated anthocyanin like peonidin, isopeonidin,

cyanidin and cyanidin-3-glucoside monoglucuronides were

reported[220, 224, 230-233]. The urinary excretion of cyanidin 3-

glucoside diglucuronide was also detected after consumption of

steamed red cabbage[234]. In the case of sulfoconjugates,

pelargonidin- and cyanidin-sulfates have been identified in the

urine[235, 236]. Anthocyanin aglycones or anthocyanidins have been

identified only in a appreciable amount in a few studies due to their

relative instability. Hassimotto et al. attributed the trace amounts of

the aglycones in small and large intestines to the action of

endogenous and microbial β-glycosidases, which hydrolyze or

deglycosylate flavonoid glycosides for subsequent glucuronidation

and sulfoconjugation[230].

The main metabolites resulted from the degradation by the

intestinal microflora are phenolic acids and aldehydes. Woodward et

al. elucidated rapid degradation of anthocyanins to phenolic acids (4-

hydroxybenzoic acid, protocatechuic acid, gallic acid) and aldehydes

(phloroglucinol aldehyde) under simulated (in vitro) physiological

conditions. In addition, the author also revealed the inversed

correlation between the number of hydroxyl groups on the B-ring and

the stability of anthocyanin molecules[237]. In an earlier study

conducted by Vitaglione and colleagues, protocatechuic acid was

found to be the major metabolite of cyanidin-glucosides in humans,

accounting for 44 percent of the ingested cyanidin-3-glucosides in the

plasma (6 hours) and 28.1 percent in the feces (24 hours) after

administration of blood orange juice[238]. However, no

protocatechuic acid was detected by Hassimotto et al. after Wistar

rats were orally administered with anthocyanin-rich extract from wild

mulberry[230].

The diverse result among studies in the area of bioavailability,

including plasma and urine concentrations as well as the availability

of certain compound can be attributed to a few factors including

source of anthocyanins, food matrix, acylation, dosage, individual

variation, analytical methodology and the sensitivity of detection

method[219, 234]. A very recent study revealed the effect of dosage,

acylation and plant matrix on the bioavailability of anthocyanin in

purple carrot juice. Highest peak anthocyanin plasma concentration

was obtained with 250mL of carrot juice containing 380.2µM of

anthocyanin compared to 2 other lower doses of 50mL (76.1µM) and

150mL (228.1µM). Two major findings in this report were

nonacylated anthocyanins were more bioavailable than acylated

anthocyanins and secondly, increased administration dose resulted in

a increased absorption but decreased absorption efficiency[234]. On

an even greater administration dosage of 714µM equivalent

anthocyanin published in later publications, no peak plasma

concentration was observed suggesting saturation of anthocyanin

occurred at dosage range from 250 to 350µM[234, 239]. These

results highlighted the fact that increased dosage resulted in improved

peak plasma concentration up until a saturation point. In addition,

sample preparation techniques and the molecule structure can also

contribute to the overall recovery of anthocyanins. Woodward et al.

demonstrated that solid-phase extraction (SPE) adopted by different

studies was seriously affected by hydroxylation on the B-ring and

resulted in compound degradation and lost of anthocyanins[237].

Similar to other polyphenols, anthocyanins affect a huge array of

biological activities and many of the potentials are related to their

inherent antioxidant capacities[71]. Anthocyanins and the aglycones

antioxidant activities are equipotent to quercetin and catechin gallates

(EGCg), especially with the aglycone cyanidin. Similar to other

antioxidants, catechol structure of the B-ring like cyanidin drastically

affects the overall radical-scavenging capacity of the molecules; for

example pelargonidin with the missing 3‟-hydroxyl group had a

lower overall antioxidant capacity by more than 3-fold compared to

cyanidin[115]. These results were further confirmed by Fukumoto et

al. who also reported improved antioxidant activity with increased

number of hydroxyl groups on the same ring whereas decreased

activity with the glycosylation of anthocyanidins[240]. However, in

vitro and in vivo studies have presented anthocyanins compelling

potentials on modulating nitric oxide(NO) production[241, 242],

protecting cells against oxidative stress[243] and oxidative

damage[244], inhibiting lipid peroxidation[245], reducing reactive

oxygen species (ROS)[246].

Anthocyanins‟ anti-carcinogenic, anti cancer and

chemopreventive activities were extensively assessed in in vitro,

animal cancer models and also controlled human dietary intervention

studies. In vitro tests have been performed on different human cancer

cell lines under different experimental conditions to show

anthocyanins‟ anti-proliferative, pro-apoptotic and pro-oxidant

potentials by regulating gene and protein expression[71, 219]. Hafeez

et al. showed that delphinidin was able to induce apoptosis on human

prostate cancer cells both in vitro and in vivo. Delphinidin treatment

of human prostate cancer LNCaP, C4-2, 22Rnu1, and PC3 cells gave

rise to cell growth inhibition and induced apoptosis in a dose-

dependent manner by activation of caspases. Similar results were also

presented in vivo in athymic nude mice implanted with PC3 cells, in

which delphinidin significantly inhibited tumor growth[247]. In rats,

black raspberries and anthocyanin-rich fractions were shown to

prevent esophageal tumorigenesis by inhibiting cell proliferation,

inflammation, and angiogenesis as well as inducing cell death in both

preneoplastic and papillomatous esophageal tissues[248].

Mirtoselect, an anthocyanin-rich standardized bilberry extract as well

as isolated cyanidin-3-glucoside were tested on ApcMin mouse models

of intestinal carcinogenesis for 12 weeks at a concentration of up to

0.3 percent in the diet. As a result, the number of intestinal adenomas

was decreased by 45 and 30 percent, respectively compared to the

untreated mouse[249]. Similarly, the chemopreventive role of

Mirtocyan (previously Mirtoselect) was later elucidated on 25

colorectal cancer patients. The consumption of up to 2g of

anthocyanins daily for 7 days before surgery inhibited the tumor

tissue proliferation by 7 percent compared to pre-intervention

values[250].

Several studies looking into anthocyanins‟ cardioprotective

effects were intended to investigate the underlying mechanisms in the

vascular system. Human umbilical vein endothelial cells (HUVECs)

were pretreated with Aronox, an anthocyanin-rich extract from

Aronia melanocarpa E before 7β-hydroxycholesterol-induced

apoptosis treatment. Aronox significantly decreased apoptosis by

inhibiting cytochrome c release, reversing both the down-regulation

of Bcl-2 and up-regulation of caspase-3[251]. In another study, pure

cyanidin- and peonidin-3-glucosides were treated on CD40-mediated

endothelial activation and apoptosis in HUVECs. Similarly, both

anthocyanins inhibited CD40-induced apoptosis, JNK and p38

activation whereas endothelial activation was also prevented by

limiting the production of pro-inflammatory cytokines and matrix

metalloproteinases[252]. Anthocyanins‟ cardioprotective effects were

also confirmed in vivo. Wistar rats fed with anthocyanin-rich diet for

8 weeks successfully protected the myocardium from ischemia-

reperfusion injury ex vivo as well as in vivo by possibly improving the

heart endogenous antioxidant defenses[253]. Atherosclerosis-induced

mice fed with anthocyanin-rich extracts from black rice for 20 weeks

had significantly improved lipid profile by decreasing serum

triglyceride, total cholesterol and non-HDL cholesterol, and hence,

resulted in a smaller atherosclerotic plaque area[254]. In dyslipidemic

patients, the intake of berry-derived anthocyanin supplement was

capable of increasing HDL-cholesterol concentrations, decreasing

LDL-cholesterol concentrations and improved the cellular cholesterol

efflux to serum[255].

Significant life-prolonging effect of anthocyanin was also

demonstrated by Butelli et al.. The group engineered tomato intended

to boost the suboptimal anthocyanin level. By expressing two

transcription factors of snapdragon in tomato, they successfully

induced the high accumulation of anthocyanin in tomato to the level

found in berries, which enhanced the overall antioxidant capacity of

tomato fruit by 3-fold and resulted in fruit with intense purple

coloration in both peel and flesh. The life span of cancer-susceptible

Trp53(-/-) mice fed with 10 percent of the engineered tomato powder

was significantly enhanced with average lifespan of 182.2 days

compared to the control diet (142 days) and red tomato diet

(145.9days)[256]. Anti-inflammatory effects exhibited by dietary

anthocyanins have also been evaluated. Recently, a study by DeFuria

et al. utilizing mice on high-fat diet showed that blueberry powder

anthocyanin displayed protective potential against adipose tissue

inflammation by attenuated the up-regulation of inflammatory genes

including tumor necrosis factor-α, interleukin-6, monocyte

chemoattractant protein 1 and inducible nitric oxide synthase[257].

Evidences suggesting anthocyanin vision improvement potential have

also been demonstrated. Matsumoto et al. verified the effects of

purified cyanidin 3-glucoside and cyanidin 3-rutinoside on

accelerating the regeneration of rhodopsin, most probably through

improved formation of a regeneration intermediate[258]. In a human

study, administration of high doses of anthocyanoside oligomer daily

for 4 weeks, improved morphoscopic objective contrast sensitivity in

myopia subjects[259]. In addition to the plethora of bioactivities

discussed, anthocyanins also possess anti-obesity and anti-

diabetic[257, 260], neuroprotective[246, 261], and gastroprotective

properties[262].

Despite all the health benefits, Galvano et al. doubted the

relevance of in vitro culture experiments performed exclusively with

anthocyanin aglycones and the significance of in vivo studies

evaluating the bioactivity anthocyanins possess. This is mainly due to

the nature of anthocyanins at physiological pH that can be easily

degraded to protocatechuic acid and other benzoic acids, resulting in

the low amount of actual anthocyanins to act on cells as well as

tissues, and promote their health benefits[263]. Similarly, there are

also reports declaring absolutely no effect on certain reported

bioactivities. Moller et al. showed no effect of blackcurrant juice or

an anthocyanin drink prepared from blackcurrant anthocyanin

concentrate on DNA damage markers in 57 healthy volunteers[264].

Possible explanation to this observation was that the antioxidant

activity might only show protective effect on oxidatively stressed

subjects.

STILBENES

Trans-resveratrol is by far the most extensively investigated and

reported stilbene, as compared to other analogs, like its cis

counterpart, pinosylvin (trans-3,5-dihydroxystilbene) and piceatannol

(trans-3,5,3',4'-tetrahydroxystilbene). Pharmacokinetic studies

indicate that circulating resveratrol in the plasma is extensively

metabolized in human body and the oral bioavaibility of resveratrol is

close to zero[265], being restricted by limited absorption, limited

chemical stability, and degradation by intestinal microflora and

intestinal enzymes[94, 95, 266]. In Walle et al.‟s bioavailability

study, when 14C-resveratrol doses of 25 mg were orally administered

to six healthy volunteers, the peak resveratrol and metabolite plasma

concentration was 491 ng/mL or equivalent to 2µM after an hour,

followed by a second peak of 1.3 µM and plasma concentrations

declined exponentially thereafter[267]. Similarly, Yu et al. reported

that virtually no unconjugated form of resveratrol was found in the

plasma or urine samples[268]. Resveratol is mainly distributed to

various tissues in its conjugated forms in humans, and resveratrol

glucuronides as well as sulfates predominate the plasma[267, 269].

The fact that resveratrol remains intact after incubation with human

liver microsomes shows that phase I enzymes are inactive in

resveratrol metabolism[268, 270]. Phase II enzymes, which are active

in conjugation reaction, are the ones that actively metabolizing the

intact resveratrol and resulting in the formation of the conjugates.

Resveratrol received little interest until 1992, when it was

postulated to explain the “French paradox”, the phenomenon that

describes the inverse correlation between the highly saturated fat

consumption and the number of cardiovascular diseases in France

compared to the USA. This led to the hypothesis that consumption of

relatively high amount of red wine might lead to cardioprotective

effects. Since then, dozens of publications have revealed different

human related health benefits associated with resveratrol, including

antioxidant, anticancer, anti-inflammatory, antiviral, anti-aging and

life-prolonging activities[14, 271-273].

Out of all the health associated benefits resveratrol possesses,

the most intriguing properties are its anti-aging and life-prolonging

potentials. A discovery in the 1930s revealed the inverse relationship

between calorie intake and lifespan, and recently, sirtuin, a highly

conserved class of NAD+-dependent deacetylase, has been shown to

play a significant role in calorie restriction[274, 275]. Up to 20000

compounds were screened for sirtuin activator compounds (STAC)

using fluorescent deacetylation assay and resveratrol was found to be

the most potent sirtuin activator, up to 14-fold improvement[271].

Howitz et al. also demonstrated resveratrol potential in yeast on

mimicking calorie restriction, increasing DNA stability and

eventually extending their lifespan by up to 70 percent[271]. Besides,

resveratrol has been demonstrated to extend the lifespan of

evolutionary distant species such as C. elegans, D. melanogaster and

vertebrate fish[276-278]. In a more recent publication by Baur et al.,

mice models were used to study the life prolonging properties of

resveratrol. They were divided into 3 main groups: standard diet mice

(SD), high calorie diet mice (HC) and high calorie diet mice with

resveratrol (HCR). After 114 weeks, resveratrol successfully reduced

the risk of death from HC by 31 percent to a point similar to the SD

or in other words, resveratrol successfully shifted the physiology of

HC towards that of mice on a SD without the need of calorie

restriction. Furthermore, resveratrol opposed the effects of high

calorie diet in 144 out of 153 significantly altered pathways and was

found to improve insulin sensitivity, improve liver histology, increase

mitochondrial number, etc[14]. It was later found that resveratrol did

not actually extending the lifespan of mice, but it delayed age-related

deterioration, such as reduction of osteoporosis, cataracts, vascular

dysfunction and declined in motor coordination[279].

There are a few limitations involved in the anti-aging effect of

resveratrol. First, these results are yet to be replicated in human and a

human clinical trial that is specifically for anti-aging study would

take a considerable long period of time. An alternative to the

difficulties is instead of anti-aging, focuses could be directed to the

potentials on age-related diseases, such as diabetes, heart attack and

cancer. Second, to achieve the equivalent dose of resveratrol fed to

the high calorie diet mice that resulted in a longer lifespan[271, 276-

278], a equivalent human dose of about 1000 bottles/day of red wine

or 60L/day of red wine with high resveratrol concentration is

required, which is unfeasible. Therefore, more efforts are needed to

be laid on elucidating the “French Paradox” because the apparent

cardioprotective potentials are probably unachievable through daily

consumption of wine. The only way to achieve the anti-aging and

cardioprotective dose has to be done through pharmacology

approach, for instance with resveratrol concentrated capsules or

supplements. However, clinical studies using high concentrated

polyphenols have showed potential toxicity effects in treated patients.

Williams showed that up to 700mg/kg of Resvida (high purity trans-

resveratrol) fed in rats daily for 90 days caused no adverse effects,

such as toxicity and it was also found that high concentration of

resveratrol was well-tolerated both in vivo and in vitro (rat

hepatocytes and Caco-2 cells)[280]. Human clinical studies also

showed no serious adverse effect after ingesting a single dose of up to

5g of resveratrol[281].

Besides strong evidence of its anti-aging and life-prolonging

properties, resveratrol also showed potential benefits in the treatment

of diabetes, neurodegenerative disorder and cancer. In an animal

study, streptozotocin-induced rats pretreated with resveratrol showed

an enhancement in catalase activities, nitric oxide and zinc levels, and

a decrease in lipid peroxidation product malondialdehyde (an

oxidative stress marker) and copper concentrations[282]. In addition,

resveratrol helped maintain insulin sensitivity in diet-induced obese

mice[283]. Regarding neurological disorders, Yousuf et al.

underlined the significance of resveratrol in the preservation of

ischemic neurovascular units and its ability in the treatment of

ischemic stroke in cerebral ischemia/reperfusion(I/R)-induced

mitochondrial dysfunctions in rats[284]. Also in Alzheimer‟s disease,

resveratrol reduced the plaque formation in a region specific manner,

including media cortex (-48 percent), striatum (-89 percent) and

hypothalamus (-90percent) after 45 days administration of resveratrol

in Alzheimer‟s disease transgenic mice[285].

Resveratrol also thrives in its anticancer activity at the initiation,

and progression stages[270]. This has been shown in both animal and

in vitro studies. Resveratrol suppressed prostate cancer in transgenic

adenocarcinoma mouse prostate when 625 mg resveratrol was

administered per kg diet for 28 weeks[286]. Similarly, resveratrol

reduced both the number and weight of the lung metastases in rat

models[287]. In human in vitro studies, a 48 hour treatment with

100µM resveratrol activated the “death pathway” in human colorectal

cancer DLD1 and HT29 cells[288]. Resveratrol also inhibited tumor

growth of in mouse xenograft models of human neuroblastoma. It

was reported that the loss of mitochondrial membrane potential was

an early response to resveratrol. When the isolated mitochondria were

treated with resveratrol, depolarization occurred, and mitochondria

released cytochrome c and Smac/Diablo and subsequently activated

the caspases[289]. Zhou et al. utilized human hepatocellular

carcinoma-derived HepG2 cells as a model to study resveratrol effect

on cell growth, cell cycle progression and apoptosis. At low

concentration (6.25 to 25µM), resveratrol did not cause cytotoxicity

or cell apoptosis but it slowed down cell cycle progression by

prolonging the synthesis phase (S phase). The slowing down of the

cell cycle at this stage allocated more time to repair damaged DNA,

hence reduced the chance of tumorigenesis and mutagenesis.

However, S-phase arrest was not observed at high concentration (50

to 100 µM); indeed resveratrol induced apoptosis and growth

inhibition mediated by mitochondria pathway[290].

Resveratrol has also been reported to show cardioprotective

effects. Recently, Yang et al. underlined the protective effect of

resveratrol in monocrotaline (MCT)-induced right ventricular

hypertrophy in rats by improving various detriment caused by MCT,

such as increased right ventricular wall thickness, systolic pressure

and hypertrophy, mitochondria swollen and cardiomyocyte

apoptosis[291]. In doxorubicin-induced cardiomyocyte death,

resveratrol also reversed the oxidative stress and cell death induced

by doxorubicin through manipulation of mitochondrial function[292].

Other possible therapeutic potentials of resveratrol are anti-

bacterial[293], anti-viral[294], anti-asthmatic[295],etc.

There were also articles reporting the lack of bioactivity on

several area of interest. 10ppm of resveratrol showed no activity on

pancreatic carcinogenesis in either the initiation or post-initiation

stages of pancreatic carcinogenesis models in hamsters, which

possibly due to the insufficient dosage used for pancreatic

protection[296]. In two eight-weeks long rat feeding experiments,

rats fed with either high resveratrol diet (300mg/kg body weight) or

low resveratrol diet (50mg/kg) revealed no effect on different

chemopreventive parameters, except for the total antioxidant

activity[297]. Researchers attributed the lack of effect to the

formation of resveratrol conjugates, which subsequently lowered the

resveratrol bioavailability.

CONCLUSION AND FUTURE PERSPECTIVES

Polyphenols are common in our daily diet and ample analytical

studies have provided good indications of individual flavonoids and

stilbene distribution in nature with flavanones found predominantly

in citrus fruits, flavones in parsley, isoflavones in leguminous plant,

flavonols in onion, flavanols in green tea and cocoa, anthocyanins in

berries, and stilbenes in red wine. Voluminous studies have identified

the tremendous health potentials of polyphenols varying from

cardioprotective, anti-carcinogenic, vasoprotective to lifespan

prolonging and anti-aging shown both in vivo and in vitro (Fig. [3]).

In order to benefit from these health potentials that polyphenols

offer, bioavailability studies are essential. Absorption, distribution,

metabolism and excretion (ADME) studies allow precise

investigation of the actual metabolites presented in the plasma and

urine, their peak plasma concentration (Cmax), the duration to reach

the respective peak (tmax), the duration the compound remains in the

body before excretion and the percentage of excretion compared to

the ingested amount. As presented in this review, most of the

flavonoids and stilbene showed low bioavailability mainly due to

efflux transporter and more importantly extensive metabolism

through glucuronidation, sulfation and methylation[98]. The free

hydroxyls exist in polyphenols are major targets for these UDP-

glucuronosyltransferases (UGTs), sulfotransferases (SULTs) and

catechol-O-methyl transferases (COMTs). In addition to flavonoids

metabolites, phenolics, carboxylic acids and carbon dioxide are

detected as a result human gut microbial degradation. Simons et al.

studied in vitro flavonoids degradation using cultivated gut

microflora[298]. The degradation rates for 5,7,4‟-trihydroxyl

flavonoids, such as naringenin, genistein, apigenin, and kaempferol

were significantly higher than other structural motifs. The structure-

activity relationship studies were generated to examine the effect of

A, B, C rings and substitution patterns on the degradation kinetics.

No rate differences were observed with A-ring variations; on the B-

ring, 4‟-hydroxyl was crucial for rapid degradation but only with the

present of 5- and 7-hydroxyls; on the C-ring, existence of the double

bond at the 2-3 position and 3-hydroxyl did not affect the rate of

microbial degradation while substitution of B-ring at position-3

(genistein) instead of position-2 (apigenin), did not affect the

degradation rate. Interestingly, daidzin (genistein glycosylated at

position-7) was rapidly hydrolyzed to daidzein while puerarin

(genistein glycosylated at position-8) was not. Puerarin was the only

tested compound that was resistant to degradation[298]. In

conclusion, polyphenol aglycones and glucosides that predominate in

food sources are not the primary compounds circulating the plasma

and subsequently acting on target tissues. Instead, the glucuronidated,

sulfoconjugated and methylated derivatives as well as the phenolic

acids, resulting from intestinal, hepatic conjugation and microflora

degradation, are the ones that possibly give rise to the reported health

potentials. The low bioavailability of flavonoids and stilbene can be

mainly attributed to the hydroxyl substitutions on the A and B rings.

Furthermore, sugar moieties, number of hydroxyls and position of

glycoside substitution can greatly affect the overall bioavailability of

polyphenols.

Figure 3. Summary of flavonoid and

stilbene bioefficacy and protective

potentials against various diseases in

humans.

In vitro studies looking into health benefits are useful in

identifying potential target molecules and understanding individual

mechanism of action. However, to identify therapeutic potential,

animal and human studies are more appropriate because most

concentrations used in vitro are significantly higher and unrealistic

compared to the actual concentration distributed to the target tissues

in vivo. This factor can explain the results of numerous human and

animal studies that cannot be extrapolated from in vitro observations.

This review provides a comprehensive discussion on the

bioavailability as well as the bioefficacy of stilbene and 6 subclasses

of flavonoids. In terms of bioavailability, they are generally low but

uncertainties still exist particularly in some contradictory results in

absorption, metabolism and tissue distribution that are yet to be

clarified. Also, given the contradictory in vivo results reported

previously, more efforts need to be laid on elucidating the issues

specifically on the bioactivity, toxicity and the adverse effects of

individual compounds.

Future in vitro studies exploring the benefits of polyphenols

should include main metabolites, and the concentration treated has to

be close to the concentration detected in plasma or at least

physiological viable. In vivo experiments should investigate the use

of well-defined mixtures of pure compounds in addition to whole

food or food extracts. This could possibly assist in identifying not

only the true effects of individual compounds, but also the possible

synergistic effects of different analogs that might exist. In terms of

bioavailability studies, future experiments can focus on more

strategies to improve bioavailability of different compounds, in

addition to the ones mentioned in this review: dispersing in food

matrix, methyl-capping exposed hydroxyls, combining with

thickening agent (pectin), prolong supplement, etc. Methyl-capping is

one promising approach that involves modifying the tested

compound. It has been shown to improve bioavailability but at the

same time decrease antioxidant potential. Improved polyphenols‟

bioavailability could help maintain a higher plasma concentration for

distribution to the targets and possibly improved bioactivity. Finally,

future studies should relate both the bioavailability and bioefficacy,

mainly on in vivo studies instead of addressing each of them

independently. Pharmacokinetics-pharmacodynamics relationships

are crucial and have to be clarified to avoid question on whether the

compounds themselves or their derivatives that are offering the

plethora of pharmacological benefits.

ACKNOWLEDGEMENTS

Chin Giaw Lim acknowledges the help of master student Lynn Wong

with the figures.

REFERENCES

[1] Lepiniec, L.; Debeaujon, I.; Routaboul, J.-M.; Baudry, A.; Pourcel, L.; Nesi, N.; Caboche, M., Genetics and biochemistry of seed flavonoids. Annual review of plant biology 2006, 57, 405-30.

[2] Gould, K. S.; Lister, C., Flavonoid functions in plants. Flavonoids 2006, 397-441.

[3] Jones, K. M.; Kobayashi, H.; Davies, B. W.; Taga, M. E.; Walker, G. C., How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Micro 2007, 5 (8), 619-633.

[4] Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C., The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000, 52 (4), 673-751.

[5] Chemler, J. A.; Koffas, M. A. G., Metabolic engineering for plant natural product biosynthesis in microbes. Current Opinion in Biotechnology 2008, 19 (6), 597-605.

[6] Watts, K.; Lee, P.; Schmidt-Dannert, C., Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli. BMC Biotechnology 2006, 6 (1), 22.

[7] King, R. E.; Bomser, J. A.; Min, D. B., Bioactivity of Resveratrol. Comprehensive Reviews in Food Science and Food Safety 2006, 5 (3), 65-70.

[8] Justesen, U.; Knuthsen, P.; Leth, T., Quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. Journal of Chromatography A 1998, 799 (1-2), 101-110.

[9] Peterson, J. J.; Beecher, G. R.; Bhagwat, S. A.; Dwyer, J. T.; Gebhardt, S. E.; Haytowitz, D. B.; Holden, J. M., Flavanones in grapefruit, lemons, and limes: A compilation and review of the data from the analytical literature. Journal of Food Composition and Analysis 2006, 19 (Supplement 1), S74-S80.

[10] Kyle, J. A. M.; Duthie, G. G., Flavonoids in foods. Flavonoids 2006, 219-262.

[11] Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jimenez, L., Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004, 79 (5), 727-747.

[12] Fukutake, M.; Takahashi, M.; Ishida, K.; Kawamura, H.; Sugimura, T.; Wakabayashi, K., Quantification of genistein and genistin in soybeans and soybean products. Food and Chemical Toxicology 1996, 34 (5), 457-461.

[13] Hollman, P. C. H.; Arts, I. C. W., Flavonols, flavones and flavanols - nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture 2000, 80 (7), 1081-1093.

[14] Baur, J. A.; Pearson, K. J.; Price, N. L.; Jamieson, H. A.; Lerin, C.; Kalra, A.; Prabhu, V. V.; Allard, J. S.; Lopez-Lluch, G.; Lewis, K.; Pistell, P. J.; Poosala, S.; Becker, K. G.; Boss, O.; Gwinn, D.; Mingyi, W.; Ramaswamy, S.; Fishbein, K. W.; Spencer, R. G.; Lakatta, E. G., Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006, 444 (7117), 337-342.

[15] Opie, L. H.; Lecour, S., The red wine hypothesis: from concepts to protective signalling molecules. Eur Heart J 2007, 28 (14), 1683-1693.

[16] Austin, M. B.; Bowman, M. E.; Ferrer, J. L.; Schroder, J.; Noel, J. P., An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chem Biol 2004, 11 (9), 1179-94.

[17] Morita, H.; Noguchi, H.; Schroder, J.; Abe, I., Novel polyketides synthesized with a higher plant stilbene synthase. European Journal of Biochemistry 2001, 268 (13), 3759-3766.

[18] Chemier, J. A.; Fowler, Z. L.; Koffas, M. A.; Leonard, E., Trends in microbial synthesis of natural products and biofuels. Adv Enzymol Relat Areas Mol Biol 2009, 76, 151-217.

[19] Akashi, T.; Aoki, T.; Ayabe, S., Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis. Plant Physiol 2005, 137 (3), 882-91.

[20] Biddle, M. M.; Lin, M.; Scheidt, K. A., Catalytic Enantioselective Synthesis of Flavanones and Chromanones. Journal of the American Chemical Society 2007, 129 (13), 3830-3831.

[21] Su, W. W., Bioreactor engineering for recombinant protein production using plant cell suspension culture. Focus on Biotechnology 2008, 6 (Plant Tissue Culture Engineering), 135-159.

[22] Wellmann, E., Ultraviolet dose-dependent induction of enzymes related to flavonoid biosynthesis in cell suspension cultures of parsley. FEBS Letters 1975, 51 (1), 105-7.

[23] Meyer, J. E.; Pepin, M. F.; Smith, M. A. L., Anthocyanin production from Vaccinium pahalae: limitations of the physical microenvironment. Journal of Biotechnology 2002, 93 (1), 45-57.

[24] Zhong, J. J.; Seki, T.; Kinoshita, S.; Yoshida, T., Effect of light irradiation on anthocyanin production by suspended culture of Perilla frutescens. Biotechnology and Bioengineering 1991, 38 (6), 653-8.

[25] Leonard, E.; Koffas, M. A. G., Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli. Applied and Environmental Microbiology 2007, 73 (22), 7246-7251.

[26] Hannemann, F.; Bichet, A.; Ewen, K. M.; Bernhardt, R., Cytochrome P 450 systems - biological variations of electron transport chains. Biochimica et Biophysica Acta, General Subjects 2007, 1770 (3), 330-344.

[27] Tomas-Barberan, F. A.; Garcia-Conesca, M. T.; Larrosa, M.; Cerda, B.; Gonzalez-Barrio, R.; Bermudez-Soto, M. J.; Gonzalez-Sarrias, A.; Espin, J. C., Bioavailability, metabolism, and bioactivity of food ellagic acid and related polyphenols. Recent Advances in Polyphenol Research 2008, 1, 263-277.

[28] Zhang, L.; Zuo, Z.; Lin, G., Intestinal and Hepatic Glucuronidation of Flavonoids. Molecular Pharmaceutics 2007, 4 (6), 833-845.

[29] Walle, T., Absorption and metabolism of flavonoids. Free Radic Biol Med 2004, 36 (7), 829-37.

[30] Prasain, J. K.; Barnes, S., Metabolism and Bioavailability of Flavonoids in Chemoprevention: Current Analytical Strategies and Future Prospectus. Molecular Pharmaceutics 2007, 4 (6), 846-864.

[31] Walle, T.; Vincent, T. S.; Walle, U. K., Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells. Biochem Pharmacol 2003, 65 (10), 1603-10.

[32] Brett, G. M.; Hollands, W.; Needs, P. W.; Teucher, B.; R. Dainty, J.; Davis, B. D.; Brodbelt, J. S.; Kroon, P. A., Absorption, metabolism and excretion of flavanones from single portions of orange fruit and juice and effects of anthropometric variables and contraceptive pill use on flavanone excretion. British Journal of Nutrition 2009, 101 (05), 664-675.

[33] Manach, C.; Morand, C.; Gil-Izquierdo, A.; Bouteloup-Demange, C.; Remesy, C., Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur J Clin Nutr 2003, 57 (2), 235-242.

[34] Mata-Bilbao Mde, L.; Andres-Lacueva, C.; Roura, E.; Jauregui, O.; Escribano, E.; Torre, C.; Lamuela-Raventos, R. M., Absorption and pharmacokinetics of grapefruit flavanones in beagles. Br J Nutr 2007, 98 (1), 86-92.

[35] Bugianesi, R.; Catasta, G.; Spigno, P.; D'Uva, A.; Maiani, G., Naringenin from Cooked Tomato Paste Is Bioavailable in Men. J. Nutr. 2002, 132 (11), 3349-3352.

[36] Stalmach, A. l.; Mullen, W.; Pecorari, M.; Serafini, M.; Crozier, A., Bioavailability of C-Linked Dihydrochalcone and Flavanone Glucosides in Humans Following Ingestion of Unfermented and Fermented Rooibos Teas. Journal of Agricultural and Food Chemistry 2009.

[37] Hollman, P. C. H.; Bijsman, M. N. C. P.; van Gameren, Y.; Cnossen, E. P. J.; de Vries, J. H. M.; Katan, M. B., The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radical Research 1999, 31 (6), 569 - 573.

[38] Nielsen, I. L. F.; Chee, W. S. S.; Poulsen, L.; Offord-Cavin, E.; Rasmussen, S. E.; Frederiksen, H.; Enslen, M.; Barron, D.;

Horcajada, M.-N.; Williamson, G., Bioavailability Is Improved by Enzymatic Modification of the Citrus Flavonoid Hesperidin in Humans: A Randomized, Double-Blind, Crossover Trial. J. Nutr. 2006, 136 (2), 404-408.

[39] Kanaze, F. I.; Bounartzi, M. I.; Georgarakis, M.; Niopas, I., Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr 2006, 61 (4), 472-477.

[40] Clifford, M.; Brown, J. E., Dietary flavonoids and health - broadening the perspective. Flavonoids 2006, 319-370.

[41] Felgines, C.; Texier, O.; Morand, C.; Manach, C.; Scalbert, A.; Regerat, F.; Remesy, C., Bioavailability of the flavanone naringenin and its glycosides in rats. Am J Physiol Gastrointest Liver Physiol 2000, 279 (6), G1148-1154.

[42] Heo, H. J.; Kim, D.-O.; Shin, S. C.; Kim, M. J.; Kim, B. G.; Shin, D.-H., Effect of Antioxidant Flavanone, Naringenin, from Citrus junos on Neuroprotection. Journal of Agricultural and Food Chemistry 2004, 52 (6), 1520-1525.

[43] Pari, L.; Gnanasoundari, M., Influence of Naringenin on Oxytetracycline Mediated Oxidative Damage in Rat Liver. Basic & Clinical Pharmacology & Toxicology 2006, 98 (5), 456-461.

[44] Andrade, J. E.; Burgess, J. R., Effect of the Citrus Flavanone Naringenin on Oxidative Stress in Rats. Journal of Agricultural and Food Chemistry 2007, 55 (6), 2142-2148.

[45] Tanaka, T.; Makita, H.; Kawabata, K.; Mori, H.; Kakumoto, M.; Satoh, K.; Hara, A.; Sumida, T.; Ogawa, H., Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis 1997, 18 (5), 957-965.

[46] Tanaka, T.; Kohno, H.; Murakami, M.; Shimada, R.; Kagami, S.; Sumida, T.; Azuma, Y.; Ogawa, H., Suppression of azoxymethane-induced colon carcinogenesis in male F344 rats by mandarin juices rich in beta-cryptoxanthin and hesperidin. International Journal of Cancer 2000, 88 (1), 146-150.

[47] Kohno, H.; Taima, M.; Sumida, T.; Azuma, Y.; Ogawa, H.; Tanaka, T., Inhibitory effect of mandarin juice rich in [beta]-cryptoxanthin and hesperidin on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary tumorigenesis in mice. Cancer letters 2001, 174 (2), 141-150.

[48] Aranganathan, S.; Panneer Selvam, J.; Sangeetha, N.; Nalini, N., Modulatory efficacy of hesperetin (citrus flavanone) on xenobiotic-metabolizing enzymes during 1,2-dimethylhydrazine-induced colon carcinogenesis. Chemico-Biological Interactions 2009, 180 (2), 254-261.

[49] Ekambaram, G.; Rajendran, P.; Magesh, V.; Sakthisekaran, D., Naringenin reduces tumor size and weight lost in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in rats. Nutrition Research 2008, 28 (2), 106-112.

[50] Falcao, M. J. C.; Pouliquem, Y. B. M.; Lima, M. A. S.; Gramosa, N. V.; Costa-Lotufo, L. V.; Militao, G. C. G.; Pessoa, C.; Odorico de Moraes, M.; Silveira, E. R., Cytotoxic Flavonoids from Platymiscium floribundum. Journal of Natural Products 2005, 68 (3), 423-426.

[51] Zhang, S.-p.; Zhou, Y.-j.; Liu, Y.; Cai, Y.-q., Effect of liquiritigenin, a flavanone existed from Radix glycyrrhizae on pro-apoptotic in SMMC-7721 cells. Food and Chemical Toxicology 2009, 47 (4), 693-701.

[52] Vafeiadou, K.; Vauzour, D.; Lee, H. Y.; Rodriguez-Mateos, A.; Williams, R. J.; Spencer, J. P. E., The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury. Archives of Biochemistry and Biophysics 2009, 484 (1), 100-109.

[53] Bodet, C.; La, V. D.; Epifano, F.; Grenier, D., Naringenin has anti-inflammatory properties in macrophage and ex vivo human whole-blood models. J Periodontal Res 2008, 43 (4), 400-7.

[54] Inês Amaro, M.; Rocha, J.; Vila-Real, H.; Eduardo-Figueira, M.; Mota-Filipe, H.; Sepodes, B.; Ribeiro, M. H., Anti-inflammatory activity of naringin and the biosynthesised naringenin by naringinase immobilized in microstructured materials in a model of DSS-induced colitis in mice. Food Research International 2009, 42 (8), 1010-1017.

[55] Hwang, T.-L.; Li, G.-L.; Lan, Y.-H.; Chia, Y.-C.; Hsieh, P.-W.; Wu, Y.-H.; Wu, Y.-C., Potent inhibition of superoxide anion production in activated human neutrophils by isopedicin, a bioactive component of the Chinese medicinal herb Fissistigma oldhamii. Free Radical Biology and Medicine 2009, 46 (4), 520-528.

[56] Jeon, S.-M.; Kim, H. K.; Kim, H.-J.; Do, G.-M.; Jeong, T.-S.; Park, Y. B.; Choi, M.-S., Hypocholesterolemic and antioxidative effects of naringenin and its two metabolites in high-cholesterol fed rats. Translational Research 2007, 149 (1), 15-21.

[57] Rajadurai, M.; Stanely Mainzen Prince, P., Preventive effect of naringin on isoproterenol-induced cardiotoxicity in Wistar rats: an in vivo and in vitro study. Toxicology 2007, 232 (3), 216-225.

[58] Szkudelska, K.; Nogowski, L.; Nowicka, E.; Szkudelski, T., In vivo metabolic effects of naringenin in the ethanol consuming rat and the effect of naringenin on adipocytes in vitro. Journal Of Animal Physiology And Animal Nutrition 2007, 91 (3-4), 91-99.

[59] Franke, A. A.; Cooney, R. V.; Henning, S. M.; Custer, L. J., Bioavailability and Antioxidant Effects of Orange Juice Components in Humans. Journal of Agricultural and Food Chemistry 2005, 53 (13), 5170-5178.

[60] Ghanim, H.; Mohanty, P.; Pathak, R.; Chaudhuri, A.; Sia, C. L.; Dandona, P., Orange Juice or Fructose Intake Does Not Induce Oxidative and Inflammatory Response. Diabetes Care 2007, 30 (6), 1406-1411.

[61] Ortiz-Andrade, R. R.; Sanchez-Salgado, J. C.; Navarrete-Vazquez, G.; Webster, S. P.; Binnie, M.; Garcia-Jimenez, S.; Leon-Rivera, I.; Cigarroa-Vazquez, P.; Villalobos-Molina, R.; Estrada-Soto, S., Antidiabetic and toxicological evaluations of naringenin in normoglycaemic and NIDDM rat models and its implications on extra-pancreatic glucose regulation. Diabetes Obes Metab 2008, 10 (11), 1097-104.

[62] Izumi, T.; Piskula, M. K.; Osawa, S.; Obata, A.; Tobe, K.; Saito, M.; Kataoka, S.; Kubota, Y.; Kikuchi, M., Soy Isoflavone Aglycones Are Absorbed Faster and in Higher Amounts than Their Glucosides in Humans. J. Nutr. 2000, 130 (7), 1695-1699.

[63] Kano, M.; Takayanagi, T.; Harada, K.; Sawada, S.; Ishikawa, F., Bioavailability of Isoflavones after Ingestion of Soy Beverages in Healthy Adults. J. Nutr. 2006, 136 (9), 2291-2296.

[64] Cassidy, A.; Brown, J. E.; Hawdon, A.; Faughnan, M. S.; King, L. J.; Millward, J.; Zimmer-Nechemias, L.; Wolfe, B.; Setchell, K. D. R., Factors Affecting the Bioavailability of Soy Isoflavones in Humans after Ingestion of Physiologically Relevant Levels from Different Soy Foods. J. Nutr. 2006, 136 (1), 45-51.

[65] Setchell, K. D. R.; Brown, N. M.; Desai, P.; Zimmer-Nechemias, L.; Wolfe, B. E.; Brashear, W. T.; Kirschner, A. S.; Cassidy, A.; Heubi, J. E., Bioavailability of Pure Isoflavones in Healthy Humans and Analysis of Commercial Soy Isoflavone Supplements. J. Nutr. 2001, 131 (4), 1362S-1375.

[66] Richelle, M.; Pridmore-Merten, S.; Bodenstab, S.; Enslen, M.; Offord, E. A., Hydrolysis of Isoflavone Glycosides to Aglycones by {beta}-Glycosidase Does Not Alter Plasma and Urine Isoflavone Pharmacokinetics in Postmenopausal Women. J. Nutr. 2002, 132 (9), 2587-2592.

[67] Zubik, L.; Meydani, M., Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 2003, 77 (6), 1459-1465.

[68] Setchell, K. D.; Brown, N. M.; Zimmer-Nechemias, L.; Brashear, W. T.; Wolfe, B. E.; Kirschner, A. S.; Heubi, J. E., Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr 2002, 76 (2), 447-453.

[69] Clarke, D. B.; Lloyd, A. S.; Botting, N. P.; Oldfield, M. F.; Needs, P. W.; Wiseman, H., Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C3]isoflavone internal standards. Analytical Biochemistry 2002, 309 (1), 158-172.

[70] Vergne, S. b.; Sauvant, P.; Lamothe, V. r.; Chantre, P.; Asselineau, J.; Perez, P.; Durand, M. n.; Moore, N.; Bennetau-Pelissero, C., Influence of ethnic origin (Asian v. Caucasian) and background diet on the bioavailability of dietary isoflavones. British Journal of Nutrition 2006, Forthcoming (-1), 1-12.

[71] Espín, J. C.; García-Conesa, M. T.; Tomás-Barberán, F. A., Nutraceuticals: Facts and fiction. Phytochemistry 2007, 68 (22-24), 2986-3008.

[72] Squadrito, F.; Altavilla, D.; Crisafulli, A.; Saitta, A.; Cucinotta, D.; Morabito, N.; D'Anna, R.; Corrado, F.; Ruggeri, P.; Frisina, N.; Squadrito, G., Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study. The American Journal of Medicine 2003, 114 (6), 470-476.

[73] Siow, R. C. M.; Li, F. Y. L.; Rowlands, D. J.; de Winter, P.; Mann, G. E., Cardiovascular targets for estrogens and phytoestrogens: Transcriptional regulation of nitric oxide synthase and antioxidant defense genes. Free Radical Biology and Medicine 2007, 42 (7), 909-925.

[74] Limer, J. L.; Speirs, V., Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Res. 2004, 6 (3), 119-127.

[75] Lamartiniere, C. A., Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 2000, 71 (6), 1705S-1707.

[76] Mai, Z.; Blackburn, G. L.; Zhou, J.-R., Soy phytochemicals synergistically enhance the preventive effect of tamoxifen on the growth of estrogen-dependent human breast carcinoma in mice. Carcinogenesis 2007, 28 (6), 1217-1223.

[77] Lakshman, M.; Xu, L.; Ananthanarayanan, V.; Cooper, J.; Takimoto, C. H.; Helenowski, I.; Pelling, J. C.; Bergan, R. C., Dietary genistein inhibits metastasis of human prostate cancer in mice. Cancer Res. 2008, 68 (6), 2024-2032.

[78] Akhter, M.; Iwasaki, M.; Yamaji, T.; Sasazuki, S.; Tsugane, S., Dietary isoflavone and the risk of colorectal adenoma: a case-control study in Japan. Br J Cancer 2009, 100 (11), 1812-1816.

[79] Mezei, O.; Banz, W. J.; Steger, R. W.; Peluso, M. R.; Winters, T. A.; Shay, N., Soy Isoflavones Exert Antidiabetic and Hypolipidemic Effects through the PPAR Pathways in Obese Zucker Rats and Murine RAW 264.7 Cells. J. Nutr. 2003, 133 (5), 1238-1243.

[80] Kim, D. W.; Yoo, K.-Y.; Lee, Y.-B.; Lee, K.-H.; Sohn, H.-S.; Lee, S.-J.; Cho, K.-H.; Shin, Y. K.; Hwang, I. K.; Won, M.-H.; Kim, D.-W., Soy Isoflavones Mitigate Long-Term Femoral and Lumbar

Vertebral Bone Loss in Middle-Aged Ovariectomized Mice. Journal of Medicinal Food 2009, 12 (3), 536-541.

[81] Kawakita, S.; Marotta, F.; Naito, Y.; Gumaste, U.; Jain, S.; Tsuchiya, J.; Minelli, E., Effect of an isoflavones-containing red clover preparation and alkaline supplementation on bone metabolism in ovariectomized rats. Clin Interv Aging 2009, 4 (1), 91-100.

[82] Thorp, A. A.; Sinn, N.; Buckley, J. D.; Coates, A. M.; Howe, P. R. C., Soya isoflavone supplementation enhances spatial working memory in men. British Journal of Nutrition 2006, Forthcoming (-1), 1-7.

[83] Monteiro, S.; de Mattos, C.; Ben, J.; Netto, C.; Wyse, A., Ovariectomy impairs spatial memory: prevention and reversal by a soy isoflavone diet. Metabolic Brain Disease 2008, 23 (3), 243-253.

[84] Zhuo, X.-G.; Melby, M. K.; Watanabe, S., Soy Isoflavone Intake Lowers Serum LDL Cholesterol: A Meta-Analysis of 8 Randomized Controlled Trials in Humans. J. Nutr. 2004, 134 (9), 2395-2400.

[85] Taku, K.; Umegaki, K.; Sato, Y.; Taki, Y.; Endoh, K.; Watanabe, S., Soy isoflavones lower serum total and LDL cholesterol in humans: a meta-analysis of 11 randomized controlled trials. Am J Clin Nutr 2007, 85 (4), 1148-1156.

[86] Taku, K.; Umegaki, K.; Ishimi, Y.; Watanabe, S., Effects of extracted soy isoflavones alone on blood total and LDL cholesterol: Meta-analysis of randomized controlled trials. Ther Clin Risk Manag 2008, 4 (5), 1097-103.

[87] Kreijkamp-Kaspers, S.; Kok, L.; Grobbee, D. E.; de Haan, E. H. F.; Aleman, A.; van der Schouw, Y. T., Dietary Phytoestrogen Intake and Cognitive Function in Older Women. J Gerontol A Biol Sci Med Sci 2007, 62 (5), 556-562.

[88] Kreijkamp-Kaspers, S.; Kok, L.; Bots, M. L.; Grobbee, D. E.; Lampe, J. W.; van der Schouw, Y. T., Randomized controlled trial of the effects of soy protein containing isoflavones on vascular function in postmenopausal women. Am J Clin Nutr 2005, 81 (1), 189-195.

[89] Cassidy, A.; Albertazzi, P.; Nielsen, I. L.; Hall, W.; Williamson, G.; Tetens, I.; Atkins, S.; Cross, H.; Manios, Y.; Wolk, A.; Steiner, C.; Branca, F., Critical review of health effects of soyabean phyto-oestrogens in post-menopausal women. Proceedings of the Nutrition Society 2006, 65 (01), 76-92.

[90] Shimoi, K.; Okada, H.; Furugori, M.; Goda, T.; Takase, S.; Suzuki, M.; Hara, Y.; Yamamoto, H.; Kinae, N., Intestinal absorption of luteolin and luteolin 7-O-[beta]-glucoside in rats and humans. FEBS Letters 1998, 438 (3), 220-224.

[91] Wittemer, S. M.; Ploch, M.; Windeck, T.; Müller, S. C.; Drewelow, B.; Derendorf, H.; Veit, M., Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans. Phytomedicine 2005, 12 (1-2), 28-38.

[92] Meyer, H.; Bolarinwa, A.; Wolfram, G.; Linseisen, J., Bioavailability of apigenin from apiin-rich parsley in humans. Annals Of Nutrition & Metabolism 2006, 50 (3), 167-172.

[93] Walle, T.; Otake, Y.; Brubaker, J. A.; Walle, U. K.; Halushka, P. V., Disposition and metabolism of the flavonoid chrysin in normal volunteers. British Journal of Clinical Pharmacology 2001, 51 (2), 143-146.

[94] Galijatovic, A.; Otake, Y.; Walle, U. K.; Walle, T., Extensive metabolism of the flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 1999, 29 (12), 1241 - 1256.

[95] Walle, U. K.; Galijatovic, A.; Walle, T., Transport of the flavonoid chrysin and its conjugated metabolites by the human intestinal cell line Caco-2. Biochemical Pharmacology 1999, 58 (3), 431-438.

[96] Walle, T.; Ta, N.; Kawamori, T.; Wen, X.; Tsuji, P. A.; Walle, U. K., Cancer chemopreventive properties of orally bioavailable flavonoids--methylated versus unmethylated flavones. Biochem Pharmacol 2007, 73 (9), 1288-96.

[97] Walle, T., Methylation of Dietary Flavones Greatly Improves Their Hepatic Metabolic Stability and Intestinal Absorption. Molecular Pharmaceutics 2007, 4 (6), 826-832.

[98] Wen, X.; Walle, T., Methylated Flavonoids Have Greatly Improved Intestinal Absorption and Metabolic Stability. Drug Metab Dispos 2006, 34 (10), 1786-1792.

[99] Tsuji, P. A.; Winn, R. N.; Walle, T., Accumulation and metabolism of the anticancer flavonoid 5,7-dimethoxyflavone compared to its unmethylated analog chrysin in the Atlantic killifish. Chemico-Biological Interactions 2006, 164 (1-2), 85-92.

[100] Michels, G.; Wätjen, W.; Niering, P.; Steffan, B.; Thi, Q. H. T.; Chovolou, Y.; Kampkötter, A.; Bast, A.; Proksch, P.; Kahl, R., Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells. Toxicology 2005, 206 (3), 337-348.

[101] Jeyabal, P. V.; Syed, M. B.; Venkataraman, M.; Sambandham, J. K.; Sakthisekaran, D., Apigenin inhibits oxidative stress-induced macromolecular damage in N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinogenesis in Wistar albino rats. Mol Carcinog 2005, 44 (1), 11-20.

[102] Czy, J.; zdot; Zbigniew Madeja; Uwe Irmer; Wlodzimierz Korohoda; Dieter F. Hülser, Flavonoid apigenin inhibits motility and invasiveness of carcinoma cells <I>in vitro</I>. International Journal of Cancer 2005, 114 (1), 12-18.

[103] Lee, L. T.; Huang, Y. T.; Hwang, J. J.; Lee, P. P.; Ke, F. C.; Nair, M. P.; Kanadaswam, C.; Lee, M. T., Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells. Anticancer Res 2002, 22 (3), 1615-27.

[104] Shukla, S.; Gupta, S., Apigenin suppresses insulin-like growth factor I receptor signaling in human prostate cancer: An in vitro and in vivo study. Molecular Carcinogenesis 2009, 48 (3), 243-252.

[105] Kaur, P.; Shukla, S.; Gupta, S., Plant flavonoid apigenin inactivates Akt to trigger apoptosis in human prostate cancer: an in vitro and in vivo study. Carcinogenesis 2008, 29 (11), 2210-2217.

[106] Vargo, M. A.; Voss, O. H.; Poustka, F.; Cardounel, A. J.; Grotewold, E.; Doseff, A. I., Apigenin-induced-apoptosis is mediated by the activation of PKCdelta and caspases in leukemia cells. Biochem Pharmacol 2006, 72 (6), 681-92.

[107] Choi, E. J.; Kim, G. H., Apigenin Induces Apoptosis through a Mitochondria/Caspase-Pathway in Human Breast Cancer MDA-MB-453 Cells. J Clin Biochem Nutr 2009, 44 (3), 260-5.

[108] Jin, B.-h.; Qian, L.-b.; Chen, S.; Li, J.; Wang, H.-p.; Bruce, I. C.; Lin, J.; Xia, Q., Apigenin protects endothelium-dependent relaxation of rat aorta against oxidative stress. European Journal of Pharmacology 2009, 616 (1-3), 200-205.

[109] Ma, X.; Li, Y. F.; Gao, Q.; Ye, Z. G.; Lu, X. J.; Wang, H. P.; Jiang, H. D.; Bruce, I. C.; Xia, Q., Inhibition of superoxide anion-mediated impairment of endothelium by treatment with luteolin and apigenin in rat mesenteric artery. Life Sci 2008, 83 (3-4), 110-7.

[110] Yamagata, K.; Miyashita, A.; Matsufuji, H.; Chino, M., Dietary flavonoid apigenin inhibits high glucose and tumor necrosis factor [alpha]-induced adhesion molecule expression in human endothelial cells. The Journal of Nutritional Biochemistry In Press, Corrected Proof.

[111] Shin, E. K.; Kwon, H.-S.; Kim, Y. H.; Shin, H.-K.; Kim, J.-K., Chrysin, a natural flavone, improves murine inflammatory

bowel diseases. Biochemical and Biophysical Research Communications 2009, 381 (4), 502-507.

[112] Geraets, L.; Haegens, A.; Brauers, K.; Haydock, J. A.; Vernooy, J. H. J.; Wouters, E. F. M.; Bast, A.; Hageman, G. J., Inhibition of LPS-induced pulmonary inflammation by specific flavonoids. Biochemical and Biophysical Research Communications 2009, 382 (3), 598-603.

[113] Duraj, J.; Zazrivcova, K.; Bodo, J.; Sulikova, M.; Sedlak, J., Flavonoid quercetin, but not apigenin or luteolin, induced apoptosis in human myeloid leukemia cells and their resistant variants. Neoplasma 2005, 52 (4), 273-9.

[114] Janssen, K.; Mensink, R.; Cox, F.; Harryvan, J.; Hovenier, R.; Hollman, P.; Katan, M., Effects of the flavonoids quercetin and apigenin on hemostasis in healthy volunteers: results from an in vitro and a dietary supplement study. Am J Clin Nutr 1998, 67 (2), 255-262.

[115] Pietta, P.-G., Flavonoids as Antioxidants. Journal of Natural Products 2000, 63 (7), 1035-1042.

[116] Michels, G.; Mohamed, G. A.; Weber, N.; Chovolou, Y.; Kampkötter, A.; Wätjen, W.; Proksch, P., Effects of methylated derivatives of Luteolin isolated from Cyperus alopecuroides in rat H4IIE hepatoma cells*. Basic & Clinical Pharmacology & Toxicology 2006, 98 (2), 168-172.

[117] Williams, R. J.; Spencer, J. P. E.; Rice-Evans, C., Flavonoids: antioxidants or signalling molecules? Free Radical Biology and Medicine 2004, 36 (7), 838-849.

[118] Akao, Y.; itoh, T.; Ohguchi, K.; Iinuma, M.; Nozawa, Y., Interactive effects of polymethoxy flavones from Citrus on cell growth inhibition in human neuroblastoma SH-SY5Y cells. Bioorganic & Medicinal Chemistry 2008, 16 (6), 2803-2810.

[119] Wen, X.; Walle, T., Preferential induction of CYP1B1 by benzo[a]pyrene in human oral epithelial cells: impact on DNA adduct formation and prevention by polyphenols. Carcinogenesis 2005, 26 (10), 1774-81.

[120] Wen, X.; Walle, U. K.; Walle, T., 5,7-Dimethoxyflavone downregulates CYP1A1 expression and benzo[a]pyrene-induced DNA binding in Hep G2 cells. Carcinogenesis 2005, 26 (4), 803-9.

[121] Cai, H.; Sale, S.; Schmid, R.; Britton, R. G.; Brown, K.; Steward, W. P.; Gescher, A. J., Flavones as Colorectal Cancer Chemopreventive Agents--Phenol-O-Methylation Enhances Efficacy. Cancer Prev Res 2009, 2 (8), 743-750.

[122] Hollman, P.; de Vries, J.; van Leeuwen, S.; Mengelers, M.; Katan, M., Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr 1995, 62 (6), 1276-1282.

[123] Graefe, E.; Wittig, J.; Mueller, S.; Riethling, A.; Uehleke, B.; Drewelow, B.; Pforte, H.; Jacobasch, G.; Derendorf, H.; Veit, M., Pharmacokinetics and bioavailability of quercetin glycosides in humans. J Clin Pharmacol 2001, 41 (5), 492-499.

[124] Wiczkowski, W.; Romaszko, J.; Bucinski, A.; Szawara-Nowak, D.; Honke, J.; Zielinski, H.; Piskula, M. K., Quercetin from Shallots (Allium cepa L. var. aggregatum) Is More Bioavailable Than Its Glucosides. J. Nutr. 2008, 138 (5), 885-888.

[125] Day, A. J.; Mellon, F.; Barron, D.; Sarrazin, G.; Morgan, M. R.; Williamson, G., Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res 2001, 35 (6), 941-52.

[126] Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C., Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005, 81 (1), 230S-242.

[127] Kawai, Y.; Saito, S.; Nishikawa, T.; Ishisaka, A.; Murota, K.; Terao, J., Different Profiles of Quercetin Metabolites in Rat Plasma: Comparison of Two Administration Methods. Biosci. Biotechnol. Biochem. 2009, 73 (3), 517-523.

[128] Bieger, J.; Cermak, R.; Blank, R.; de Boer, V. C. J.; Hollman, P. C. H.; Kamphues, J.; Wolffram, S., Tissue distribution of quercetin in pigs after long-term dietary supplementation. Journal of Nutrition 2008, 138 (8), 1417-1420.

[129] M. Tamura; H. Nakagawa; T. Tsushida; K. Hirayama; K. Itoh, Effect of Pectin Enhancement on Plasma Quercetin and Fecal Flora in Rutin-Supplemented Mice. Journal of Food Science 2007, 72 (9), S648-S651.

[130] Nishijima, T.; Iwai, K.; Saito, Y.; Takida, Y.; Matsue, H., Chronic Ingestion of Apple Pectin Can Enhance the Absorption of Quercetin. Journal of Agricultural and Food Chemistry 2009, 57 (6), 2583-2587.

[131] Muthukumaran, S.; Sudheer, A. R.; Menon, V. P.; Nalini, N., Protective effect of quercetin on nicotine-induced prooxidant and antioxidant imbalance and DNA damage in Wistar rats. Toxicology 2008, 243 (1-2), 207-215.

[132] Maia Campos, P. M.; Gianeti, M. D.; Kanashiro, A.; Lucisano-Valim, Y. M.; Gaspar, L. R., In vitro antioxidant and in vivo photoprotective effects of an association of bioflavonoids with liposoluble vitamins. Photochem Photobiol 2006, 82 (3), 683-8.

[133] Zhao, J.; Jin, X.; Yaping, E.; Zheng, Z. S.; Zhang, Y. J.; Athar, M.; DeLeo, V. A.; Mukhtar, H.; Bickers, D. R.; Wang, Z. Y., Photoprotective effect of black tea extracts against UVB-induced phototoxicity in skin. Photochem Photobiol 1999, 70 (4), 637-44.

[134] Lee, S.; Kim, Y. J.; Kwon, S.; Lee, Y.; Choi, S. Y.; Park, J.; Kwon, H. J., Inhibitory effects of flavonoids on TNF-alpha-induced IL-8 gene expression in HEK 293 cells. BMB Rep 2009, 42 (5), 265-70.

[135] Bureau, G.; Longpre, F.; Martinoli, M. G., Resveratrol and quercetin, two natural polyphenols, reduce apoptotic neuronal cell death induced by neuroinflammation. J. Neurosci. Res. 2008, 86 (2), 403-410.

[136] Hamalainen, M.; Nieminen, R.; Vuorela, P.; Heinonen, M.; Moilanen, E., Anti-inflammatory effects of flavonoids: genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and NF-kappa B activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-kappa B activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages. Mediat. Inflamm. 2007, 10.

[137] Murakami, A.; Ashida, H.; Terao, J., Multitargeted cancer prevention by quercetin. Cancer Lett 2008, 269 (2), 315-25.

[138] Kim, K.-S.; Rhee, K.-H.; Yoon, J.-H.; Lee, J. G.; Lee, J.-H.; Yoo, J.-B., Ginkgo biloba extract (EGb 761) induces apoptosis by the activation of caspase-3 in oral cavity cancer cells. Oral Oncol. 2005, 41 (4), 383-389.

[139] Kang, J. W.; Kim, J. H.; Song, K.; Kim, S. H.; Yoon, J. H.; Kim, K. S., Kaempferol and quercetin, components of Ginkgo biloba extract (EGb 761), induce caspase-3-dependent apoptosis in oral cavity cancer cells. Phytother Res 2009.

[140] Tan, J.; Wang, B.; Zhu, L., Regulation of survivin and Bcl-2 in HepG2 cell apoptosis induced by quercetin. Chem Biodivers 2009, 6 (7), 1101-10.

[141] Shan, B. E.; Wang, M. X.; Li, R. Q., Quercetin inhibit human SW480 colon cancer growth in association with inhibition of cyclin D1 and survivin expression through Wnt/beta-catenin signaling pathway. Cancer Invest 2009, 27 (6), 604-12.

[142] Warren, C. A.; Paulhill, K. J.; Davidson, L. A.; Lupton, J. R.; Taddeo, S. S.; Hong, M. Y.; Carroll, R. J.; Chapkin, R. S.;

Turner, N. D., Quercetin May Suppress Rat Aberrant Crypt Foci Formation by Suppressing Inflammatory Mediators That Influence Proliferation and Apoptosis. Journal of Nutrition 2009, 139 (1), 101-105.

[143] Volate, S. R.; Davenport, D. M.; Muga, S. J.; Wargovich, M. J., Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin, curcumin, silymarin, ginseng and rutin). Carcinogenesis 2005, 26 (8), 1450-1456.

[144] Kwon, K. H.; Murakami, A.; Tanaka, T.; Ohigashi, H., Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression. Biochemical Pharmacology 2005, 69 (3), 395-406.

[145] Du, G.; Lin, H.; Wang, M.; Zhang, S.; Wu, X.; Lu, L.; Ji, L.; Yu, L., Quercetin greatly improved therapeutic index of doxorubicin against 4T1 breast cancer by its opposing effects on HIF-1alpha in tumor and normal cells. Cancer Chemother Pharmacol 2009.

[146] Morris, J. D. H.; Pramanik, R.; Zhang, X.; Carey, A.-M.; Ragavan, N.; Martin, F. L.; Muir, G. H., Selenium- or quercetin-induced retardation of DNA synthesis in primary prostate cells occurs in the presence of a concomitant reduction in androgen-receptor activity. Cancer Letters 2006, 239 (1), 111-122.

[147] Lee, D.-H.; Szczepanski, M.; Lee, Y. J., Role of Bax in quercetin-induced apoptosis in human prostate cancer cells. Biochemical Pharmacology 2008, 75 (12), 2345-2355.

[148] Chow, J. M.; Shen, S. C.; Huan, S. K.; Lin, H. Y.; Chen, Y. C., Quercetin, but not rutin and quercitrin, prevention of H2O2-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages. Biochem Pharmacol 2005, 69 (12), 1839-51.

[149] Bournival, J.; Quessy, P.; Martinoli, M. G., Protective Effects of Resveratrol and Quercetin Against MPP(+) -Induced Oxidative Stress Act by Modulating Markers of Apoptotic Death in Dopaminergic Neurons. Cell Mol Neurobiol 2009.

[150] Hou, Y.; Aboukhatwa, M. A.; Lei, D. L.; Manaye, K.; Khan, I.; Luo, Y., Anti-depressant natural flavonols modulate BDNF and beta amyloid in neurons and hippocampus of double TgAD mice. Neuropharmacology 2009.

[151] Akaishi, T.; Morimoto, T.; Shibao, M.; Watanabe, S.; Sakai-Kato, K.; Utsunomiya-Tate, N.; Abe, K., Structural requirements for the flavonoid fisetin in inhibiting fibril formation of amyloid beta protein. Neurosci Lett 2008, 444 (3), 280-5.

[152] Ansari, M. A.; Abdul, H. M.; Joshi, G.; Opii, W. O.; Butterfield, D. A., Protective effect of quercetin in primary neurons against A[beta](1-42): relevance to Alzheimer's disease. The Journal of Nutritional Biochemistry 2009, 20 (4), 269-275.

[153] Mojzisova, G.; Sarissky, M.; Mirossay, L.; Martinka, P.; Mojzis, J., Effect of flavonoids on daunorubicin-induced toxicity in H9c2 Cardiomyoblasts. Phytother Res 2009, 23 (1), 136-9.

[154] Lodi, F.; Jimenez, R.; Moreno, L.; Kroon, P. A.; Needs, P. W.; Hughes, D. A.; Santos-Buelga, C.; Gonzalez-Paramas, A.; Cogolludo, A.; Lopez-Sepulveda, R.; Duarte, J.; Perez-Vizcaino, F., Glucuronidated and sulfated metabolites of the flavonoid quercetin prevent endothelial dysfunction but lack direct vasorelaxant effects in rat aorta. Atherosclerosis 2009, 204 (1), 34-39.

[155] Kampkotter, A.; Timpel, C.; Zurawski, R. F.; Ruhl, S.; Chovolou, Y.; Proksch, P.; Watjen, W., Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin. Comp Biochem Physiol B Biochem Mol Biol 2008, 149 (2), 314-23.

[156] Sen, G.; Mukhopadhyay, S.; Ray, M.; Biswas, T., Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity. J Antimicrob Chemother 2008, 61 (5), 1066-75.

[157] Davis, J. M.; Murphy, E. A.; McClellan, J. L.; Carmichael, M. D.; Gangemi, J. D., Quercetin reduces susceptibility to influenza infection following stressful exercise. Am J Physiol Regul Integr Comp Physiol 2008, 295 (2), R505-9.

[158] Bischoff, S. C., Quercetin: potentials in the prevention and therapy of disease. Curr Opin Clin Nutr Metab Care 2008, 11 (6), 733-40.

[159] Hertog, M. G.; Sweetnam, P. M.; Fehily, A. M.; Elwood, P. C.; Kromhout, D., Antioxidant flavonols and ischemic heart disease in a Welsh population of men: the Caerphilly Study. Am J Clin Nutr 1997, 65 (5), 1489-94.

[160] Bae, S.-C.; Jung, W.-J.; Lee, E.-J.; Yu, R.; Sung, M.-K., Effects of Antioxidant Supplements Intervention on the Level of Plasma Inflammatory Molecules and Disease Severity of Rheumatoid Arthritis Patients. J Am Coll Nutr 2009, 28 (1), 56-62.

[161] Kääriäinen, T. M.; Piltonen, M.; Ossola, B.; Kekki, H.; Lehtonen, S.; Nenonen, T.; Lecklin, A.; Raasmaja, A.; Männistö, P. T., Lack of robust protective effect of quercetin in two types of 6-hydroxydopamine-induced parkinsonian models in rats and dopaminergic cell cultures. Brain Research 2008, 1203, 149-159.

[162] Ossola, B.; Kääriäinen, T. M.; Raasmaja, A.; Männistö, P. T., Time-dependent protective and harmful effects of quercetin on 6-OHDA-induced toxicity in neuronal SH-SY5Y cells. Toxicology 2008, 250 (1), 1-8.

[163] Ferry, D. R.; Smith, A.; Malkhandi, J.; Fyfe, D. W.; deTakats, P. G.; Anderson, D.; Baker, J.; Kerr, D. J., Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition. Clin. Cancer Res. 1996, 2 (4), 659-668.

[164] Chow, H. H.; Cai, Y.; Alberts, D. S.; Hakim, I.; Dorr, R.; Shahi, F.; Crowell, J. A.; Yang, C. S.; Hara, Y., Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 2001, 10 (1), 53-8.

[165] Lee, M. J.; Maliakal, P.; Chen, L.; Meng, X.; Bondoc, F. Y.; Prabhu, S.; Lambert, G.; Mohr, S.; Yang, C. S., Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev 2002, 11 (10 Pt 1), 1025-32.

[166] Donovan, J. L.; Crespy, V.; Oliveira, M.; Cooper, K. A.; Gibson, B. B.; Williamson, G., (+)-Catechin is more bioavailable than (-)-catechin: relevance to the bioavailability of catechin from cocoa. Free Radic Res 2006, 40 (10), 1029-34.

[167] Nakagawa, K.; Okuda, S.; Miyazawa, T., Dose-dependent incorporation of tea catechins, (-)-epigallocatechin-3-gallate and (-)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem 1997, 61 (12), 1981-5.

[168] Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm, D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L., Procyanidin dimer B2 [epicatechin-(4 beta-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 2002, 76 (4), 798-804.

[169] Spencer, J. P. E.; Schroeter, H.; Shenoy, B.; S. Srai, S. K.; Debnam, E. S.; Rice-Evans, C., Epicatechin Is the Primary Bioavailable Form of the Procyanidin Dimers B2 and B5 after Transfer across the Small Intestine. Biochemical and

Biophysical Research Communications 2001, 285 (3), 588-593.

[170] Donovan, J. L.; Bell, J. R.; Kasim-Karakas, S.; German, J. B.; Walzem, R. L.; Hansen, R. J.; Waterhouse, A. L. In Catechin is present as metabolites in human plasma after consumption of red wine, Amer Inst Nutrition: 1999; pp 1662-1668.

[171] Van Amelsvoort, J. M.; Van Hof, K. H.; Mathot, J. N.; Mulder, T. P.; Wiersma, A.; Tijburg, L. B., Plasma concentrations of individual tea catechins after a single oral dose in humans. Xenobiotica 2001, 31 (12), 891-901.

[172] Donovan, J. L.; Kasim-Karakas, S.; German, J. B.; Waterhouse, A. L., Urinary excretion of catechin metabolites by human subjects after red wine consumption. British Journal of Nutrition 2002, 87 (01), 31-37.

[173] Natsume, M.; Osakabe, N.; Oyama, M.; Sasaki, M.; Baba, S.; Nakamura, Y.; Osawa, T.; Terao, J., Structures of (-)-epicatechin glucuronide identified from plasma and urine after oral ingestion of (-)-epicatechin: differences between human and rat. Free Radical Biology and Medicine 2003, 34 (7), 840-849.

[174] Lu, H.; Meng, X. F.; Yang, C. S., Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate. Drug Metab. Dispos. 2003, 31 (5), 572-579.

[175] Rios, L. Y.; Gonthier, M. P.; Remesy, C.; Mila, I.; Lapierre, C.; Lazarus, S. A.; Williamson, G.; Scalbert, A., Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects. Am J Clin Nutr 2003, 77 (4), 912-8.

[176] Ferruzzi, M. G.; Lobo, J. K.; Janle, E. M.; Whittaker, N.; Cooper, B.; Simon, J. E.; Wu, Q. L.; Welch, C.; Ho, L.; Weaver, C.; Pasinetti, G. M., Bioavailability of Gallic Acid and Catechins from Grape Seed Polyphenol Extract is Improved by Repeated Dosing in Rats: Implications for Treatment in Alzheimer's Disease. J Alzheimers Dis 2009.

[177] Serafini, M.; Bugianesi, R.; Maiani, G.; Valtuena, S.; De Santis, S.; Crozier, A., Plasma antioxidants from chocolate. Nature 2003, 424 (6952), 1013-1013.

[178] Roura, E.; Andres-Lacueva, C.; Estruch, R.; Mata-Bilbao, M. L.; Izquierdo-Pulido, M.; Waterhouse, A. L.; Lamuela-Raventos, R. M., Milk does not affect the bioavailability of cocoa powder flavonoid in healthy human. Ann Nutr Metab 2007, 51 (6), 493-8.

[179] Mullen, W.; Borges, G.; Donovan, J. L.; Edwards, C. A.; Serafini, M.; Lean, M. E.; Crozier, A., Milk decreases urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans. Am J Clin Nutr 2009, 89 (6), 1784-91.

[180] Yan, X.; Zhang, J.-j.; Li, X.; Lei, Z.; Dong, S.; Hui, L., Green tea polyphenols inhibit cognitive impairment induced by chronic cerebral hypoperfusion via modulating oxidative stress. The Journal of Nutritional Biochemistry 2009, In Press, Corrected Proof.

[181] Yokozawa, T.; Nakagawa, T.; Kitani, K., Antioxidative Activity of Green Tea Polyphenol in Cholesterol-Fed Rats. Journal of Agricultural and Food Chemistry 2002, 50 (12), 3549-3552.

[182] Kimura, M.; Umegaki, K.; Kasuya, Y.; Sugisawa, A.; Higuchi, M., The relation between single/double or repeated tea catechin ingestions and plasma antioxidant activity in humans. Eur J Clin Nutr 2002, 56 (12), 1186-93.

[183] Rozan, P.; Hidalgo, S.; Nejdi, A.; Bisson, J. F.; Lalonde, R.; Messaoudi, M., Preventive antioxidant effects of cocoa polyphenolic extract on free radical production and cognitive performances after heat exposure in Wistar rats. J Food Sci 2007, 72 (3), S203-6.

[184] Wang, J. F.; Schramm, D. D.; Holt, R. R.; Ensunsa, J. L.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L., A dose-response effect from chocolate consumption on plasma epicatechin and oxidative damage. J Nutr 2000, 130 (8S Suppl), 2115S-9S.

[185] Pearson, D. A.; Schmitz, H. H.; Lazarus, S. A.; Keen, C. L.; Lester, P., Inhibition of in Vitro low-density lipoprotein oxidation by oligomeric procyanidins present in chocolate and cocoas. In Methods in Enzymology, Academic Press: 2001; Vol. Volume 335, pp. 350-360.

[186] Keen, C. L.; Holt, R. R.; Oteiza, P. I.; Fraga, C. G.; Schmitz, H. H., Cocoa antioxidants and cardiovascular health. Am J Clin Nutr 2005, 81 (1 Suppl), 298S-303S.

[187] Mehrinfar, R.; Frishman, W. H., Flavanol-rich cocoa: a cardioprotective nutraceutical. Cardiol Rev 2008, 16 (3), 109-15.

[188] Hamed, M. S.; Gambert, S.; Bliden, K. P.; Bailon, O.; Singla, A.; Antonino, M. J.; Hamed, F.; Tantry, U. S.; Gurbel, P. A., Dark chocolate effect on platelet activity, C-reactive protein and lipid profile: a pilot study. South Med J 2008, 101 (12), 1203-8.

[189] Tinahones, F. J.; Rubio, M. A.; Garrido-Sanchez, L.; Ruiz, C.; Gordillo, E.; Cabrerizo, L.; Cardona, F., Green Tea Reduces LDL Oxidability and Improves Vascular Function. J Am Coll Nutr 2008, 27 (2), 209-213.

[190] Murphy, K. J.; Chronopoulos, A. K.; Singh, I.; Francis, M. A.; Moriarty, H.; Pike, M. J.; Turner, A. H.; Mann, N. J.; Sinclair, A. J., Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr 2003, 77 (6), 1466-1473.

[191] Yamakuchi, M.; Bao, C.; Ferlito, M.; Lowenstein, C. J., Epigallocatechin gallate inhibits endothelial exocytosis. Biological Chemistry 2008, 389 (7), 935-941.

[192] Kavanagh, K. T.; Hafer, L. J.; Kim, D. W.; Mann, K. K.; Sherr, D. H.; Rogers, A. E.; Sonenshein, G. E., Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. J Cell Biochem 2001, 82 (3), 387-98.

[193] Kushima, Y.; Iida, K.; Nagaoka, Y.; Kawaratani, Y.; Shirahama, T.; Sakaguchi, M.; Baba, K.; Hara, Y.; Uesato, S., Inhibitory effect of (-)-epigallocatechin and (-)-epigallocatechin gallate against heregulin beta1-induced migration/invasion of the MCF-7 breast carcinoma cell line. Biol Pharm Bull 2009, 32 (5), 899-904.

[194] Siddiqui, I. A.; Raisuddin, S.; Shukla, Y., Protective effects of black tea extract on testosterone induced oxidative damage in prostate. Cancer Letters 2005, 227 (2), 125-132.

[195] Jourdain, C.; Tenca, G.; Deguercy, A.; Troplin, P.; Poelman, D., In-vitro effects of polyphenols from cocoa and beta-sitosterol on the growth of human prostate cancer and normal cells. Eur J Cancer Prev 2006, 15 (4), 353-61.

[196] Lee, A. H.; Fraser, M. L.; Binns, C. W., Tea, coffee and prostate cancer. Mol Nutr Food Res 2009, 53 (2), 256-65.

[197] Ran, Z. H.; Xu, Q.; Tong, J. L.; Xiao, S. D., Apoptotic effect of Epigallocatechin-3-gallate on the human gastric cancer cell line MKN45 via activation of the mitochondrial pathway. World J Gastroenterol 2007, 13 (31), 4255-9.

[198] Suzuki, E.; Yorifuji, T.; Takao, S.; Komatsu, H.; Sugiyama, M.; Ohta, T.; Ishikawa-Takata, K.; Doi, H., Green Tea Consumption and Mortality among Japanese Elderly People: The Prospective Shizuoka Elderly Cohort. Ann Epidemiol 2009.

[199] Adachi, S.; Shimizu, M.; Shirakami, Y.; Yamauchi, J.; Natsume, H.; Matsushima-Nishiwaki, R.; To, S.; Weinstein, I. B.; Moriwaki, H.; Kozawa, O., (-)-Epigallocatechin gallate downregulates EGF receptor via phosphorylation at

Ser1046/1047 by p38 MAP kinase in colon cancer cells. Carcinogenesis 2009.

[200] Milligan, S. A.; Burke, P.; Coleman, D. T.; Bigelow, R. L.; Steffan, J. J.; Carroll, J. L.; Williams, B. J.; Cardelli, J. A., The green tea polyphenol EGCG potentiates the antiproliferative activity of c-Met and epidermal growth factor receptor inhibitors in non-small cell lung cancer cells. Clin Cancer Res 2009, 15 (15), 4885-94.

[201] Li, Y.; Zhang, T.; Jiang, Y.; Lee, H.-F.; Schwartz, S. J.; Sun, D., (-)-Epigallocatechin-3-gallate Inhibits Hsp90 Function by Impairing Hsp90 Association with Cochaperones in Pancreatic Cancer Cell Line Mia Paca-2. Molecular Pharmaceutics 2009, 6 (4), 1152-1159.

[202] Yasuda, Y.; Shimizu, M.; Sakai, H.; Iwasa, J.; Kubota, M.; Adachi, S.; Osawa, Y.; Tsurumi, H.; Hara, Y.; Moriwaki, H., (-)-Epigallocatechin gallate prevents carbon tetrachloride-induced rat hepatic fibrosis by inhibiting the expression of the PDGFRbeta and IGF-1R. Chem Biol Interact 2009.

[203] Kaufmann, R.; Henklein, P.; Settmacher, U., Green tea polyphenol epigallocatechin-3-gallate inhibits thrombin-induced hepatocellular carcinoma cell invasion and p42/p44-MAPKinase activation. Oncol Rep 2009, 21 (5), 1261-7.

[204] Saul, N.; Pietsch, K.; Menzel, R.; Stürzenbaum, S. R.; Steinberg, C. E. W., Catechin induced longevity in C. elegans: From key regulator genes to disposable soma. Mechanisms of Ageing and Development 2009, 130 (8), 477-486.

[205] Abbas, S.; Wink, M., Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans. Planta Med 2009, 75 (3), 216-21.

[206] Jeon, H. Y.; Kim, J. K.; Kim, W. G.; Lee, S. J., Effects of oral epigallocatechin gallate supplementation on the minimal erythema dose and UV-induced skin damage. Skin Pharmacol Physiol 2009, 22 (3), 137-41.

[207] Kim, C. Y.; Lee, C.; Park, G. H.; Jang, J. H., Neuroprotective effect of epigallocatechin-3-gallate against beta-amyloid-induced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity. Arch Pharm Res 2009, 32 (6), 869-81.

[208] Lee, Y. K.; Yuk, D. Y.; Lee, J. W.; Lee, S. Y.; Ha, T. Y.; Oh, K. W.; Yun, Y. P.; Hong, J. T., (-)-Epigallocatechin-3-gallate prevents lipopolysaccharide-induced elevation of beta-amyloid generation and memory deficiency. Brain Research 2009, 1250, 164-174.

[209] Wang, H.; Wen, Y.; Du, Y.; Yan, X.; Guo, H.; Rycroft, J. A.; Boon, N.; Kovacs, E. M.; Mela, D. J., Effects of Catechin Enriched Green Tea on Body Composition. Obesity (Silver Spring) 2009.

[210] Lee, M. S.; Kim, C. T.; Kim, Y., Green tea (-)-epigallocatechin-3-gallate reduces body weight with regulation of multiple genes expression in adipose tissue of diet-induced obese mice. Ann Nutr Metab 2009, 54 (2), 151-7.

[211] Grassi, D.; Desideri, G.; Necozione, S.; Lippi, C.; Casale, R.; Properzi, G.; Blumberg, J. B.; Ferri, C., Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. J Nutr 2008, 138 (9), 1671-6.

[212] Tsuneki, H.; Ishizuka, M.; Terasawa, M.; Wu, J. B.; Sasaoka, T.; Kimura, I., Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004, 4, 18.

[213] Ried, K.; Frank, O. R.; Stocks, N. P., Dark chocolate or tomato extract for prehypertension: a randomised controlled trial. BMC Complement Altern Med 2009, 9, 22.

[214] Datla, K. P.; Zbarsky, V.; Rai, D.; Parkar, S.; Osakabe, N.; Aruoma, O. I.; Dexter, D. T., Short-Term Supplementation with Plant Extracts Rich in Flavonoids Protect Nigrostriatal Dopaminergic Neurons in a Rat Model of Parkinson's Disease. J Am Coll Nutr 2007, 26 (4), 341-349.

[215] Kikuchi, N.; Ohmori, K.; Shimazu, T.; Nakaya, N.; Kuriyama, S.; Nishino, Y.; Tsubono, Y.; Tsuji, I., No association between green tea and prostate cancer risk in Japanese men: the Ohsaki Cohort Study. Br J Cancer 2006, 95 (3), 371-373.

[216] Bonkovsky, H. L., Hepatotoxicity associated with supplements containing Chinese green tea (Camellia sinensis). Ann Intern Med 2006, 144 (1), 68-71.

[217] Stevens, T.; Qadri, A.; Zein, N. N., Two Patients with Acute Liver Injury Associated with Use of the Herbal Weight-Loss Supplement Hydroxycut. Annals of Internal Medicine 2005, 142 (6), 477-478.

[218] McCann, D.; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.; Grimshaw, K.; Kitchin, E.; Lok, K.; Porteous, L.; Prince, E.; Sonuga-Barke, E.; Warner, J. O.; Stevenson, J., Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo-controlled trial. Lancet 2007, 370 (9598), 1560-7.

[219] Galvano, F.; La Fauci, L.; Vitaglione, P.; Fogliano, V.; Vanella, L.; Felgines, C., Bioavailability, antioxidant and biological properties of the natural free-radical scavengers cyanidin and related glycosides. Ann Ist Super Sanita 2007, 43 (4), 382-93.

[220] Kay, C. D.; Mazza, G.; Holub, B. J., Anthocyanins Exist in the Circulation Primarily as Metabolites in Adult Men. J. Nutr. 2005, 135 (11), 2582-2588.

[221] Mertens-Talcott, S. U.; Rios, J.; Jilma-Stohlawetz, P.; Pacheco-Palencia, L. A.; Meibohm, B.; Talcott, S. T.; Derendorf, H., Pharmacokinetics of Anthocyanins and Antioxidant Effects after the Consumption of Anthocyanin-Rich Açai Juice and Pulp (Euterpe oleracea Mart.) in Human Healthy Volunteers. Journal of Agricultural and Food Chemistry 2008, 56 (17), 7796-7802.

[222] Milbury, P. E.; Cao, G.; Prior, R. L.; Blumberg, J., Bioavailablility of elderberry anthocyanins. Mechanisms of Ageing and Development 2002, 123 (8), 997-1006.

[223] Galvano, F.; La Fauci, L.; Lazzarino, G.; Fogliano, V.; Ritieni, A.; Ciappellano, S.; Battistini, N. C.; Tavazzi, B.; Galvano, G., Cyanidins: metabolism and biological properties. The Journal of Nutritional Biochemistry 2004, 15 (1), 2-11.

[224] Garcia-Alonso, M.; Minihane, A.-M.; Rimbach, G.; Rivas-Gonzalo, J. C.; de Pascual-Teresa, S., Red wine anthocyanins are rapidly absorbed in humans and affect monocyte chemoattractant protein 1 levels and antioxidant capacity of plasma. The Journal of Nutritional Biochemistry 2009, 20 (7), 521-529.

[225] Miyazawa, T.; Nakagawa, K.; Kudo, M.; Muraishi, K.; Someya, K., Direct intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3,5-diglucoside, into rats and humans. J Agric Food Chem 1999, 47 (3), 1083-91.

[226] Lehtonen, H.-M.; Rantala, M.; Suomela, J.-P.; Viitanen, M.; Kallio, H., Urinary Excretion of the Main Anthocyanin in Lingonberry (Vaccinium vitis-idaea), Cyanidin 3-O-Galactoside, and Its Metabolites. Journal of Agricultural and Food Chemistry 2009, 57 (10), 4447-4451.

[227] Frank, T.; Sonntag, S.; Strass, G.; Bitsch, I.; Bitsch, R.; Netzel, M., Urinary pharmacokinetics of cyanidin glycosides in healthy young men following consumption of elderberry juice. Int J Clin Pharmacol Res 2005, 25 (2), 47-56.

[228] Matsumoto, H.; Inaba, H.; Kishi, M.; Tominaga, S.; Hirayama, M.; Tsuda, T., Orally administered delphinidin 3-rutinoside

and cyanidin 3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms. J Agric Food Chem 2001, 49 (3), 1546-51.

[229] Tian, Q. G.; Giusti, M. M.; Stoner, G. D.; Schwartz, S. J., Urinary excretion of black raspberry (Rubus occidentalis) anthocyanins and their metabolites. Journal of Agricultural and Food Chemistry 2006, 54 (4), 1467-1472.

[230] Hassimotto, N. M. A.; Genovese, M. I.; Lajolo, F. M., Absorption and metabolism of cyanidin-3-glucoside and cyanidin-3-rutinoside extracted from wild mulberry (Morus nigra L.) in rats. Nutrition Research 2008, 28 (3), 198-207.

[231] Charron, C. S.; Clevidence, B. A.; Britz, S. J.; Novotny, J. A., Effect of Dose Size on Bioavailability of Acylated and Nonacylated Anthocyanins from Red Cabbage (Brassica oleracea L. Var. capitata). Journal of Agricultural and Food Chemistry 2007, 55 (13), 5354-5362.

[232] Hollands, W.; Brett, G. M.; Dainty, J. R.; Teucher, B.; Kroon, P. A., Urinary excretion of strawberry anthocyanins is dose dependent for physiological oral doses of fresh fruit. Mol Nutr Food Res 2008, 52 (10), 1097-105.

[233] Wu, X.; Pittman, H. E., III; Prior, R. L., Pelargonidin Is Absorbed and Metabolized Differently than Cyanidin after Marionberry Consumption in Pigs. J. Nutr. 2004, 134 (10), 2603-2610.

[234] Charron, C. S.; Kurilich, A. C.; Clevidence, B. A.; Simon, P. W.; Harrison, D. J.; Britz, S. J.; Baer, D. J.; Novotny, J. A., Bioavailability of Anthocyanins from Purple Carrot Juice: Effects of Acylation and Plant Matrix. Journal of Agricultural and Food Chemistry 2009, 57 (4), 1226-1230.

[235] Felgines, C.; Talavera, S.; Gonthier, M. P.; Texier, O.; Scalbert, A.; Lamaison, J. L.; Remesy, C., Strawberry anthocyanins are recovered in urine as glucuro- and sulfoconjugates in humans. J Nutr 2003, 133 (5), 1296-301.

[236] Felgines, C.; Talavera, S.; Texier, O.; Gil-Izquierdo, A.; Lamaison, J. L.; Remesy, C., Blackberry anthocyanins are mainly recovered from urine as methylated and glucuronidated conjugates in humans. J Agric Food Chem 2005, 53 (20), 7721-7.

[237] Woodward, G.; Kroon, P.; Cassidy, A.; Kay, C., Anthocyanin Stability and Recovery: Implications for the Analysis of Clinical and Experimental Samples. Journal of Agricultural and Food Chemistry 2009, 57 (12), 5271-5278.

[238] Vitaglione, P.; Donnarumma, G.; Napolitano, A.; Galvano, F.; Gallo, A.; Scalfi, L.; Fogliano, V., Protocatechuic Acid Is the Major Human Metabolite of Cyanidin-Glucosides. J. Nutr. 2007, 137 (9), 2043-2048.

[239] Kurilich, A. C.; Clevidence, B. A.; Britz, S. J.; Simon, P. W.; Novotny, J. A., Plasma and Urine Responses Are Lower for Acylated vs Nonacylated Anthocyanins from Raw and Cooked Purple Carrots. Journal of Agricultural and Food Chemistry 2005, 53 (16), 6537-6542.

[240] Fukumoto, L. R.; Mazza, G., Assessing Antioxidant and Prooxidant Activities of Phenolic Compounds†Journal of Agricultural and Food Chemistry 2000, 48 (8), 3597-3604.

[241] Bao, L.; Yao, X.-S.; Yau, C.-C.; Tsi, D.; Chia, C.-S.; Nagai, H.; Kurihara, H., Protective Effects of Bilberry (Vaccinium myrtillus L.) Extract on Restraint Stress-Induced Liver Damage in Mice. Journal of Agricultural and Food Chemistry 2008, 56 (17), 7803-7807.

[242] Metzger, B. T.; Barnes, D. M.; Reed, J. D., Purple Carrot (Daucus carota L.) Polyacetylenes Decrease Lipopolysaccharide-Induced Expression of Inflammatory Proteins in Macrophage and Endothelial Cells. Journal of Agricultural and Food Chemistry 2008, 56 (10), 3554-3560.

[243] Lyall, K. A.; Hurst, S. M.; Cooney, J.; Jensen, D.; Lo, K.; Hurst, R. D.; Stevenson, L. M., Short-term blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharide-stimulated inflammatory responses. Am J Physiol Regul Integr Comp Physiol 2009, 297 (1), R70-81.

[244] Palikova, I.; Valentova, K.; Oborna, I.; Ulrichova, J., Protectivity of Blue Honeysuckle Extract against Oxidative Human Endothelial Cells and Rat Hepatocyte Damage. J Agric Food Chem 2009.

[245] Goupy, P.; Bautista-Ortin, A. B.; Fulcrand, H.; Dangles, O., Antioxidant activity of wine pigments derived from anthocyanins: hydrogen transfer reactions to the dpph radical and inhibition of the heme-induced peroxidation of linoleic acid. J Agric Food Chem 2009, 57 (13), 5762-70.

[246] Chen, G.; Bower, K. A.; Xu, M.; Ding, M.; Shi, X.; Ke, Z. J.; Luo, J., Cyanidin-3-glucoside reverses ethanol-induced inhibition of neurite outgrowth: role of glycogen synthase kinase 3 Beta. Neurotox Res 2009, 15 (4), 321-31.

[247] Hafeez, B. B.; Siddiqui, I. A.; Asim, M.; Malik, A.; Afaq, F.; Adhami, V. M.; Saleem, M.; Din, M.; Mukhtar, H., A dietary anthocyanidin delphinidin induces apoptosis of human prostate cancer PC3 cells in vitro and in vivo: involvement of nuclear factor-kappaB signaling. Cancer Res 2008, 68 (20), 8564-72.

[248] Wang, L.-S.; Hecht, S. S.; Carmella, S. G.; Yu, N.; Larue, B.; Henry, C.; McIntyre, C.; Rocha, C.; Lechner, J. F.; Stoner, G. D., Anthocyanins in Black Raspberries Prevent Esophageal Tumors in Rats. Cancer Prev Res 2009, 2 (1), 84-93.

[249] Cooke, D.; Schwarz, M.; Boocock, D.; Winterhalter, P.; Steward, W. P.; Gescher, A. J.; Marczylo, T. H., Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis--relationship with tissue anthocyanin levels. Int J Cancer 2006, 119 (9), 2213-20.

[250] Thomasset, S.; Berry, D. P.; Cai, H.; West, K.; Marczylo, T. H.; Marsden, D.; Brown, K.; Dennison, A.; Garcea, G.; Miller, A.; Hemingway, D.; Steward, W. P.; Gescher, A. J., Pilot Study of Oral Anthocyanins for Colorectal Cancer Chemoprevention. Cancer Prev. Res. 2009, 2 (7), 625-633.

[251] Zapolska-Downar, D.; Nowicka, G.; Sygitowicz, G.; Jarosz, M., Anthocyanin-Rich Aronox Extract from Aronia melanocarpa E Protects against 7b-Hydroxycholesterol-Induced Apoptosis of Endothelial Cells. Annals of Nutrition and Metabolism 2008, 53 (3-4), 283-294.

[252] Xia, M.; Ling, W.; Zhu, H.; Ma, J.; Wang, Q.; Hou, M.; Tang, Z.; Guo, H.; Liu, C.; Ye, Q., Anthocyanin attenuates CD40-mediated endothelial cell activation and apoptosis by inhibiting CD40-induced MAPK activation. Atherosclerosis 2009, 202 (1), 41-47.

[253] Toufektsian, M. C.; de Lorgeril, M.; Nagy, N.; Salen, P.; Donati, M. B.; Giordano, L.; Mock, H. P.; Peterek, S.; Matros, A.; Petroni, K.; Pilu, R.; Rotilio, D.; Tonelli, C.; de Leiris, J.; Boucher, F.; Martin, C., Chronic dietary intake of plant-derived anthocyanins protects the rat heart against ischemia-reperfusion injury. J Nutr 2008, 138 (4), 747-52.

[254] Xia, X.; Ling, W.; Ma, J.; Xia, M.; Hou, M.; Wang, Q.; Zhu, H.; Tang, Z., An anthocyanin-rich extract from black rice enhances atherosclerotic plaque stabilization in apolipoprotein E-deficient mice. J Nutr 2006, 136 (8), 2220-5.

[255] Qin, Y.; Xia, M.; Ma, J.; Hao, Y.; Liu, J.; Mou, H.; Cao, L.; Ling, W., Anthocyanin supplementation improves serum LDL- and HDL-cholesterol concentrations associated with the inhibition of cholesteryl ester transfer protein in dyslipidemic subjects. Am J Clin Nutr 2009, 90 (3), 485-92.

[256] Butelli, E.; Titta, L.; Giorgio, M.; Mock, H. P.; Matros, A.; Peterek, S.; Schijlen, E.; Hall, R. D.; Bovy, A. G.; Luo, J.; Martin, C., Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat. Biotechnol. 2008, 26 (11), 1301-1308.

[257] DeFuria, J.; Bennett, G.; Strissel, K. J.; Perfield, J. W., 2nd; Milbury, P. E.; Greenberg, A. S.; Obin, M. S., Dietary blueberry attenuates whole-body insulin resistance in high fat-fed mice by reducing adipocyte death and its inflammatory sequelae. J Nutr 2009, 139 (8), 1510-6.

[258] Matsumoto, H.; Nakamura, Y.; Tachibanaki, S.; Kawamura, S.; Hirayama, M., Stimulatory Effect of Cyanidin 3-Glycosides on the Regeneration of Rhodopsin. Journal of Agricultural and Food Chemistry 2003, 51 (12), 3560-3563.

[259] Lee, J.; Lee, H. K.; Kim, C. Y.; Hong, Y. J.; Choe, C. M.; You, T. W.; Seong, G. J., Purified high-dose anthocyanoside oligomer administration improves nocturnal vision and clinical symptoms in myopia subjects. British Journal of Nutrition 2005, 93 (06), 895-899.

[260] Seymour, E. M.; Singer, A. A. M.; Kirakosyan, A.; Urcuyo-Llanes, D. E.; Kaufman, P. B.; Bolling, S. F., Altered hyperlipidemia, hepatic steatosis, and hepatic peroxisome proliferator-activated receptors in rats with intake of tart cherry. Journal of Medicinal Food 2008, 11 (2), 252(8).

[261] Chen, G.; Luo, J., Anthocyanins: Are They Beneficial in Treating Ethanol Neurotoxicity? Neurotox Res 2009.

[262] Li, C.-Y.; Xu, H.-D.; Zhao, B.-T.; Chang, H.-I.; Rhee, H.-I., Gastroprotective effect of cyanidin 3-glucoside on ethanol-induced gastric lesions in rats. Alcohol 2008, 42 (8), 683-687.

[263] Galvano, F.; Frigiola, A.; Gazzolo, D.; Biondi, A.; Malaguarnera, M.; Li Volti, G., Endothelial protective effects of anthocyanins: the underestimated role of their metabolites. Ann Nutr Metab 2009, 54 (2), 158-9.

[264] Møller, P.; Loft, S.; Alfthan, G.; Freese, R., Oxidative DNA damage in circulating mononuclear blood cells after ingestion of blackcurrant juice or anthocyanin-rich drink. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2004, 551 (1-2), 119-126.

[265] Wenzel, E.; Somoza, V., Metabolism and bioavailability of <I>trans</I>-resveratrol. Molecular Nutrition & Food Research 2005, 49 (5), 472-481.

[266] Day, A. J.; DuPont, M. S.; Ridley, S.; Rhodes, M.; Rhodes, M. J. C.; Morgan, M. R. A.; Williamson, G., Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver [beta]-glucosidase activity. FEBS Letters 1998, 436 (1), 71-75.

[267] Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis, J. E., Jr.; Walle, U. K., High absorption but very low bioavailibity of oral resveratrol in humans. Drug Metab Dispos 2004, 32 (12), 1377-1382.

[268] Yu, C.; Shin, Y. G.; Chow, A.; Li, Y.; Kosmeder, J. W.; Lee, Y. S.; Hirschelman, W. H.; Pezzuto, J. M.; Mehta, R. G.; van Breemen, R. B., Human, Rat, and Mouse Metabolism of Resveratrol. Pharmaceutical Research 2002, 19 (12), 1907-1914.

[269] Soleas, G. J.; Angelini, M.; Grass, L.; Diamandis, E. P.; Goldberg, D. M.; Lester, P., Absorption of trans-resveratrol in rats. In Methods in Enzymology, Academic Press: 2001; Vol. Volume 335, pp. 145-154.

[270] Udenigwe, C. C.; Ramprasath, V. R.; Aluko, R. E.; Jones, P. J. H., Potential of resveratrol in anticancer and anti-inflammatory therapy. Nutrition Reviews 2008, 66 (8), 445.

[271] Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L.-L.; Scherer, B.; Sinclair, D. A., Small molecule

activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425 (6954), 191-196.

[272] Baur, J. A.; Sinclair, D. A., Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov 2006, 5 (6), 493-506.

[273] Signorelli, P.; Ghidoni, R., Resveratrol as an anticancer nutrient: molecular basis, open questions and promises. The Journal of Nutritional Biochemistry 2005, 16 (8), 449-466.

[274] Baur, J. A., Obesity: Do Grapes Hold the Answer? [Miscellaneous]. Pediatric Research 2007, 61 (6), 633.

[275] Kaeberlein, M.; Rabinovitch, P. S., MEDICINE: Grapes versus gluttony. Nature 2006, 444 (7117), 280-281.

[276] Viswanathan, M.; Kim, S. K.; Berdichevsky, A.; Guarente, L., A Role for SIR-2.1 Regulation of ER Stress Response Genes in Determining C. elegans Life Span. Developmental Cell 2005, 9 (5), 605-615.

[277] Wood, J. G.; Rogina, B.; Lavu, S.; Howitz, K.; Helfand, S. L.; Tatar, M.; Sinclair, D., Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 2004, 430 (7000), 686-689.

[278] Valenzano, D. R.; Terzibasi, E.; Genade, T.; Cattaneo, A.; Domenici, L.; Cellerino, A., Resveratrol Prolongs Lifespan and Retards the Onset of Age-Related Markers in a Short-Lived Vertebrate. Current Biology 2006, 16 (3), 296-300.

[279] Pearson, K. J.; Baur, J. A.; Lewis, K. N.; Peshkin, L.; Price, N. L.; Labinskyy, N.; Swindell, W. R.; Kamara, D.; Minor, R. K.; Perez, E.; Jamieson, H. A.; Zhang, Y.; Dunn, S. R.; Sharma, K.; Pleshko, N.; Woollett, L. A.; Csiszar, A.; Ikeno, Y.; Le Couteur, D.; Elliott, P. J.; Becker, K. G.; Navas, P.; Ingram, D. K.; Wolf, N. S.; Ungvari, Z.; Sinclair, D. A.; de Cabo, R., Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. Cell Metab 2008, 8 (2), 157-68.

[280] Williams, L. D.; Burdock, G. A.; Edwards, J. A.; Beck, M.; Bausch, J., Safety studies conducted on high-purity trans-resveratrol in experimental animals. Food and Chemical Toxicology 2009, 47 (9), 2170-2182.

[281] Boocock, D. J.; Faust, G. E. S.; Patel, K. R.; Schinas, A. M.; Brown, V. A.; Ducharme, M. P.; Booth, T. D.; Crowell, J. A.; Perloff, M.; Gescher, A. J.; Steward, W. P.; Brenner, D. E., Phase I Dose Escalation Pharmacokinetic Study in Healthy Volunteers of Resveratrol, a Potential Cancer Chemopreventive Agent. Cancer Epidemiol Biomarkers Prev 2007, 16 (6), 1246-1252.

[282] Aribal-Kocatürk, P.; Özelçi Kavas, G.; İren Büyükkağnici, D., Pretreatment Effect of Resveratrol on Streptozotocin-Induced Diabetes in Rats. Biological Trace Element Research 2007, 118 (3), 244-249.

[283] Lagouge, M.; Argmann, C.; Gerhart-Hines, Z.; Meziane, H.; Lerin, C.; Daussin, F.; Messadeq, N.; Milne, J.; Lambert, P.; Elliott, P.; Geny, B.; Laakso, M.; Puigserver, P.; Auwerx, J., Resveratrol Improves Mitochondrial Function and Protects against Metabolic Disease by Activating SIRT1 and PGC-1[alpha]. Cell 2006, 127 (6), 1109-1122.

[284] Yousuf, S.; Atif, F.; Ahmad, M.; Hoda, N.; Ishrat, T.; Khan, B.; Islam, F., Resveratrol exerts its neuroprotective effect by modulating mitochondrial dysfunctions and associated cell death during cerebral ischemia. Brain Research 2009, 1250, 242-253.

[285] Karuppagounder, S. S.; Pinto, J. T.; Xu, H.; Chen, H. L.; Beal, M. F.; Gibson, G. E., Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease. Neurochem Int 2009, 54 (2), 111-8.

[286] Harper, C. E.; Patel, B. B.; Wang, J.; Arabshahi, A.; Eltoum, I. A.; Lamartiniere, C. A., Resveratrol suppresses prostate

cancer progression in transgenic mice. Carcinogenesis 2007, 28 (9), 1946-1953.

[287] Busquets, S.; Ametller, E.; Fuster, G.; Olivan, M.; Raab, V.; Argilés, J. M.; López-Soriano, F. J., Resveratrol, a natural diphenol, reduces metastatic growth in an experimental cancer model. Cancer Letters 2007, 245 (1-2), 144-148.

[288] Trincheri, N. F.; Nicotra, G.; Follo, C.; Castino, R.; Isidoro, C., Resveratrol induces cell death in colorectal cancer cells by a novel pathway involving lysosomal cathepsin D. Carcinogenesis 2007, 28 (5), 922-931.

[289] Van Ginkel, P. R.; Sareen, D.; Subramanian, L.; Walker, Q.; Darjatmoko, S. R.; Lindstrom, M. J.; Kulkarni, A.; Albert, D. M.; Polans, A. S., Resveratrol inhibits tumor growth of human neuroblastoma and mediates apoptosis by directly targeting mitochondria. Clin. Cancer Res. 2007, 13 (17), 5162-5169.

[290] Zhou, R.; Fukui, M.; Choi, H. J.; Zhu, B. T., Induction of a reversible, non-cytotoxic S-phase delay by resveratrol: implications for a mechanism of lifespan prolongation and cancer protection. British Journal of Pharmacology 2009, 9999 (9999).

[291] Yang, D. L.; Zhang, H. G.; Xu, Y. L.; Gao, Y. H.; Yang, X. J.; Hao, X. Q.; Li, X. H., Resveratrol Inhibits Right Ventricular Hypertrophy Induced By Monocrotaline In Rats. Clin Exp Pharmacol Physiol 2009.

[292] Brookins Danz, E. D.; Skramsted, J.; Henry, N.; Bennett, J. A.; Keller, R. S., Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway. Free Radical Biology and Medicine 2009, 46 (12), 1589-1597.

[293] Jung, C. M.; Heinze, T. M.; Schnackenberg, L. K.; Mullis, L. B.; Elkins, S. A.; Elkins, C. A.; Steele, R. S.; Sutherland, J. B., Interaction of dietary resveratrol with animal-associated bacteria. FEMS Microbiol Lett 2009, 297 (2), 266-73.

[294] Berardi, V.; Ricci, F.; Castelli, M.; Galati, G.; Risuleo, G., Resveratrol exhibits a strong cytotoxic activity in cultured cells and has an antiviral action against polyomavirus: potential clinical use. J Exp Clin Cancer Res 2009, 28, 96.

[295] Lee, M.; Kim, S.; Kwon, O.-K.; Oh, S.-R.; Lee, H.-K.; Ahn, K., Anti-inflammatory and anti-asthmatic effects of resveratrol, a polyphenolic stilbene, in a mouse model of allergic asthma. International Immunopharmacology 2009, 9 (4), 418-424.

[296] Kuroiwa, Y.; Nishikawa, A.; Kitamura, Y.; Kanki, K.; Ishii, Y.; Umemura, T.; Hirose, M., Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters. Cancer Letters 2006, 241 (2), 275-280.

[297] Wenzel, E.; Soldo, T.; Erbersdobler, H.; Somoza, V., Bioactivity and metabolism of trans-resveratrol orally administered to Wistar rats. Mol Nutr Food Res 2005, 49 (5), 482-94.

[298] Simons, A. L.; Renouf, M.; Hendrich, S.; Murphy, P. A., Human gut microbial degradation of flavonoids: structure-function relationships. J Agric Food Chem 2005, 53 (10), 4258-63.