Journal of Chromatography B, 1006 (2015) 1–7

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Analysis of stereochemistry and biosynthesis of epicatechin in tea

plants by chiral phase high performance liquid chromatography
Yumei Qian a,b , Xianqian Zhao c , Lei Zhao d , Lilan Cui a , Li Liu a , Xiaolan Jiang a , Yajun Liu c ,
Liping Gao c,∗∗ , Tao Xia a,∗
a
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Rd., Hefei 230036 Anhui, China
b
School of Biological and Food Engineering, Suzhou University, 49 Middle Bianhe Rd., Suzhou 234000 Anhui, China
c
School of Life Science, Anhui Agricultural University, 130 West Changjiang Rd., Hefei 230036 Anhui, China
d
College of Horticulture, Qingdao Key Laboratory of Genetic Improvement and Breeding in Horticultural Plants, Qingdao Agricultural University,
700Changcheng Rd., Qingdao 266109 Shandong, China

a r t i c l e i n f o a b s t r a c t

Article history: Tea (Camellia sinensis) is rich in flavan-3-ols (catechins), especially epicatechin (EC), which is the
Received 28 June 2015 predominant extension unit of polymeric proanthocyanidins (PAs). However, studies assessing EC’s stere-
Received in revised form 16 October 2015 ochemistry are scarce. Here, a high performance liquid chromatography column using amylose tris-(3,
Accepted 16 October 2015
5-dimethylphenylcarbamate) immobilized on silica-gel as chiral stationary phases (CSPs) was applied to
Available online 23 October 2015
explore its stereochemistry and biosynthetic pathway in tea plants. The results revealed (−)-epicatechin
[(−)-EC] was the predominant di-hyroxy-non-galloylated-catechins, while (+)-epicatechin [(+)-EC] was
Keywords:
not detected. Interestingly, (−)-EC was the only product obtained from cyanidin using the partially puri-
High performance liquid chromatography
Chiral phase
fied native C. sinensis anthocyanidin reductase (CsANR) in the presence of reduction nicotinamide adenine
Stereochemistry dinucleotide phosphate (NADPH); meanwhile, (+)-EC was the main product using recombinant CsANR
Biosynthesis in the same conditions. In addition, (−)-EC could be obtained from (+)-catechin [(+)-C] using recombi-
Epicatechin nant CsANR, which displayed C3 -epimerase activity in the presence of oxidation nicotinamide adenine
Tea dinucleotide phosphate (NADP+ ). But the partially purified native CsANR did not possess this function.
Finally, (−)-EC could result from the de-gallate acid reaction of epicatechin gallate (ECG) catalyzed by a
novel partially purified native galloylated catechins hydrolase (GCH) from tea leaves. In summary, (−)-
EC is likely the product of native protein from the tea plants, and (+)-EC is only produced in a reaction
catalyzed by recombinant CsANR in vitro.
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction Previous studies have found that 2R-flavan-3-ols are mainly

Tea [Camellia sinensis (L.) O. Kuntze], originated in China, is one
of the most widely cultivated and consumed beverages throughout
the world. Flavan-3-ols (catechins) are the major class of secondary
metabolites in tea plants; they are responsible for tea quality and
beneficial to human health [1–4].
Owing to two asymmetric atoms at the C2 and C3 positions, four
heterogeneous flavan-3-ol types are known, including (2R, 3R)-cis-
flavan-3-ols (i.e., (−)-epi-form), (2S, 3S)-cis-flavan-3-ols (i.e., (+)-
epi-form), (2S, 3R)-trans-flavan-3-ols (i.e., (−)-form), and (2R, 3S)-
trans-flavan-3-ols (i.e., (+)-form) (Fig. 1A).
∗ Corresponding author. Fax: +86 551 65785729.
∗∗ Corresponding author. Fax: +86 551 65785729.
E-mail addresses: gaolp62@126.com (L. Gao), xiatao62@126.com (T. Xia).
of two types (i.e., epi-form and non epi-form) that consist
of (−)-epigallocatechin gallate [(−)-EGCG], (−)-epigallocatechin
[(−)-EGC], (−)-epicatechin gallate [(−)-ECG], (−)-EC, (+)-C and (+)-
gallocatechin [(+)-GC] in tea plants, using the two-way paper
chromatography technology [5]. The content of epicatechins (ECs),
the collective term for (−)-EC, (−)-EGCG, (−)-EGC, and (−)-ECG,
could be affected by different cultivation conditions; for instance,
the content of ECs decreased by shading [6]. However, ECs con-
stitute the main components of total catechins in tea leaves [7]
(Fig. 1B). 2S-flavan-3-ols, being non-native flavan-3-ols, such as
(−)-gallocatechin gallate [(−)-GCG], (−)-gallocatechin [(−)-GC],
(−)-catechin [(−)-C], and (−)-catechin gallate [(−)-CG], were only
detected in manufactured tea. For example, Chen et al. reported
that (−)-GCG levels could reach as much as 50% of total catechins
in some tea drinks, using HPLC [8]; in addition, Gotti et al. reported
that (−)-GC and (−)-C were detected in tea infusion at 85 ◦ C for

http://dx.doi.org/10.1016/j.jchromb.2015.10.024
1570-0232/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
2 Y. Qian et al. / J. Chromatogr. B 1006 (2015) 1–7
Fig. 1. Stereochemistry of flavan-3-ols. (A) Four types of flavan-3-ols; (B) typical epicatechins in tea plants.
5 min via a chiral cyclodextrin-micellar electrokinetic chromatog-
raphy (CD-MEKC) method [9].
High performance liquid chromatography well known for direct
enantioseparation based on different CSPs (i.e., chiral HPLC) has
received increasing attention due to its advantages, and the
polysaccharide-based CSPs are most powerful, thanks to their
high loading capacity and wide applicability [10,11]. To our
knowledge, studies using chiral HPLC with amylose tris-(3,5-
dimethylphenylcarbamate) immobilized on silica-gel to identify
the stereochemistry of flavan-3-ols in fresh tea leaves are scare.
For (−)-EC biosynthesis, there may be three biosynthetic path-
ways in tea plants. The main one is de novo synthesis (Fig. 2). Xie
et al. described anthocyanidins as the most effective substrates, and
anthocyanidin reductase (ANR) as key enzymes encoded by impor-
tant genes; for instance, BANYULS (BAN) from Arabidopsis thaliana
and Medicago truncatul could convert cyanidin into (−)-EC in the
presence of NADPH [12]. Further study revealed (−)-C as a minor
by-product in the above enzyme reaction system, straight from
(−)-EC after a non-enzymatic C2 -epimerization [13].
Recently, new developments have been proposed for the func-
tion of ANR. Gargouri et al. reported that (+)-EC and (−)-C are
produced from cyanidin by using recombinant Vitis vinifera antho-
cyanidin reductase (VvANR) in the presence of NADPH [14]. Pang
also reported that recombinant CsANR could convert cyanidin to
the same products as above in vitro [15]. However, (+)-EC and (−)-C
are not detected in the tea plant; specifically, (+)-EC is seldom found
in the plant kingdom [16]. So far, previous studies have reported
the stereochemistry of EC obtained from cyanidin by using recom-
binant ANR in vitro, but did not report that by native ANR in vivo.
The second biosynthetic pathway is assumed that (−)-EC
results from (+)-C by epimerization. Extensive studies have shown
that epimerization of flavan-3-ols can be classified into two
types, namely non-enzymatic and enzymatic epimerization. Non-
enzymatic epimerization often occurs at the C2 position under
extreme external environment conditions such as high temper-
ature or pH higher than 6.0 [17–20]. Under moderate physical
conditions, non-enzymatic epimerization between (−)-EC and (−)-
C could be observed only after incubation of (−)-EC in 100 mM
Tris–HCl buffer with pH 7 at temperatures above 30 ◦ C for 30 min,
but not in MES buffer [13]. Meanwhile, enzymatic epimerization
occurs at the C3 position. Interestingly, recombinant VvANR could
act as a pure C3 -epimerase of 2R-flavan-3-ols in reverse direction,
but not 2S-flavan-3-ols in the presence of excess NADP+ ; in other
words, (−)-EC was obtained from (+)-C by enzymatic epimeriza-
tionin vitro [14].
In the third biosynthesis pathway, (−)-EC can result from ECG
via de-gallate acid reaction. Our previous study indicated that a
novel native protein, named galloylated catechins hydrolase (GCH),
could catalyze ECG into EC [21]. This paper, it was identified by
using chiral HPLC that (−)-EC could be obtained from de-gallate
acid reaction.
According to previous reports, several questions arise as to
(−)-EC biosynthesis in tea plants. For example, what is EC’s stereo-
chemistry in tea plants? Do the above mentioned three biosynthetic
pathways of (−)-EC really exist in tea plants? Here, we used chiral
HPLC to determine the stereochemistry of EC, and found (−)-EC,
not (+)-EC, in tea plants. Subsequently, we demonstrated that (−)-
EC was the only product of the partially purified native CsANR
from fresh tea leaves using cyanidin as substrates, and the atypi-
cal flavan-3-ol (+)-EC was the main product of recombinant CsANR
in the same conditions. In addition, (−)-EC could be obtained from
(+)-C via C3 -epimerization catalyzed by recombinant CsANRin vitro.
 Y. Qian et al. / J. Chromatogr. B 1006 (2015) 1–7
Fig. 2. Biosynthesis pathway of (−)-EC in tea plants. PAL: phenylalanine ammonia
lyase; C4H: cinnamate 4-hydroxylase; 4CL: 4-coumaroyl-CoA ligase; CHS: chal-
cone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3 H:
flavonoid 3 -hydroxylase; DFR: dihydroflavonol 4-reductase; LAR: leucoanthocyani-
din reductase; ANS: anthocyanidin synthase; ANR: anthocyanidin reductase; ECGT,
epicatechin glucosyltransferase; GCH, galloylated catechins hydrolase.
However, native C3 -epimerase was not detected in tea plants.
Finally, (−)-EC was obtained from ECG after catalysis by the par-
tially purified GCH from fresh tea leaves via de-gallate acid reaction.
2. Materials and methods
2.1. Plant materials
Young shoots (the bud, first and second leaves) of tea plants
[C. sinensis(L.) O. Kuntze] were selected from the Experimental Tea
Garden, Anhui Agricultural University, Anhui, China, during early
summer. All samples were immediately frozen in liquid nitrogen
and stored at −80 ◦ C until used.
2.2. Chemicals and reagents
The compounds (−)-C, (+)-C, (−)-EC, and ECG were purchased
from Sigma (St. Louis, MO, USA). NADPH tetrasodium salt and
NADP+ sodium salt were obtained from Roche Diagnostics (Indi-
anapolis, IN, USA). HPLC grade methanol, acetonitrile, acetic acid,
ethyl acetate, n-hexane, ethanol and 2-propanol were purchased
from Tedia Co., Ltd. (Fairfield, OH, USA). Cyanidin chloride was
procured from Axxora Co., Ltd. (Lausanne, Switzerland). Analyti-
cal reagents, including trichloromethane, formic acid, concentrated
3
hydrochloric acid, vanillin, and other solvents used for extraction
were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shang-
hai, China).
2.3. Extraction and purification of catechins
Total catechins were extracted and purified as previous method
[22], with a slight modification. Briefly, 1 g of sample was ground
to powder in liquid nitrogen and extracted with 10 mL methanol
for 10 min in an ultrasonic sonicator at 4 ◦ C. After centrifugation
at 5000 g for 10 min at 4 ◦ C, the supernatants were collected for
total catechins extraction. The residues were re-extracted twice as
described above. Total catechins were purified on thin-layer chro-
matography (TLC, GF254 TLC sheet, 5 × 20 cm; Biotechnology Co.,
Hefei, China) using chloroform:methanol:formic acid (28:10:1, v/v)
as eluent, and detected with 1% vanillin–HCl (w/v). EC and C chro-
matography spots were identified by Rf values and comparison
with standards; these chromatography spots were collected and
dissolved in methanol. After centrifugation at 5000 g for 10 min at
4 ◦ C, the supernatants were filtered through a 0.22 ␮m membrane
for further chromatography analysis.
2.4. Reverse phase chromatography analysis of (epi)-catechins
The EC and C in methanol were separated and identified
by Reverse phase chromatography (RP-HPLC) on a Phenomenex
Synergi 4 ␮ Fusion-RP80 column (5 ␮m, 4.6 × 250 mm). RP-HPLC
analysis was carried out as reported by Liu et al. [23]. The solvent
system consisted of 1% (v/v) acetic acid (A) and 100% acetonitrile
(B). A linear gradient at a flow rate of 1.0 mL/min was set after injec-
tion (5 ␮L) as follows: B from 13 to 30% (v/v) in 30 min, and B from
30 to13 % (v/v) for 30–35 min. Ultraviolet spectra were recorded on
a Waters 600UV array detector (Waters Corporation, Milford, MA,
USA) at 280 nm. The compounds were identified by UV spectra and
chromatographic characteristics with authentic standards.
2.5. Chiral phase chromatography analysis of (epi)-catechins
The stereoisomers of (+)-C and (−)-EC were separated on chi-
ral HPLC column (catalog no. 80325; Chiral Technologies; 5 ␮m,
4.6 × 250 mm) with amylose tris-(3,5-dimethylphenylcarbamate)
immobilized on silica-gel. The analytical chiral HPLC was per-
formed as described by Pang et al. [15] with some modifications.
Solvents A (0.5% acetic acid in hexane) and B (0.5% acetic acid in
ethanol) were used at a flow rate of 1 mL/min with the follow-
ing gradient: 0–10 min, 15% B; 10–20 min, 15–20% B; 20–23 min,
20–50% B; 23–38 min, 50% B; 38–45 min, 50–15% B. The other con-
ditions were the same as RP-HPLC.
To construct standard curves, the standards of (+)-C and (−)-
EC were dissolved and diluted in methanol to a series of different
concentrations (i.e., (+)-C with ranges from 0.50 to 0.01 mg/mL and
(−)-EC with ranges from 0.35 to 0.02 mg/mL). The content of the
stereoisomers of (+)-C and (−)-EC in fresh tea leaves were calcu-
lated by standard curves.
2.6. Enzyme extraction and assays
Native proteins from fresh tea leaves were extracted in accor-
dance with the method of Zhang et al. [24]. Ammonium sulfate
precipitation was used for preliminary purification of the native
proteins. The fractions of 0–40% and 40–70% ammonium sulfate
were collected as partially purified native CsANR and CsGCH pro-
teins respectively for enzymatic assay. The recombinant CsANR
expressed in Escherichia coli was prepared and purified according
to the previous study [25].
4 Y. Qian et al. / J. Chromatogr. B 1006 (2015) 1–7
Fig. 3. Analysis of stereochemistry of (epi)-catechin in fresh leaves. (A) Isolation and purification of the total flavan-3-ols from fresh leaves via TLC; (B) RP-HPLC analysis of
the first chromatographic spot on TLC; (C) further chiral HPLC analysis of the first chromatographic spot on TLC; (D) reverse phase chromatography of standards of C and EC;
(E) chiral phase chromatography of the standards of (−)-C, (+)-C and (−)-EC.
ANR assay reactions were incubated at 45 ◦ C for 0.5 h in a
total volume of 1 mL 50 mM phosphate buffer (pH 6.5), contain-
ing 0.5 mM cyanidin chloride, 2 mM NADPH, and 0.8 mg partially
purified native ANR protein or 0.2 mg recombinant CsANR protein.
Recombinant CsANR epimerization was carried out at 30 ◦ C for
0.5 h in a total volume of 1 mL 50 mM phosphate buffer (pH 7.5),
containing 2.5 mM NADP+ , 0.25 mM (+)-C or (−)-EC, and 0.2 mg
recombinant CsANR protein.
Non-enzymatic epimerization was performed at 80 ◦ C for 0.5 h
in a total volume of 1 mL aqueous solution containing 0.25 mM (+)-C
or (−)-EC.
De-gallate acid reaction was incubated at 30 ◦ C for 0.5 h in a total
volume of 1 mL 50 mM phosphate buffer (pH 7.0), containing 8 mM
ECG, 4 mM ascorbic acid, and 0.08 mg partially purified native GCH
protein.
All enzymatic reactions were initiated by adding the appropriate
enzyme solution, and terminated by addition of ethyl acetate. The
enzymatic products were identified via the above RP-HPLC or chiral
HPLC.
3. Results and discussion
3.1. Qualitative and quantitative analysis of (epi)-catechins
stereochemistry in fresh tea leaves
In recent decades, the importance of stereochemistry has
received a great deal of attention, and chiral HPLC as an effective
method has been applied for enantioseparation [26–30]. Here, chi-
ral HPLC was used to identify the stereochemistry of catechins in
fresh tea leaves.
First, total catechins were extracted from fresh tea leaves and
dissolved in methanol, and subsequently isolated and purified by
TLC (see Fig. 3A). According to standards, the first chromatographic
spot on TLC represented the mixture of EC and C, and was col-
lected for further identification by RP-HPLC and chiral HPLC (see
Fig. 3B and C). Based on the (−)-EC and (+)-C authentic standards
shown in Fig. 3E, (−)-EC and (+)-C existed mainly in fresh tea leaves,
with a predominance of (−)-EC according to quantitative analysis.
The content of (−)-EC was 5.49 ± 0.53 mg/g DW and that of (+)-C
was 0.52 ± 0.03 mg/g DW. However, the (+)-EC and (−)-C were not
detected.
3.2. Identification of (−)-EC from enzymatic reduction reaction
Multiple studies have shown that EC is the product of ANR using
cyanidin as substrate [12,17,24,25,31–33]. However, few stud-
ies have focused on stereochemistry of these enzymatic products
[14,15].
To investigate the stereochemistry of EC produced from cyani-
din in the tea plants, the products from the reaction catalyzed by
the native CsANR from fresh tea leaves were isolated and purified
by TLC. Subsequently, the purified products were collected and fur-
ther analyzed using chiral HPLC. As shown in Fig. 4A, (−)-EC was
the only product obtained from cyanidin using partially purified
native CsANR in the presence of NADPH.
Under the above reaction conditions, recombinant CsANR
mainly converted cyanidin into products of (+)-EC and (−)-C
(Fig. 4B), and a minor product (−)-EC. These findings con-
firmed that (−)-EC resulted from CsANR reduction reaction in tea
plants.However, the catalytic mechanisms of native CsANR in tea
plants and recombinant CsANR in vitro are different.
3.3. Identification of (−)-EC from epimerization
Epimerization may constitute another biosynthetic pathway of
(−)-EC, and ANR is probably an epimerase. Gargouri et al. reported
that recombinant VvANR acted not only as reductase, but also as
C3 -epimerase in vitro [14,34].
In order to address whether CsANR has a double function,
recombinant CsANR was incubated in phosphate buffer at pH 7.5
and 30 ◦ C for 0.5 h using (−)-EC or (+)-C as substrate respectively
in the presence of NADP+ . Interestingly, (−)-EC and (+)-C were
converted into their respective C3 -epimers, (+)-C and (−)-EC (see
Fig. 5A and B); in addition, C3 -epimerization was not detected with-
out CsANR or with boiled CsANR protein (date not shown). These
results indicated that recombinant CsANR also acts as C3 -epimerase
in the presence of NADP+ in vitro. In other words, (−)-EC could
derive from (+)-C as well. However, C3 -epimerase reaction cat-
alyzed by native CsANR from fresh leaves was not detected, and
 Y. Qian et al. / J. Chromatogr. B 1006 (2015) 1–7 5

Fig. 4. Chiral HPLC analysis of products resulted from cyanidin by partially purified native CsANR or recombinant CsANR in the presence of NADPH. (A) Products resulted
from the partially purified native CsANR catalysis after purification via TLC; (B) products of the recombinant CsANR.

Fig. 5. Chiral HPLC analysis of products using (epi)-catechin as substrates in recombinant CsANR or non-enzymatic reaction respectively. (A) Products of recombinant CsANR
using (+)-C as substrate in the presence of NADP+; (B) products of recombinant CsANR using (−)-EC as substrate in the presence of NADP+; (C) products from (+)-C incubated
in water at 80 ◦ C; (D) products from (−)-EC incubated in water at 80◦ C.

non-enzymatic epimerization did not happen at the C3 position
in vitro.
Previous studies assessing non-enzymatic epimerization at the
C2 position have shown that the reciprocal transformation between
EC and C often occurs during food processing mainly due to high
temperatures [13,16,18,19]. Here, epimerization at the C2 position
was not detected at 30 ◦ C or 45 ◦ C in different solutions (Tris–HCl
buffer, Phosphate buffer and purified water) for different heating
times (from 0.5 h to 5 h) with pH from 6.0 to 7.5 (date not shown).
However, epimerization at the C2 position was observed at 80 ◦ C
for 0.5 h using (−)-EC or (+)-C as substrate respectively in purified
water (see Fig. 5C and D), that was consistent with previous reports
[18,19,35].
From the above results, we inferred that (−)-EC could derive
from (−)-C by C2 - epimerization in extreme environmental condi-
tions such as high temperature for short time treatment, but did
not occur in physiological conditions (e.g., 30 ◦ C or 45 ◦ C).
3.4. Identification of (−)-EC from de-gallate acid reaction
The third possible biosynthetic pathway of (−)-EC is
by way of de-gallate acid reaction of (−)-ECG. Previous
research has indicated that galloylated catechins, including
(−)- EGCG and (−)-ECG, comprise up to 76% of total catechins in
tea plants [36]. Importantly, Liu et al. discovered a novel native
enzyme involved in the biosynthetic pathway of galloylated
catechins [23]. Our preliminary experiments also revealed another
novel native protein belonging to hydrolytic enzymes, which could
convert ECG to EC and gallic acid (GA) by de-gallate acid reaction
[21] (see Fig. 6A).
We also analyzed the stereochemistry of de-gallate acid reaction
products using chiral HPLC after purification via TLC, and found that
(−)-EC was indeed the product of enzyme catalytic de-gallate acid
reaction (see Fig. 6B). Therefore, (−)-EC could result from (−)-ECG
via de-gallate acid reaction.
4. Conclusions
Flavan-3-ols are widely distributed in tea plants, fruits, and
vegetables, and exist in monomeric and polymeric forms [37–43].
Previous studies have shown that monomeric (epi)-catechin was
the building block of polymeric flavan-3-ols, commonly known
as PAs (also named condensed tannins), which are involved in
important physiological functions in plants and beneficial to human
6 Y. Qian et al. / J. Chromatogr. B 1006 (2015) 1–7
Fig. 6. Analysis of products from ECG via de-gallate acid reaction catalyzed by the partially purified native GCH from fresh tea leaves. (A) RP-HPLC analysis of products
resulted from ECG; (B) further Chiral HPLC analysis of the chromatographic spot of EC after purification via TLC.
health [44–49]. In tea plants, our previous study indicated that EC
was the basic unit for polymeric PAs which are abundant in roots
[50]. Traditionally, EC is considered the product of ANR using cyani-
din as substrate [12,31]. So far, few studies have focused on EC
stereochemistry [12–15,51].
In this work, we found that (−)-EC was the predominant di-
hydroxy-non-galloylated-flavan-3-ol in tea leaves, while (+)-EC
was not detected. To better understand the biosynthesis of (−)-EC,
the chiral HPLC column was used to identify the stereochemistry
of EC catalyzed by native CsANR in tea plants and recombinant
CsANR in vitro. Indeed, (−)-EC was the only product resulted from
native CsANR reaction, while (+)-EC was the main product from
recombinant CsANR reaction. Although (−)-EC as a minor prod-
uct was detected in the recombinant CsANR reaction, based on the
results reported by Xie et al., it might derive from (−)-C via non-
enzymatic epimerization at the C2 position [13]. In addition, (−)-EC
could result from (+)-C by recombinant CsANR which possesses C3 -
epimerase activity in vitro. However, the partially purified native
CsANR from tea leaves did not display this function. Based on the
above description, we could infer that CsANR functions are quite
complex, and the mechanisms of CsANR in tea plants were different
from those in vitro. Finally, (−)-EC probably results from de-gallate
acid reaction of (−)-ECG by a novel partially purified hydrolytic
enzyme in the tea plants. To summarize, (−)-EC is probably a prod-
uct of the native protein from the tea plants, while (+)-EC is only
produced by recombinant CsANR in vitro.
Acknowledgements
This work was supported by the Natural Science Foundation of
China (31570694, 31470689, 31300577, 31200229, and 31170282),
the Specialized Research Fund for the Doctoral Program of Higher
Education (20133418130001), Anhui Major Demonstration Project
for Leading Talent Team on Tea Chemistry and Health and the
Biology Key Subject Construction of Anhui and Science Open Foun-
dation of Suzhou University (2010YKF26). We are grateful to Dr.
Xianzhi He for correcting the English grammar in the manuscript
and for his kind suggestions.
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