Biotransformation of tea polyphenols by gut microbiota

JOURNAL OF FUNCTIONAL FOODS

x x x ( 2 0 1 4 ) x x x –x x x

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Biotransformation of tea polyphenols by gut microbiota Huadong Chen, Shengmin Sang* Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081, USA

A R T I C L E I N F O

A B S T R A C T

Article history:

Tea is one of the most widely consumed beverages worldwide and has received increasing

Available online xxxx

attention from researchers and food industries for various reasons mainly related to its health benefits. Polyphenols, such as catechins for green tea and theaflavins and thearub-

Keywords:

igins for black tea, are considered to be the main active components of tea. Recently, there

Tea

is increasing awareness that the beneficial health effects of tea could be partly contributed

Catechins

by breakdown products of its polyphenols formed in the gut. Different studies have been

Theaflavins

carried out to understand the formation of microbially derived metabolites of tea compo-

Microbiota

nents and their bioactivities. In general, tea catechins are typically transformed to specific

Biotransformation

hydroxyphenyl-c-valerolactones, which could be further metabolized to smaller phenolic

Bioactivity

acids by gut flora. This review summarizes the current knowledge on the metabolism of major tea components by gut microbiota and the bioactivities of their metabolites. Ó 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Tea is one of the most widely consumed beverages in the world and is second only to water in popularity as a beverage. More than 300 different kinds of tea are produced from the leaves of Camellia sinensis by different manufacturing processes. Tea is manufactured in three basic forms: green tea, oolong tea, and black tea. Green tea is prepared by precluding the oxidation of green leaf polyphenols. During black tea production, oxidation is promoted so that most of these substances are oxidized. Oolong tea is a partially oxidized product. Of the approximately 2.5 million metric tons of dried tea manufactured, only 20% of the tea production worldwide is green tea, less than 2% is oolong tea and about 78% is black tea. In general, catechins are the most abundant polyphenols in green tea. The main pigments in black tea are theaflavins and thearubigins, which are formed by the oxidation and polymerization of catechins during the process known as fermentation. Although the thearubigins account up to 60% of

the dry weight of black tea extract, the chemistry of thearubigins is still unclear (Haslam, 2003; Sang, Lambert, Ho, & Yang, 2011). The beneficial health effects of tea have been demonstrated in animal experiments and in some human studies including the prevention of cancer and heart disease (Havsteen, 2002; Mennen, Walker, Bennetau-Pelissero, & Scalbert, 2005; Yang, & Landau, 2000). In general, these effects are attributed to the polyphenol compounds in tea. Recently, more evidence on beneficial effects of tea polyphenols has partially attributed them to their breakdown products formed by the gut microbiota. In general, following tea intake, the major components like catechins, theaflavins and thearubigins reach gastrointestinal tract. Studies have suggested that the extent of absorption of dietary polyphenols in the small intestine is about 10–20%, while the largest part reaches the large intestine and is subject to colonic microbial breakdown (Kuhnle et al., 2000; Spencer, Schroeter, Rechner, & RiceEvans, 2001; Spencer et al., 1999), followed by absorption in

* Corresponding author. Tel.: +1 704 250 5710; fax: +1 704 250 5709. E-mail addresses: [email protected], [email protected] (S. Sang). 1756-4646/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2014.01.013

Please cite this article in press as: Chen, H., & Sang, S., Biotransformation of tea polyphenols by gut microbiota, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2014.01.013

2

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the blood-stream or excretion in the faeces (Feng, 2006; Roowi et al., 2010). Degradation of these polyphenols by gut microbiota results in new metabolites. Gut microbiota can hydrolyze glycosides, glucuronides, sulphates, amides, esters, and lactones. They also carry out ring-cleavage, reduction, decarboxylation, demethylation, and dehydroxylation reactions (Selma, Espin, & Tomas-Barberan, 2009). Once absorbed, the microbial breakdown products reach the liver, in which phase II conjugation reactions can occur and lead to glucuronidated, sulphated, and methylated conjugates or a combination thereof. These conjugates then circulate in the blood, may undergo enterohepatic circulation, and then finally leave the body by excretion in the urine and faeces (van Duynhoven et al., 2011). On the other hand, phenolic compounds and their metabolites contribute to the maintenance of gastrointestinal health by interaction with epithelial cells, and largely by modulating the gut microbial composition. Polyphenols may act as promoting factors of growth, proliferation, or survival for beneficial gut bacteria. The effects of dietary polyphenols on the modulation of the intestinal ecology and on the growth of gut microbial species have been reviewed (Hervert-Hernandez & Goni, 2011; Selma et al., 2009). The aim of this contribution is to summarize the current knowledge on the metabolism of the major tea components by gut microbiota and the bioactivities of these microbially derived metabolites.

2. Biotransformation microbiota

of

tea

catechins

by

Catechins, usually accounting for 30–42% of the dry weight of the solids in brewed green tea (Balentine, Wiseman, & Bouwens, 1997; Sang et al., 2011), are members of a more general class of flavonoid, the flavan-3-ols. They are characterized by di- or trihydroxyl group substitution of the B ring and the meta-5,7-dihydroxy substitution of the A ring. There are four major catechins in tea: ()-epigallocatechin gallate (EGCG), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG), and ()-epicatechin (EC), in which EGCG is the most abundant one and may account for 50–80% of the total catechins in tea. Studies have demonstrated that the absorption of catechins in the small intestine is relatively low, which implies that the majority of ingested catechins will reach the large intestine where they are subjected to colonic microbial breakdown, followed by absorption in the blood-stream or excretion in the faeces. The metabolism of ()-EGCG, ()-EGC, ()-ECG, ()-EC, and their corresponding stereoisomers (+)GCG, (+)-GC, (+)-CG, (+)-C by microbiota has been well studied in in vitro and in vivo models (Tables 1 and 2).

2.1.

(+)-Catechin (C) and ()-epicatechin (EC)

Griffith reported 3-hydroxyphenylpropionic acid (3-HPPA) in the urine of rats fed a diet with (+)-C, and then suggested that the formation of this metabolite could be dependent on the action of intestinal microflora (Griffiths, 1962). This suggestion was confirmed by his later work in which he compared metabolites from rats fed a (+)-C diet with and without added antibiotics (Griffiths, 1964). Since then more and more in vitro and in vivo studies on the metabolism of (+)-C and ()-EC have been

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reported. Many in vitro models were developed to investigate the metabolism of (+)-C and ()-EC, in which most commonly used are faecal samples or bacteria from rats, pigs, and humans. Significant variations among different species were observed due to different experimental conditions, incubation time, detection approach, and composition of the bacteria, albeit the metabolism pathway shares certain similarities. Even within the same species, the result can be quite different due to the composition of the gut microbiota varies substantially among individuals (Tappenden & Deutsch, 2007), consequently, each intestinal microbial community can be expected to display its own characteristic metabolic profile. Strong interindividual variations were found after incubation of black tea with gut microflora from 10 healthy human subjects (Gross et al., 2010). Recently, Takagaki and Nanjo (2013) identified several new metabolites of (+)-C or ()-EC generated by rat intestinal microbiota and updated the proposed metabolic pathway of (+)-C and ()-EC. The compounds (+)-C and ()-EC possess the same planar structure but different optical properties. A similar microbial metabolism profile between (+)-C and ()-EC has been found (Takagaki & Nanjo, 2013). However, Tzounis et al. (2008) reported the formation of microbial metabolites from (+)-C required its initial conversion to (+)-EC. Van’t Slot and Humpf (2009) also found that the incubation of (+)-C and ()-EC yielded comparable results and no differences in the microbial degradation between epimers were detectable. So far, four different strains from human intestinal bacteria have been identified to process the ability to metabolize (+)-C and ()EC. Wang et al. (2001) reported that Eubacterium sp. strain SDG-2 converted (+)-C to 1-(3 0 ,4 0 -dihydroxyphenyl)-3(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol, but converted ()-EC to 1-(3 0 ,4 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol and 1-(3 0 -hydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol, which means Eubacterium sp. strain SDG-2 has the ability of p-dehydroxylation in the B ring of ()-EC but not in (+)-C. Recently, Kutschera, Engst, Blaut, and Braune (2011) revealed that both (+)-C and ()-EC could be biotransformed to 1-(3 0 ,4 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol by Eggerthella lenta rK3. However, the conversion of (+)-C proceeded five times faster than that of ()-EC. Flavonifractor plautii aK2 further converted 1-(3 0 ,4 0 dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol to d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone and d-(3 0 ,4 0 -dihydroxyphenyl)-c-valeric acid. Flavonifractor plautii DSM 6740 catalyzed the identical reaction indicating it is not strain specific. The in vivo study on the metabolism of (+)-C has been investigated in rats, monkeys, pigs, and humans. Following the absorption of these metabolites, some of them may be Omethylated, glucuronidated, and sulphated, and the acids may be oxidized to benzoic acid derivatives, which may then be conjugated with glycine giving hippuric acid derivatives. For example, oral administration of (+)-C to men resulted in rapid metabolism and excretion of this compound, and the major phenolic acid metabolite is 3-HPPA and the major lactone metabolites include d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone and d-(3 0 -hydroxyphenyl)-c-valerolactone (Das, 1971). The phenolic compounds in the urine are excreted in both free and conjugated forms, including their glucuronides and sulphates (Das, 1971). In general, different species showed similar

Please cite this article in press as: Chen, H., & Sang, S., Biotransformation of tea polyphenols by gut microbiota, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2014.01.013

Metabolite

Model

(+)-C or ()-EC

1-(4 0 -Hydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 1-(3 0 -Hydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 1-(3 0 ,4 0 -Dihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol d-(3 0 ,4 0 -Dihydroxyphenyl)-c-valeric acid

Rat (Takagaki & Nanjo, 2013)

d-(3 0 -Hydroxyphenyl)-c-valeric acid 5-(3 0 ,4 0 -Dihydroxyphenyl)-4-oxo-valeric acid 5-(3 0 -Hydroxyphenyl)-4-oxo-valeric acid d-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone

Rat (Takagaki & Nanjo, Rat (Takagaki & Nanjo, Rat (Takagaki & Nanjo, Rat (Takagaki & Nanjo, Schantz et al., 2010)

d-(3 0 -Hydroxyphenyl)-c-valerolactone d-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 -Dihydroxyphenyl)-pentanoic acid 5-(3 0 -Hydroxyphenyl)-pentanoic acid 3,4-DHPPA

Rat (Takagaki & Nanjo, 2013) Human (Schantz et al., 2010) Rat (Takagaki & Nanjo, 2013), Human (Meselhy et al., 1997) Rat (Takagaki & Nanjo, 2013) Pig (van’t Slot and Humpf, 2009), Rat (Takagaki & Nanjo, 2013) Rat (Takagaki & Nanjo, 2013), Human (Meselhy et al., 1997; Roowi et al., 2010) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009), human (Roowi et al., 2010) Pig (van’t Slot and Humpf, 2009)

3-HPPA 4-HPAA 3-HBA 4-HBA

(+)-GC or ()-EGC

Phloroglucinol 1-(3 0 ,5 0 -Dihydroxyphenyl)- 3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone Phloroglucinol 3,4-DHPPA 3-HPPA 4-HPPA 3,4-DHPAA 3-HPAA 4-HPAA 3-HBA 4-HBA

Responsible microbiota

Rat (Groenewoud & Hundt, 1984; Takagaki & Nanjo, 2013), Human (Meselhy et al., 1997) Rat (Groenewoud & Hundt, 1984; Takagaki & Nanjo, 2013), Human (Meselhy et al., 1997) Rat (Takagaki & Nanjo, 2013), human (Roowi et al., 2010)

2013) 2013) 2013) 2013), Human (Roowi et al., 2010;

Eubacterium sp. strain SDG-2 (Wang et al., 2001) Eubacterium sp. strain SDG-2 (Wang et al., 2001), Eggerthella lenta rK3 (Kutschera et al., 2011) Flavonifractor plautii aK2 (Kutschera et al., 2011), Flavonifractor plautii DSM 6740 (Kutschera et al., 2011)

Flavonifractor plautii aK2 (Kutschera et al., 2011), Flavonifractor plautii DSM 6740 (Kutschera et al., 2011)

Eubacterium sp. strain SDG-2 (Wang et al., 2001) Human (Roowi et al., 2010; Schantz et al., 2010), Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009), human (Roowi et al., 2010), Pig (van’t Slot and Humpf, 2009) Pig (van’t Slot and Humpf, 2009)

x x x ( 2 0 1 4 ) x x x –x x x

Compound

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3

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Table 1 – Biotransformation of major tea polyphenols by gut microbiota in vitro.

4

Compound

Metabolite

Model

(+)-CG or ()-ECG

()-EC Gallic acid Pyrogallol 1-(3 0 -Hydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 1-(3 0 ,4 0 -Dihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone 5-(3 0 -Hydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 -Dihydroxyphenyl)-pentanoic acid 5-(3 0 -Hydroxyphenyl)-pentanoic acid 5-(3 0 -Methoxyphenyl)-pentanoic acid 200 ,300 -Dihydroxypenyl-3-(3 0 ,4 0 -dihydroxyphenyl)propanoate 3,4-DHPPA 3-HPPA EGC

Human Human Human Human

(+)-GCG or ()-EGCG

et et et et

al., al., al., al.,

1997) 1997) 1997) 1997)

Human (Meselhy et al., 1997) Human Human Human Human Human Human

(Meselhy (Meselhy (Meselhy (Meselhy (Meselhy (Meselhy

et et et et et et

al., al., al., al., al., al.,

1997) 1997) 1997) 1997) 1997) 1997)

Human (Meselhy et al., 1997) Human (Meselhy et al., 1997) Human (Roowi et al., 2010;Schantz et al., 2010), Pig (van’t Slot and Humpf, 2009), Rat (Takagaki & Nanjo, 2010)

Gallic acid

Human (Roowi et al., 2010;Schantz et al., 2010), Pig (van’t Slot and Humpf, 2009), Rat (Takagaki & Nanjo, 2010)

5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone

Human (Roowi et al., 2010), Pig (van’t Slot and Humpf, 2009), Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010)

4 0 -Dehydroxylation-EGC 1-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 1-(3 0 ,5 0 -Dihydroxyphenyl)- 3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol d-(3 0 ,5 0 -Dihydroxyphenyl)-c-valeric acid d-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valeric acid 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-pentanoic acid 5-(3 0 ,5 0 -Dihydroxyphenyl)-pentanoic acid 5-(3 0 -Hydroxyphenyl)-pentanoic acid d-(3 0 ,5 0 -Dihydroxyphenyl)-c-valerolactone Pyrogallol Pyrocatechol 4-HPAA 3,5-DHPPA Theaflavin Gallic acid Pyrogallol

Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Rat (Takagaki & Nanjo, 2010) Human (Roowi et al., 2010) Human (Roowi et al., 2010) Human (Roowi et al., 2010) Rat (Takagaki & Nanjo, 2010) Human (Chen et al., 2012) Human (Chen et al., 2012) Human (Chen et al., 2012)

Enterobacter aerogenes, Raoultella planticola, Klebsiella pneumoniae susp. pneumoniae, and Bifidobacterium longum subsp. infantis (Takagaki & Nanjo, 2010) Enterobacter aerogenes, Raoultella planticola, Klebsiella pneumoniae susp. pneumoniae, and Bifidobacterium longum subsp. infantis (Takagaki & Nanjo, 2010)

x x x ( 2 0 1 4 ) x x x –x x x

Theaflavin-3-gallate

Responsible microbiota (Meselhy (Meselhy (Meselhy (Meselhy

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Table 1 – continued

0

Theaflavin-3 -gallate

Theaflavin-3,3 0 -digallate

Green tea

Model

Responsible microbiota

Theaflavin Gallic acid Pyrogallol Theaflavin

Human Human Human Human

Theaflavin-3-gallate Theaflavin-3 0 -gallate

Human (Chen et al., 2012) Human (Chen et al., 2012)

Gallic acid

Human (Chen et al., 2012)

Pyrogallol

Human (Chen et al., 2012)

3-Methoxy-4-hydroxyphenylacetic acid 4-HPAA 3,4-DHPAA 3-HPPA Phlorogucinol Pyrogallol Coumaric acid Gallic acid 5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 ,5 0 -Rrihydroxyphenyl)-c-valerolactone 5-(3 0 -Hydroxyphenyl)-c-valerolactone Protocatechuic acid Dihydrocaffeic acid Homovanillic acid Quinic acid 3-Methoxy-4-hydroxyphenylacetic acid 4-HPAA 3,4-DHPAA 3-HPPA 2,4,6-Trihydroxybenzoic acid 4-HPPA Phlorogucinol Pyrogallol Coumaric acid Gallic acid 5-(3 0 ,4-Dihydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone 5-(3 0 -Hydroxyphenyl)-c-valerolactone Protocatechuic acid Dihydrocaffeic acid Homovanillic acid Quinic acid

Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

(Chen (Chen (Chen (Chen

et et et et

al., al., al., al.,

2012) 2012) 2012) 2012)

Lactobacillus plantarum 299v (Chen Bacillus subtilis (Chen et al., 2012) Lactobacillus plantarum 299v (Chen Lactobacillus plantarum 299v (Chen Bacillus subtilis (Chen et al., 2012) Lactobacillus plantarum 299v (Chen Bacillus subtilis (Chen et al., 2012) Lactobacillus plantarum 299v (Chen Bacillus subtilis (Chen et al., 2012)

et al., 2012), et al., 2012) et al., 2012), et al., 2012), et al., 2012),

(Gao et al., 2006) (Gao et al., 2006) (Gao et al., 2006) (Gao et al., 2006) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Gao et al., 2006) (Gao et al., 2006) (Gao et al., 2006) (Gao et al., 2006) (Gao et al., 2006) (Gross et al., 2010) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012) (Dall’Asta et al., 2012)

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Black tea

Metabolite

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Abbreviations: 3,4-DHPPA, 3,4-dihydroxyphenylpropionic acid; 3-HPPA, 3-hydroxyphenylpropionic acid; 4-HPPA, 4-hydroxyphenylpropionic acid; 3-HBA, 3-hydroybenzoic acid; 4-HBA, 4-hydroybenzoic acid; 3,4-DHPAA, 3,4-dihydroxyphenylacetic acid; 3-HPAA, 3-hydroxyphenylacetic acid; 4-HPAA, 4-hydroxyphenylacetic acid.

5

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Compound

6

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x x x ( 2 0 1 4 ) x x x –x x x

Table 2 – Biotransformation of major tea polyphenols by gut microbiota in vivo. (+)-C or ()-EC

3-HBA m-Hydroxyhippuric acid 3-HPPA 4-HPPA d-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone d-(3 0 -Hydroxyphenyl)-c-valerolactone

()-EGC

()-ECG

()-EGCG

m-Hydroxyphenylhydracrylic acid 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone 5-(3 0 ,5 0 -Dihydroxyphenyl)-c-valerolactone EC Gallic acid Pyrogallol 1-(3 0 ,4 0 -Dihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone 5-(3 0 -Hydroxyphenyl)-c-valerolactone 5-(3 0 ,4 0 -Dihydroxyphenyl)-valeric acid 3-HPPA (E)-3-(3-hydroxyphenyl)-acrylic acid EGC Gallic acid 1-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 1-(3 0 ,5 0 -Dihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone 5-(3 0 ,5 0 -Dihydroxyphenyl)-c-valerolactone

Theaflavin-3, 3 0 -digallate

Green tea

5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone Theaflavin Theaflavin-3-gallate Theaflavin-3 0 -gallate Gallic acid Pyrocatechol Pyrogallol 4-HBA 4-HPAA 3-HPAA 3-Methoxy-4-hydroxyphenylacetic acid Hippuric acid 3-(3-Hydroxyphenyl)-3-hydroxypropionic acid 5-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone

5-(3 0 ,4 0 -Dihydroxyphenyl)-c-valerolactone

5-(3 0 ,5 0 -Dihydroxyphenyl)-c-valerolactone

Black tea

3,4-DHBA 3-Methoxy-4-hydroxy-hippuric acid Vanillic acid Gallic acid 3,4-DHPAA

Human (Hackett, Griffiths, Broillet, & Wermeille, 1983), pig (Das & Griffiths, 1968) Human (Hackett et al., 1983) Human (Das, 1971;Hackett et al., 1983), rat (Das & Sothy, 1971; Griffiths, 1962, 1964) Rat (Das & Sothy, 1971), Monkey (Das, 1974) Rat (Das, 1971; Das & Sothy, 1971), human (Meng et al., 2002) Rat (Das, 1971; Das & Sothy, 1971), pig (Das & Griffiths, 1968), Monkey (Das, 1974) Monkey (Das, 1974) Human (Meng et al., 2002) Human (Meng et al., 2002) Human (Meng et al., 2002) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003; Takizawa, Morota, Takeda, & Aburada, 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri et al., 2003) Rat (Kohri, Matsumoto et al., 2001a; Kohri, Nanjo et al., 2001b) Rat (Kohri et al., 2001a; Kohri et al., 2001b) Rat (Kohri et al., 2001a; Kohri et al., 2001b) Rat (Kohri et al., 2001a; Kohri et al., 2001b) Rat (Kohri et al., 2001a; Kohri et al., 2001b), Human (Meng et al., 2002), mouse (Meng et al., 2002) Rat (Kohri et al., 2001a; Kohri et al., 2001b), Human (Meng et al., 2002), mouse (Meng et al., 2002) Human (Meng et al., 2002), mouse (Meng et al., 2002) Mouse (Chen et al., 2012) Mouse (Chen et al., 2012) Mouse (Chen et al., 2012) Mouse (Chen et al., 2012) Human (Roowi et al., 2010) Human (Roowi et al., 2010) Human (Pietta et al., 1998; Roowi et al., 2010) Human (Henning et al., 2013; Roowi et al., 2010) Human (Henning et al., 2013) Human (Roowi et al., 2010) Human (Roowi et al., 2010) Human (Roowi et al., 2010) Human (Del Rio et al., 2010; Henning et al., 2013; Li et al., 2000; Meng et al., 2002; Roowi et al., 2010; van der Hooft et al., 2012), mouse (Meng et al., 2002) Human (Del Rio et al., 2010; Henning et al., 2013; Li et al., 2000; Meng et al., 2002; van der Hooft et al., 2012), mouse (Meng et al., 2002) Human (Meng et al., 2002; van der Hooft et al., 2012), mouse (Meng et al., 2002) Human (Pietta et al., 1998) Human (Pietta et al., 1998) Human (Pietta et al., 1998) Human (Henning et al., 2013) Human (Henning et al., 2013)

Abbreviations: 3,4-DHBA, 3,4-dihydroxybenzoic acid; 3-HPPA, 3-hydroxyphenylpropionic acid; 4-HPPA, 4-hydroxyphenylpropionic acid; 3-HBA, 3-hydroybenzoic acid; 4-HBA, 4-hydroybenzoic acid; 3,4-DHPAA, 3,4-dihydroxyphenylacetic acid; 3-HPAA, 3-hydroxyphenylacetic acid; 4-HPAA, 4-hydroxyphenylacetic acid.

Please cite this article in press as: Chen, H., & Sang, S., Biotransformation of tea polyphenols by gut microbiota, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2014.01.013

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OH OH

2'

HO

8

HO

OH

O

5' 2

6'

4

6

OH

OH

OH

(+)-C or (-)-EC

Phlorglucinol

OH OH

2'

HO

OH

OH OH

2'

HO

5'

OH

4'' 6''

6'

2'

OH

HO

5' 1

5'

6'

6'

2

OH

2''

OH 1-(4'-Hydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH

3

OH

OH 1-(3',4'-Dihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH 1-(3'-Hydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH

OH OH

OH OH

2'

O 6'

δ-(3',4',5'-Trihydroxyphenyl)γ-valerolactone

5'

δ-(3',4'-Dihydroxyphenyl)γ-valeric acid

δ-(3',4'-Dihydroxyphenyl)γ-valerolactone

OH

6'

δ-(3'-Hydroxyphenyl)γ-valerolactone

δ-(3'-Hydroxyphenyl)γ-valeric acid

OH O

5' 6'

5'

O

2'

O

+

O

OH

6'

OH

2'

6'

5'

O

OH

OH

OH

2'

+ O

O

5'

O

OH

OH

2'

OH OH

2'

2'

O

O

5'

O

5'

6'

6'

OH

OH 5-(3'-Hydroxyphenyl)-4-oxo-valeric acid

5-(3',4'-Dihydroxyphenyl)-4-oxo-valeric acid

OH

OH OH

2'

O

2'

O

5'

5' 6'

6'

OH

OH

5-(3'-Hydroxyphenyl)pentanoic acid

5-(3',4'-Dihydroxyphenyl)pentanoic acid

OH

OH OH

O

O OH

OH

3,4-DHPPA

3-HPPA

OH

OH

O

O

O

HO 3,4-DHPAA

OH

OH

3,4-DHBA

3-HBA

OH

OH

O

OH

OH

OH

O

HO

OH 4-HPAA

4-HBA

Fig. 1 – Possible microbial metabolism pathway of (+)-C and ()-EC in the gut microbiota.

metabolic profile. In some cases, small differences may be found. For example, m-hydroxyphenylhydracrylic acid was

only identified in monkeys and humans, but not in other rodents (Feng, 2006). So far, the metabolism of ()-EC by gut

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microflora in vivo has not been reported. A summary of the metabolism of (+)-C in vivo was shown in Table 2. Based on previous in vitro and in vivo studies, a proposed microbial catabolism pathway of (+)-C and ()-EC is summarized in Fig. 1. In general, the beginning stage of microbial metabolism is to cleave the C ring to form metabolite 1-(3 0 ,4 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol. Subsequent dehydroxylation of 1-(3 0 ,4 0 dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol takes place to form 1-(3 0 -hydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol as a major metabolite and 1-(4 0 hydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol as a minor metabolite by dehydroxylation at 4 0 - and 3 0 -position, respectively. These metabolites then undergo ring fission of the phloroglucinol ring to form mainly their corresponding 4-hydroxy-5-phenylvaleric acids and 5-phenyl-c-valerolactones, which are then biotransformed to the corresponding valeric acids by the dehydroxylation at position 4. The dehydroxylation proceeded in a two-step reaction; that is, the 4-hydroxy-5-phenylvaleric acids were converted to their corresponding 4-oxo-5-phenylvaleric acids by oxidation, followed by reductive elimination of oxygen at the 4-oxo group to produce 5-phenylvaleric acids. The 5-phenylvaleric acids then undergo b-oxidation or a-oxidation to form smaller phenolic acids, such as 3,4-dihydroxyphenylpropionic acid (3,4-DHPPA), 3-hydroxyphenylpropionic acid (3-HPPA), 4hydroxyphenylpropionic acid (4-HPAA), 3-hydroybenzoic acid (3-HBA), and 4-hydroybenzoic acid (4-HBA) (Fig. 1). Besides 3,4-DHPPA, 4-HBA, and 3-HBA, phloroglucinol was also observed after incubation of ()-EC by the intestinal microbiota in the pig cecum model, which is potentially formed by the direct C ring cleavages at 1–2 and 4–10 bonds of EC (van’t Slot and Humpf, 2009). In addition, besides d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone, d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone was also reported as a metabolite of (+)-C and ()-EC in human ileostomy fluids, which indicated not only dehydroxylation but also hydroxylation could be carried out by gut bacteria (Schantz, Erk, & Richling, 2010). However, this result needs to be further verified.

2.2.

(+)-Gallocatechin (GC) and ()-epigallocatechin (EGC)

Compared with (+)-C and ()-EC, (+)-GC and ()-EGC possess one more hydroxyl group in the B ring, but the investigation on the metabolism of (+)-GC or ()-EGC was not as much as that of (+)-C or ()-EC. It has been reported that EGC can be metabolized to 1-(3 0 ,5 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol, by a human intestinal bacterium, Eubacterium sp. strain SDG-2 (Wang et al., 2001). Besides, 5-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone was also reported as a major metabolite of EGC by human faecal bacteria (Roowi et al., 2010; Schantz et al., 2010). In addition, several simple phenolic acids were formed by b-oxidation or a-oxidation, such as 3,4-DHPPA, 3-HPPA, 4-hydroxyphenylpropionic acid (4-HPPA), 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3-hydroxyphenylacetic acid (3-HPAA) acid, 3-HBA, 4-HBA as well as phloroglucinol (Roowi et al., 2010; van’t Slot and Humpf, 2009). Although the key C-ring cleaved metabolites such as 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol, d-(3 0 ,5 0 -dihydroxpenyl)-c-valerolactone,

x x x ( 2 0 1 4 ) x x x –x x x

and d-(3 0 ,4 0 -dihydroxpenyl)-c-valerolactone were not reported, we deduce they undergo this pathway to form above mentioned small phenolic acids as that of (+)-C or ()-EC (Fig. 1), Based on the reported metabolites, a microbial catabolism pathway of (+)-GC and ()-EGC is proposed in Fig. 2. Human study on the metabolism of EGC was conducted by ingesting pure EGC (Meng et al., 2002). After intake of 200 mg of pure EGC, d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone, d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone, and d-(3 0 ,5 0 dihydroxyphenyl)-c-valerolactone were detected in the urine. The urinary level of d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone reached 4.7 lM in the sample collected at 3–6 h after ingestion of EGC. Peak urinary level of d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-cvalerolactone of 14 lM was observed at 6–9 h after EGC ingestion. The compound d-(3 0 ,5 0 -dihydroxyphenyl)-c-valerolactone was observed with peak urinary value of 6.6 lM at 6–9 h after EGC ingestion (Meng et al., 2002).

2.3. (ECG)

(+)-Catechin gallate (CG) and ()-epicatechin gallate

(+)-CG and ()-ECG are the 3-O-gallate products of (+)-C and ()-EC, respectively. Since ()-ECG is the major component of tea, its biotransformation has extensively been investigated, while the biotransformation of (+)-CG has not been reported. The biotransformation of ()-ECG is discussed below. The first step of microbial metabolism of ()-ECG is hydrolysis, giving gallic acid and ()-EC. Both gallic acid and ()-EC undergo further microbial metabolism. Gallic acid is metabolized to pyrogallol (Meselhy, Nakamura, & Hattori, 1997), and the metabolism of ()-EC follows that discussed above. Meselhy et al. (1997) reported that 13 metabolites were found after incubation ()-ECG with human intestinal microbiota. They are ()-EC, 1-(3 0 ,4 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol, 1-(3 0 -hydroxyphenyl)-3-(200 ,400 ,60000 -trihydroxyphenyl)propan-2-ol,5-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone, 5-(3 0 -hydroxyphenyl)-pentanoic acid, 5-(3 0 -metho xyphenyl)-pentanoic acid, 5-(3 0 -hydroxyphenyl)-c-valerolactone, 5-(3 0 ,4 0 -dihydroxyphenyl)-pentanoic acid, 3,4-DHPPA, 3-HPPA, gallic acid, pyrogallol, and 200 ,300 -dihydroxypenyl-3(3 0 ,4 0 -dihydroxyphenyl)-propanoate. The authors suggested that formation of metabolite 200 ,300 -dihydroxypenyl-3-(3 0 ,4 0 dihydroxyphenyl)-propanoate is the result of condensation of pyrogallol and 3,4-DHPPA. In particular, 5-(3 0 -methoxyphenyl)-pentanoic acid was isolated from the reaction mixture of ()-ECG with human intestinal bacteria, which indicated momo-methylation of the aromatic hydroxyl group in 5-(3 0 -hydroxyphenyl)-pentanoic acid. The in vivo metabolism of ()-ECG has been studied in rats. After intravenous administration of ()-ECG, ()-EC,1-(3 0 ,4 0 -dihydroxyphenyl)3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol,5-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone,5-(3 0 -hydroxyphenyl)-c-valerolactone,5-(3 0 ,4 0 -dihydroxyphenyl)-valeric acid, 3-HPPA, (E)-3-(3hydroxyphenyl)-acrylic acid, gallic acid and pyrogallol were identified as the microbial metabolites of ECG (Kohri, Suzuki, & Nanjo, 2003). The most abundant metabolite in rat plasma and urine was found to be the conjugated form of pyrogallol (Kohri et al., 2003). The microbial catabolism pathway of (+)-CG and ()-ECG is proposed in Fig. 3.

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9

x x x ( 2 0 1 4 ) x x x –x x x

OH OH

2'

HO

OH

8

HO

5'

O 2 4

6

OH

OH

Phlorglucinol

(+)-GC or (-)-EGC

OH

OH

2' 8

HO 6

OH

2' 5'

OH 2

OH

6'

OH

6'

8

HO

OH

2

4

OH OH 1-(3',5'-Dihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

6

OH

2'

OH

6'

4

OH

5'

OH

O

5'

O

OH

OH

OH

6'

δ-(3',4',5'-Trihydroxyphenyl)γ-valerolactone

1-(3',4',5'-Trihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH

OH

OH

2' 2'

O

O

5'

O

5'

O

6'

6'

OH

δ-(3',4'-Dihydroxyphenyl)γ-valerolactone

δ-(3',5'-Dihydroxyphenyl)γ-valerolactone

OH OH

OH O

O

OH

OH 4-HPPA

OH

OH

3-HPPA

OH

O

O HO

O

OH 3,4-DHPPA

HO

OH

3-HPAA

OH

O HO

3,4-DHPAA

4-HPAA

OH OH O

O OH

OH

3-HBA

4-HBA

Fig. 2 – Possible microbial metabolism pathway of (+)-GC and ()-EGC.

2.4. (+)-Gallocatechin gallate epigallocatechin gallate (EGCG)

(GCG)

and

()-

()-EGCG, as the major catechin in tea, is the 3-O-gallate product of ()-EGC. As expected, the ester bonds of (+)-GCG and ()-EGCG are not stable against microbial degradation. Van’t Slot and Humpf (2009) reported that both (+)-GCG and ()EGCG were degraded by the intestinal microbiota in the pig cecum model to produce (+)-GC and ()-EGC, respectively, and gallic acid. This degradation was also proved by the human and rat intestinal flora in in vitro model (Schantz et al., 2010;Takagaki & Nanjo, 2010). After that, the metabolism of (+)-GC and ()-EGC occurs as was described above. Takagaki and Nanjo (2010) investigated the metabolism of ()-EGCG by rat intestinal bacteria in vitro and proposed three metabolic pathways. ()-EGCG is hydrolyzed to ()-EGC and gallic acid at the first step of metabolism. Of the 169 screened strains of enteric bacteria, Enterobacter aerogenes, Raoultella planticola, Kleb-

siella pneumoniae susp. pneumoniae, and Bifidobacterium longum subsp. infantis were found to have the ability to hydrolyze ()EGCG to ()-EGC and gallic acid. This step was common in all the three pathways. EGC formed from EGCG is converted to 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol by reductive opening between the 1 and 2 positions of EGC. The resulting 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 trihydroxyphenyl)propan-2-ol is transformed to 1-(3 0 ,5 0 -dihyby droxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol dehydroxylation at 4 0 position of 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol. Furthermore, the metabolite 1-(3 0 ,5 0 -dihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol undergoes ring-fission of the phloroglucinol moiety to yield 5-(3 0 ,5 0 -dihydroxyphenyl)-c-valeric acid as the major metabolite. This degradation pathway was considered to be the major route of EGCG metabolism in the in vitro study. At the same time, metabolite 5-(3 0 ,5 0 -dihydroxyphenyl)-c-valerolactone may be formed by lactonization of

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OH

OH

2'

8

HO

OH

OH

2'

O

5' 2

8

HO

6'

O

5' 2

4

6

O

OH

OH

6'

4

6

O

x x x ( 2 0 1 4 ) x x x –x x x

OH

OH EC

OH OH (+)-CG or (-)-ECG

OH

OH OH

2'

OH HO

OH

O

OH

5'

2'

OH

HO

5'

6'

6'

OH

OH

OH 1-(3',4'-Dihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH Gallic acid

OH OH 1-(3'-Hydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH OH

OH

OH 2'

OH Pyrogalloyl

O

O

OH O

5'

5'

OH

2'

O

5' 6'

6'

6'

OH 5-(3',4'-Dihydroxyphenyl)-pentanoic acid

OH 5-(3'-Hydroxyphenyl)-pentanoic acid

OH

OH O

OH OH

2'

δ-(4'-Hydroxy-3'-methoxyphenyl)γ-valerolactone

O

δ-(3'-Hydroxyphenyl)γ-valerolactone

OH

OMe 2'

6'

6'

δ-(3',4'-Dihydroxyphenyl)γ-valerolactone

O

5'

O

5'

O

O

2'

OH

2' 5' 6'

OH 2'',3''-Dihydroxypenyl-3-(3',4' -dihydroxyphenyl)-propanoate

OH

OH

OH

OH

O O

O

OH OH

OH 3,4-DHPPA

(E)-3-(3-hydroxyphenyl)acrylic acid 3-HPPA

Fig. 3 – Possible microbial metabolism pathway of (+)-CG and ()-ECG.

5-(3 0 ,5 0 -dihydroxyphenyl)-c-valeric acid right after the ring-fission. A small portion of metabolite 5-(3 0 ,5 0 -dihydroxyphenyl)-c-valeric acid is converted into 3,5-dihydroxyphenylpropionic acid, but its conversion is only very slight. In addition, a very small amount of EGC is converted to 4 0 -dehydroxylated metabolite, which appeared not to be further metabolized by the intestinal bacteria (Takagaki & Nanjo, 2010). Wang et al. (2001) have discussed the presence of 4 0 -hydroxyl group of EGC may be necessary for the reductive opening of its heterocyclic ring, which is essential for further degradation of EGC. One minor pathway is the formation of metabolite 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol from EGC. The compound 1-(3 0 ,4 0 ,5 0 trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol undergoes degradation of the phloroglucinol ring to produce d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valeric acid. This compound is converted into d-(3 0 ,5 0 -dihydroxyphenyl)-c-valeric acid by dehydroxylation at the 4 0 position. Furthermore, the metabolite d-(3 0 ,5 0 -dihydroxyphenyl)-c-valeric acid undergoes dehydroxylation at the 4 position to form 5-(3 0 ,5 0 -dihydroxy-

phenyl)-pentanoic acid, followed by transformation to 5-(3 0 ,-hydroxyphenyl)-pentanoic acid by elimination of the 5 0 -hydroxyl group in 5-(3 0 ,5 0 -dihydroxyphenyl)-pentanoic acid. Another minor pathway is the formation of 5-(3 0 ,4 0 , 5 0 -trihydroxyphenyl)-pentanoic acid by 1-(3 0 ,4 0 ,5 0 -trihydroxyvia dphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol (3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valeric acid. Finally, metabolite 5-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-pentanoic acid is converted to 5-(3 0 ,5 0 -dihydroxyphenyl)-pentanoic acid by undergoing dehydroxylation at the 4 0 position. The metabolite 5-(3 0 ,4 0 ,5 0 trihydroxyphenyl)-c-valerolactone is also produced right after ring fission of the phloroglucinol moiety of 1-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-3-(200 ,400 ,600 -trihydroxyphenyl)propan-2-ol. A proposed microbial catabolism pathway of (+)-GCG and ()EGCG is summarized in Fig. 4. The in vivo study on the metabolism of EGCG has also been investigated. It undergoes similar metabolic pathway as that of in vitro study. Kohri, Matsumoto et al. (2001a), Kohri, Nanjo et al. (2001b) showed after oral administration of EGCG to rats, most of EGCG moved into the cecum and large intestine

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OH

OH

2'

8

HO

OH

OH

OH

2'

2

6'

OH

8

HO

O

4

6

OH

O

8

HO

5'

O 2

4

OH

2'

5'

O

6

11

x x x ( 2 0 1 4 ) x x x –x x x

5'

O

OH

6'

2 6

OH

6'

4

OH

OH

OH

OH EGC

OH OH (+)-GCG or (-)-EGCG OH

OH

OH

O

OH OH

2'

OH

5'

OH

HO

OH

OH

HO

OH

6'

Gallic acid

2' 5'

OH

6'

OH

OH

OH 1-(3',4',5'-Trihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH 1-(3'-Hydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

OH OH OH OH Pyrogallol

OH OH

2'

O

OH

6'

OH 5'

OH

HO

6'

5'

O

OH OH

2'

OH

5'

O

5'

O

OH

6'

OH

6'

OH

OH OH 1-(3',4',5'-Trihydroxyphenyl)-3-(2'',4'',6''trihydroxyphenyl)propan-2-ol

δ-(3',4',5'-Trihydroxyphenyl)γ-valerolactone

2'

O

OH

OH

2'

δ-(3',5'-Dihydroxyphenyl)γ-valerolactone

δ-(3'-Hydroxyphenyl)γ-valeric acid

OH Pyrocatechol

OH 5'

O 6'

OH

OH

OH 2'

5'

O

OH

OH 5-(3',4'-Dihydroxyphenyl)-pentanoic acid

2'

2'

6'

OH

5'

O 6'

OH

OH 5-(3',5'-Dihydroxyphenyl)-pentanoic acid

5-(3'-Hydroxyphenyl)-pentanoic acid

OH OH O OH

OH 3,4-DHPPA O

OH OH 3,5-DHPPA

OH OH

O HO

3,4-DHPAA

OH

O HO 4-HPAA

Fig. 4 – Possible microbial metabolism pathway of (+)-GCG and ()-EGCG.

and then underwent degradation by intestinal bacteria to 5-(3 0 ,5 0 -dihydroxyphenyl)-c-valerolactone with EGC as an intermediate. A great part of 5-(3 0 ,5 0 -dihydroxyphenyl)-cvalerolactone is absorbed in the body, undergoing glucuronidation in the intestinal mucosa and/or liver, to form its glucuronidated metabolite, which enters the blood circula-

tion, is distributed to various tissues, and is finally excreted in the urine. After intake 200 mg of pure EGCG, the human urinary level of d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone reached 4.7 lM in the sample collected at 3–6 h after ingestion of EGCG. Peak urinary level of d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-cvalerolactone of 8.3 lM was observed at 3–6 h after EGCG

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x x x ( 2 0 1 4 ) x x x –x x x

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OH OH OH HO

O

HO

O

O OH OH

O OH

O

HO OH

HO OH

O

OH

OH

TF3'G

OH OH

O

OH

OH HO

HO

O

O

OH

O HO

O

HO

O

O

OH

O

HO OH

O OH

HO

OH

OH

PG

GA

O

OH

OH

OH

HO HO

HO

OH

TF

OH

OH

OH

OH

OH

TFDG O

OH

OH O OH

HO

O

HO

O

O OH OH OH

OH

TF3G

Fig. 5 – Possible microbial metabolism pathway of theaflavins (Chen et al., 2012).

ingestion. The compound d-(3 0 ,5 0 -dihydroxyphenyl)-c-valerolactone was observed with peak urinary values of 8.3 lM (Meng et al., 2002).

3. Biotransformation of tea theaflavins by microbiota Theaflavins, including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3 0 -gallate (TF3 0 G), and theaflavin-3,3 0 -digallate (TFDG), are the major bioactive polyphenols present in black tea. They are formed from co-oxidation of selected pairs of catechins in tea leaves during fermentation (Sang et al., 2011). Recently, theaflavins have received extensive attention due to their antioxidative, anti-inflammatory, and anti-tumor activities (Kumar, Pillare, & Maru, 2010; Sharma & Rao, 2009). However, it has been reported that theaflavins have poor systematic bioavailability. Very limited amounts of TFDG (<1 nmol/g tissue) were detected in tissue samples collected from mice treated with decaffeinated black tea (50 mg/g diet) for two weeks (Henning et al., 2006). We demonstrated that faecal microbiota from healthy human volunteers involved in the metabolism of TFDG, and found that TFDG is metabolized to TF, TF3G, TF3 0 G, gallic acid, and pyrogallol. Moreover, both TF3G and TF3 0 G are metabolized to TF, gallic acid, and pyrogallol by human microbiota (Fig. 5). In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG (Chen et al., 2012). Besides, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3 0 G, and gallic acid as the major faecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG-gavaged

GF mice. Our studies indicate that the microbiota is important for the metabolism of theaflavins in both mice and humans. In this experiment, inter-individual variation was also detected.

4. Biotransformation of other tea flavonoids by microbiota Flavonols, including quercetin, kaempferol, myricetin, and their glycosides are also present in tea. They make up about 0.5–2.5% (w/w) extract as aglycone in tea infusions. The fate of these tea polyphenols in the human body as a result of uptake in the small and large intestine has been the subject of study over the recent years. Hein, Rose, van’t Slot, Friedrich, and Humpf (2008) demonstrated that quercetin O-glycosides are almost completely metabolized by the pig intestinal microbiota. The liberated aglycone quercetin was completely metabolized to phenolic degradation products such as 3,4DHPAA, 4-HPAA, and phloroglucinol. Quercetin can also be metabolized by human faecal flora to form smaller phenolic acid, such as 3,4-DHPAA, 3-HPAA, and phloroglucinol (Aura et al., 2002; Winter, Moore, Dowell, & Bokkenheuser, 1989). In the same study, the authors reported kaempferol was metabolized to 4-HPAA by four Clostridium strains. Griffith and Smith (1972) found that myricetin can be metabolized by rat microbiota both in vitro and in vivo. Hanske, Loh, Sczesny, Blaut, and Braune (2009) investigated the impact of human intestinal microbiota metabolism of apigenin-7-glucoside by comparing germ free (GF) and human microbiota-associated (HMA) rats. Naringenin, eriodictyol, phloretin, 3,4-DHPPA, 4HPPA, 3-HPPA, and 4-hydroxycinnamic acid in their free and

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conjugated forms were formed in HMA rats but not in GF rats, with 4-HPPA being the major one (Hanske et al., 2009).

5. Biotransformation microbiota

of

tea

extract

x x x ( 2 0 1 4 ) x x x –x x x

13

consumption have been studied by metabolomics approach (van Dorsten, Daykin, Mulder, & Van Duynhoven, 2006). However, this approach is still in its infancy.

by

Besides studies on individual polyphenols in tea, microbiallyderived metabolites of tea extract have also been studied in vitro and in vivo. In theory, metabolism of tea extract is the overall effects of its individual components. Therefore, the biotransformation of tea depends on its constituents. It was difficult to determine which component was responsible for certain metabolites since it was a mixture of different polyphenols and some polyphenols possess similar metabolic pathways. In general, these metabolites can be classified into two categories: phenolic acid and hydroxyphenyl-c-valerolactone. Different phenolic acids have been found from different in vitro experiments. For example, Gao et al. (2006) used a dynamic in vitro model of the large intestine that simulates colonic fermentation by the intestinal microbiota and found the fermentation products after the infusion of green tea were 3-methoxy-4-hydroxyphenylacetic acid, 4-HPAA, 3HPPA, 3,4DHPAA, and 2,4,6-THBA. While the main product was 3-methoxy-4-hydroxyphenylacetic acid and smaller amounts of 4-HPAA, 3-HPPA, 3,4-DHPAA and 2,4,6-THBA were present after black tea infusion. Gross et al. (2010) investigated the bioconversion of black tea polyphenols in in vitro faecal batch fermentations and found these complex polyphenol mixtures were degraded to a limited number of key metabolites. The major metabolites were 3-HPPA and its hydroxylated derivatives followed by phenylacetic acid and its hydroxylated derivatives. Benzoic acids were generally produced in lower amounts. There are many factors affecting the formation of these metabolites, such as the origin of the tea polyphenols, the composition of the microbiota, the length of time of fermentation. In vivo metabolism of tea was also investigated. In particular, the ring fission metabolites 5-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone, 5-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone, and 5-(3 0 -hydroxyphenyl)-c-valerolactone were found in mouse and human urine and plasma after green tea ingestion (Li et al., 2000; Meng et al., 2002; Roowi et al., 2010). 4-HBA, 3,4-DHBA, 3-methoxy-4-hydroxy-hippuric acid, vanillic acid were identified as the major tea metabolites from the human urine samples collected at 6–48 h after drinking tea (about 400 mg tea catechins) (Pietta et al., 1998). Henning et al. (2013) showed urine concentrations of 4-HPAA, 3-HPAA, d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone and d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone were increased significantly in men drinking green tea, while 3-O-methylgallic acid was significantly increased after drinking black tea. In serum, 3,4DHPAA was significantly increased after consumption of green tea and black tea and 4-HPAA after green tea consumption. The typical metabolites of green tea and black tea are shown in Tables 1 and 2. Unraveling the complex metabolic fate of tea polyphenols requires joint deployment of in vitro and humanized mouse models and human intervention trials. Within these systems, the variation in diversity and functionality of the colonic microbiota can increasingly be captured by rapidly developing metabolomics technologies. The metabolic differences between green tea and black tea

6. Bioactivity of microbial derived metabolites of tea polyphenols The biological activities of microbial metabolites derived from the catabolism of tea have recently drawn much attention. In particular, those of phenolic acid formed from tea polyphenols have been unraveled. For example, multiple studies have revealed that both gallic acid and pyrogallol play an important role in the inhibition of cancer. It is reported that gallic acid induced apoptosis in A375.S2 human melanoma cells and suppressed lipopolysaccharide-induced nuclear factor-jB signaling by preventing ReIA acetylation in A-549 lung cancer cells (Choi et al., 2009; Lo et al., 2010). Several laboratory animal studies have shown that gallic acid can prevent cancer in different organs including colon, prostate and lung (Choi et al., 2009; Giftson, Jayanthi, & Nalini, 2010; Giftson Senapathy, Jayanthi, Viswanathan, Umadevi, & Nalini, 2011; Kaur, Velmurugan, Rajamanickam, Agarwal, & Agarwal, 2009; Kawada et al., 2001; Raina et al., 2008). In addition, pyrogall has been reported to inhibit the growth of human lung cancer Calu-6 cells via multi pathways (Han, Kim, Kim, & Park, 2008a, 2008b, 2008c, 2009). Monagas et al. (2009) reported 3,4-DHPPA and 3,4-DHPAA reduced lipopolysaccharide-stimulated cytokine secretion by human peripheral blood mononuclear cells. The bioactivities of these phenolic acids have been reviewed (Lambert, Sang, & Yang, 2007; Monagas et al., 2010; Spencer et al., 2001). Here we focus on the specific metabolites of tea polyphenols, the hydroxyphenyl-c-valerolactone.

6.1.

Antioxidant activity

The antioxidant activity of d-(3,4-dihydroxy-phenyl)-c-valerolactone and its methyl derivative d-(3-methoxy-4-hydroxyphenyl)-c-valerolactone has been tested against superoxide radicals, as well as by the ferric reducing antioxidant potential (FRAP) test (Grimm, Schafer, & Hogger, 2004). d-(3,4-Dihydroxy-phenyl)-c-valerolactone was significantly more effective than (+)-C, ascorbic acid and trolox in superoxide scavenging, whereas d-(3-methoxy-4-hydroxy-phenyl)-c-valerolactone did not exhibit scavenging activity (Grimm et al., 2004). In a redox-linked colorimetric assay, both d-(3,4-dihydroxy-phenyl)-c-valerolactone and d-(3-methoxy-4-hydroxyphenyl)-c-valerolactone displayed antioxidant activities, with d-(3,4-dihydroxy-phenyl)-c-valerolactone being significantly more potent than (+)-catechin and ascorbic acid (Grimm et al., 2004). Besides, d-(3,4-dihydroxy-phenyl)-c-valerolactone and d-(3,4-dihydroxy-phenyl)-d-valerolactone showed very similar antioxidant capacity by measuring the ORAC (oxygen radical absorbance capacity) (Sanchez-Patan et al., 2011).

6.2.

Anti-proliferative activity

The hydroxyphenyl-c-valerolactone metabolites have been synthesized and their anti-proliferative activities tested (Lambert, Rice, Hong, Hou, & Yang, 2005). In general, the methylated hydroxyphenyl-c-valerolactone was less effective

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x x x ( 2 0 1 4 ) x x x –x x x

than those of unmethylated metabolites. For example, d(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone was more effective in the inhibition of the growth of a series of immortalized and malignant human cell lines than its trimethoxylated derivative, with the exception of HCT-116 colon cancer cells, and immobilized human (INT407) and rat (IEC-6) intestinal cells, which were not sensitive to the growth-inhibitory effects of this compound. The compound d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)c-valerolactone was also more effective in the inhibition of the growth of colon (HT-29) and oesophagus (KYSE150) cancer cells than d-(3 0 ,4 0 -dihydroxyphenyl)-c-valerolactone and its mono- and dimethoxylated derivatives. However, the growth-inhibitory effects of this metabolite were lower than that of the EGCG. Treatment of KYSE150 with d-(3 0 ,4 0 ,5 0 -Trihydroxyphenyl)-c-valerolactone at 50 lM resulted in a 40% cell-growth inhibition after 48 h, whereas EGCG resulted in a 50% inhibition at 20 lM (Lambert et al., 2005).

was significantly diminished at higher concentrations of d(3,4-dihydroxy-phenyl)-c-valerolactone and in the presence of glucose, suggesting a facilitated transport of d-(3,4-dihydroxy-phenyl)-c-valerolactone via GLUT-1 transporter. This concept was further supported by structural similarities between the natural substrate a-D-glucose and the S-isomer of d-(3,4-dihydroxy-phenyl)-c-valerolactone. After cellular uptake, d-(3,4-dihydroxy-phenyl)-c-valerolactone underwent further metabolism by conjugation with glutathione. They presented strong indication for a transporter-mediated accumulation of a flavonoid metabolite in human erythrocytes and subsequent formation of a novel glutathione adduct. However, the physiologic role of the adduct remains to be elucidated.

6.3.

Numerous studies have found that gut microbiota plays an important role in the metabolism of polyphenols. Therefore, the role of gut microbiota should be further examined in order to better understand the beneficial health effects of tea. Deployment of in vitro gut models, humanized mouse models, and human intervention trials, in combination with deployment of metabolomics and microbiomics, provide a prerequisite for unraveling the role of colonic microbiota in the bioconversion of tea polyphenols. In general, these microbial metabolites are further absorbed and metabolized by phase II enzymes, to finally enter the circulation or eliminated in the urine (Fig. 6). The bioactivity of these metabolites, mainly small phenolic acid and hydroxyphenyl-c-valerolactone, remains largely unknown. Further studies on the bioactivity of these metabolites are warranted. Strong inter-individual variations on the metabolism of these compounds were found in both in vitro and in vivo experiments. System biology strategies need to be pursued for linking gut microbial diversity to nutritional phenotypes and bioactivity of the tea polyphenols. Ultimately, direct strategy for personalized nutrition based on dietary modulation of gut microbial functionality of

Catethins

Liver

Flavonols

Phase I metabolism

Flavones TFS …

Phase II metabolism

Bile

Portal vein

Small intestine

Tea intake

Colon

The compound d-(3,4-dihydroxy-phenyl)-c-valerolactone and its methyl derivative d-(3-methoxy-4-hydroxy-phenyl)-c-valerolactone had similar inhibitory effect on matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) (Grimm et al., 2004). Both metabolites also had similar efficacy in the inhibition of the secretion of MMP-9 from LPS-stimulated human monocytes (Grimm et al., 2004). On the other hand, Lambert et al. (2005) reported d-(3 0 ,4 0 ,5 0 -trihydroxyphenyl)-c-valerolactone showed inhibition of NO production in murine macrophage cells (RAW264.7) with IC50 of 20 lM, while its trimethoxylated derivative did not present any activity (Lambert et al., 2005). Besides, Uhlenhut and Hogger (2012) recently showed for the first time, that d-(3,4-dihydroxy-phenyl)-c-valerolactone inhibited nitrite production (IC50 1.3 lg/ml, 95% CI 0.96–1.70) and iNOS expression (IC50 3.8 lg/ml, 95% CI 0.99–14.35) in a concentration-dependent fashion. This exemplifies bioactivation by metabolism because the d-(3,4-dihydroxy-phenyl)-cvalerolactone precursor molecule catechin is only weakly active. However, the authors found these effects required a much higher concentration of d-(3,4-dihydroxy-phenyl)-c-valerolactone than those previously detected in human plasma samples. To understand this, they investigated a possible accumulation of d-(3,4-dihydroxy-phenyl)-c-valerolactone in cells and indeed observed high-capacity binding of this d-(3,4-dihydroxy-phenyl)-c-valerolactone to macrophages, monocytes, and endothelial cells. This binding was distinctly decreased in the presence of the influx inhibitor phloretin, suggesting the contribution of a facilitated d-(3,4-dihydroxyphenyl)-c-valerolactone transport into cells (Uhlenhut & Hogger, 2012). Since both d-(3,4-dihydroxy-phenyl)-c-valerolactone (34.9 ± 1.28%) and d-(3-methoxy-4-hydroxy-phenyl)c-valerolactone (26.4 ± 0.03%) showed lower plasma protein binding capacity (Kurlbaum & Hogger, 2011), Kurlbaum, Mulek, and Hogger (2013) hypothesized that measurable concentrations of these metabolites reside in compartments other than plasma. They found that caffeic acid, taxifolin, and ferulic acid passively bind to red blood cells, but only the bioactive metabolite d-(3,4-dihydroxy-phenyl)-c-valerolactone revealed pronounced accumulation. The partitioning of d-(3,4-dihydroxy-phenyl)-c-valerolactone into erythrocytes

Conclusion

Phenolic acids Hydroxyphenyl-γ-valerolactones ...

Anti-inflammatory effects

7.

Organs and Tissues

Blood circulation

Kidney

Urine excretion

Fecal excretion

Fig. 6 – Schematic diagram of the metabolism of tea components by gut microbiota.

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individuals or populations is a promising area for future research.

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Please cite this article in press as: Chen, H., & Sang, S., Biotransformation of tea polyphenols by gut microbiota, Journal of Functional Foods (2014), http://dx.doi.org/10.1016/j.jff.2014.01.013