Biomimetic one-pot preparation of a black tea polyphenol theasinensin A from epigallocatechin gallate by treatment with copper(II) chloride and ascorbic acid

Article abstract of DOI:10.1248/cpb.59.1183

Chromatographic separation of black tea polyphenols is too difficult to supply sufficient quantities of pure compounds for biological experiments. Thus, facile methods to prepare black tea constituents were desired. Treatment of epigallocatechin gallate w

Full text of DOI:10.1248/cpb.59.1183

September 2011
Note
Chem. Pharm. Bull. 59(9) 1183—1185 (2011)
1183
Biomimetic One-Pot Preparation of a Black Tea Polyphenol Theasinensin
A from Epigallocatechin Gallate by Treatment with Copper(II) Chloride
and Ascorbic Acid
Takuya SHII, Makoto MIYAMOTO, Yosuke MATSUO, Takashi TANAKA,* and Isao KOUNO
Graduate School of Biomedical Sciences, Nagasaki University; 1–14 Bunkyo-machi, Nagasaki 852–8521, Japan.
Received May 8, 2011; accepted June 14, 2011; published online June 17, 2011
Chromatographic separation of black tea polyphenols is too difficult to supply sufficient quantities of pure
compounds for biological experiments. Thus, facile methods to prepare black tea constituents were desired.
Treatment of epigallocatechin gallate with copper(II) chloride efficiently afforded an unstable quinone dimer, de-
hydrotheasinensin A, and subsequent treatment with ascorbic acid stereoselectively yielded theasinensin A. The
latter is a dimer with an R-biphenyl bond, one of the major polyphenols found in black tea. The method is sim-
pler and more effective than enzymatic preparation.
Key words theasinensin A; black tea; epigallocatechin gallate; dehydrotheasinensin A; copper(II) chloride
Black tea is one of the most popular beverages in the biological experiments. Therefore, development of methods
world, accounting for 70% of global tea production.1) It is for selective preparation of each black tea polyphenol is de-
produced by mechanical distortion and bruising of fresh tea sired. Theaflavins,15,16) which are characteristic pigments of
leaves and contains abundant polyphenols, generated by en- black tea with the benzotropolone moiety, can be prepared by
zymatic oxidation of tea leaf catechins. Theasinensin A (3)2—4) in vitro enzymatic and chemical oxidation of readily avail-
is a biologically active black tea polyphenol,5—7) produced by able green tea catechins.17,18) As for theasinensins, the yields
enzymatic oxidation of epigallocatechin gallate (1), the most of chemical and enzymatic preparations from tea catechins
abundant polyphenol found in fresh tea leaves.1) Experiments are low.9,19) Therefore, in this study we developed a facile and
using fresh tea leaves proved that the production mechanism biomimetic method to prepare 3 and its analogs.
of 3 is not simple oxidative coupling.8) In vitro model experi-
ments to mimic black tea production revealed that the en- Results and Discussion
zymes oxidize 1 to ortho-quinone and subsequent dimeriza-
The oxidative enzymes, tyrosinase and catecholoxidase,
tion of the quinone affords an unstable dimer, named dehy- are ubiquitous plant enzymes containing a binuclear copper
drotheasinensin A (2).9) On heating and drying of the tea center.20,21) They oxidize o-diphenols to the corresponding o-
leaves in the final stage of black tea production, 2 undergoes quinones, coupled with the reduction of molecular oxygen to
redox dismutation to give reduction products 3 and its atropi- water. To mimic the oxidation of catechins in tea leaves, 1
somer, along with a complex mixture of oxidation products, was treated with various copper salts (CuSO4, Cu(CH3COO)2,
including compounds produced by oxidative cleavage of aro- CuCO3, CuCl2) and ferrous salts (FeSO4, K3[Fe(CN)6],
matic rings9) (Chart 1). It is also known that reduction of 2 FeCl3) in aqueous MeOH and vigorously stirred to mix with
with reducing agents, such as mercaptoethanol and ascorbic oxygen from the air. HPLC comparison of the reaction mix-
acid, afford only 3, with an R-biphenyl bond. Stereoselective tures indicated that CuCl2 is the most effective for oxidation
formation of 2 from 1 was accounted for by the presence of a of 1 to 2. The amount of CuCl2 was shown to be at least 0.5
chiral center at the benzylic position of the pyrogallol molar equivalents, and the amount affected the reaction rate.
ring.10,11) Recently various health benefits of black tea Mixing of air (oxygen) into the reaction mixture regenerates
polyphenols have been reported.12,13) However, biological ac- CuCl2. The optimum concentration of MeOH in the reaction
tivities of each polyphenol component have not been studied solvent was 30%. A higher concentration of MeOH increased
sufficiently, compared with green tea polyphenols. Because uncharacterized byproducts, while a lower concentration
black tea polyphenols are a complex mixture of catechin oxi- slowed the rate of oxidation. Other solvents, such as aqueous
dation products, including polymeric substances,14) chro- acetonitrile, afforded complex mixtures. The optimum pH of
matographic separation of each polyphenol component is too the reaction mixture was shown to be 4 to 5 and addition of
difficult to supply sufficient quantities of pure compounds for buffer agents was unnecessary. At higher pH, decomposition
Chart 1. Production of Theasinensin A (3) from Epigallocatechin Gallate (1) via Dehydrotheasinensin A (2)
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: t-tanaka@nagasaki-u.ac.jp

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Vol. 59, No. 9
of product 2 was observed.9) Addition of imidazole, ethylene-
Animal experiments normally require pure samples at
diamine, and oxalic acid as ligands was not effective. The gram scale. Considering practicality and cost, the price of 1
initial oxidation step of the reaction was carried out at room as a starting material is too high and therefore the reaction
temperature and elevation of the reaction temperature in- was subsequently applied to commercially available green
creased byproducts.
tea polyphenol mixtures, which have recently become less
The oxidation product 2 is unstable and gradually decom- expensive. The extract contained mainly 1, epicatechin gal-
poses during isolation.9) Therefore, 2 was reduced without late, epigallocatechin, epicatechin, and their isomers (Fig.
separation and the reduction was successfully achieved by 2A). After treatment of the mixture with CuCl2, HPLC analy-
heating with ascorbic acid. On the heating, increase of sis (Fig. 2B) revealed that 1 and epigallocatechin were selec-
byproducts was not observed, probably because reduction tively oxidized to dehydrotheasinensins (2, 4—6)8) (Fig. 3).
proceeded preferentially and the metal ions were removed by Catechol-type catechins were not oxidized because the redox
complexation with ascorbic acid. Reduction with mercap- potential is higher than that of pyrogallol-type catechins.24,25)
toethanol instead of ascorbic acid reduced the yield of 3.
A reaction under optimized conditions is as follows: 1
(2.2 mmol) and CuCl2 (2.00 mmol) were dissolved in 30%
MeOH and vigorously stirred to mix air into the solution at
room temperature for 24 h. The HPLC profile at this stage is
shown in Fig. 1B. The reaction mixture was heated with an
excess amount of ascorbic acid and separated by Diaion
HP20 column chromatography to afford 3 (0.58 mmol, 53%).
The yield was much higher than that by enzymatic prepara-
tion of 3 (usually less than 30%19)). Further elution of the
column gave a complex mixture of dimeric and oligomeric
products. A large part of black tea polyphenols remain to be
chemically identified due to difficulty in purification.22,23)
These byproducts are probably related to the unknown black
tea polyphenols and chemical characterization is now in
progress.
Fig. 2. HPLC Profiles of the Reaction Mixture of Tea Polyphenols with
CuCl2 and Ascorbic Acid
(A) A commercial tea catechin mixture, (B) after treatment with CuCl2 (24 h), (C)
after treatment with ascorbic acid (85 °C, 15 min). GA: gallic acid, GC: gallocatechin,
EGC: epigallocatechin, Ca: catechin, EC: epicatechin, ECg: epicatechin gallate, GCg:
gallocatechin gallate, AA: ascorbic acid.
Fig. 1. HPLC Profiles of the Reaction Mixture of 1 with CuCl2 and Ascor-
bic Acid
(A) Starting material, (B) after treatment with CuCl2 (24 h), (C) after treatment with
ascorbic acid (AA) (85 °C, 15 min). Peak 2 was broadened due to equilibrium between
hydrated quinone structures, detection: 280 nm.
Fig. 3. Structures of 4—8

September 2011
1185
and CuCl2 (269 mg, 2.00 mmol) in 30% MeOH (400 ml) was vigorously
stirred to mix air into the solution at room temperature for 24 h. The HPLC
profile at this stage is shown in Fig. 1B. To the reaction mixture, an excess
amount of ascorbic acid (10 g) was added and heated at 85 °C for 15 min.
After cooling, the mixture was concentrated to evaporate MeOH and the re-
sulting aqueous solution was applied to a Diaion HP20 column (3.0 cm
i.d.ꢀ25 cm) with water. After washing the column with water to remove
reagents, gradient elution with water containing MeOH (5% stepwise gradi-
ent, each 100 ml) afforded 3 (0.533 g, 0.58 mmol, 53%).
Subsequent reduction with ascorbic acid yielded theasi-
nensins B (7) and C (8) as well as 3, which are easily separa-
ble by Diaion HP20 and Sephadex LH-20 column chro-
matography.
The concentration of theasinensins in black tea is compa-
rable to that of theaflavins.26,27) However, their biological ac-
tivities have not been fully evaluated because of difficulties
with supply of pure compounds, due to the complexity of
black tea polyphenols. Our results provide a facile and effi-
cient method for preparation of pure theasinensins. In addi-
tion, it is important that the reactions mimic the oxidation of
catechins in tea leaves during black tea production. Dehy-
drotheasinensins (2, 4—6) are key intermediates of oxidation
of epigallocatechin and its gallate, which in total account for
over 70% of tea catechins. Production of dehydrotheasi-
nensins during black tea production has been confirmed8) but
they are not detected in the final products. Understanding the
degradation of dehydrotheasinensins is essential for charac-
terization of unknown black tea polyphenols. Detailed analy-
sis of the minor reaction products will contribute to black tea
chemistry. The difference between enzymatic oxidation in
the tea leaves and the in vitro reaction with CuCl2 is that the
tea leaf enzymes preferentially oxidize catechol-type cate-
chins rather than pyrogallol-type catechins, which are chemi-
cally more sensitive to oxidation.18) Oxidative couplings be-
tween the quinones produced from catechol-type and pyro-
gallol type catechins generate theaflavins.15—17,28) In the pres-
ent experiments, the catechol-type catechins were not oxi-
dized and thus theaflavins were not detected in the reaction
products. Enzymatic oxidation of a commercially available
mixture of green tea catechins produces theaflavins as well as
theasinensins. However, the reactions are accompanied by
production of many other, uncharacterized oxidation prod-
ucts, produced by complex quinone coupling reactions as
well as further degradation of theaflavins.29,30) Chromato-
graphic separation of theaflavins and theasinensins from the
resulting complex mixture is problematic. The method for
preparing theasinensins from tea catechins described here
outperforms the enzymatic preparation in terms of selectiv-
ity, cost, and purification of the products.
Acknowledgments This work was supported by a Grant-in-Aid for
Young Scientists (B) No. 22700734 from the Japan Society for the Promo-
tion of Science and a Research Grant from the Asahi Breweries Foundation.
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General Column chromatography was performed using Diaion HP20SS
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performed on 0.2-mm-thick precoated Kieselgel 60 F254 plates (Merck)
using toluene–ethyl formate–formic acid (1 : 7 : 1, v/v) or cellulose F254
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(15 min) CH3CN in 50 mM H3PO4 (flow rate 0.8 ml/min; detection uisng a
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Oxidation of 1 and Separation of 3 A solution of 1 (1.0 g, 2.18 mmol)