Changes of major tea polyphenols and production of

Food Chemistry 170 (2015) 110–117
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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Changes of major tea polyphenols and production of four new B-ring
fission metabolites of catechins from post-fermented Jing-Wei
Fu brick tea
Yun-Fei Zhu a, Jing-Jing Chen a, Xiao-Ming Ji b, Xin Hu b, Tie-Jun Ling a, Zheng-Zhu Zhang a, Guan-Hu Bao a,⇑,
Xiao-Chun Wan a,⇑
a
b
Key Laboratory of Tea Biochemistry and Biotechnology, Anhui Agricultural University, 130 West Changjiang Road, Hefei City, Anhui Province 230036, China
Shaanxi Zhong Fu Tea Research Institute, Xianyang City, Shaanxi Province 712044, China
a r t i c l e
i n f o
Article history:
Received 18 April 2014
Received in revised form 3 August 2014
Accepted 14 August 2014
Available online 23 August 2014
Chemical compounds studied in this article:
Gallic acid (GA, PubChem CID: 370)
(+)-Catechin (C, PubChem CID: 9064)
()-Epicatechin (EC, PubChem CID: 72276)
()-Epigallocatechin gallate (EGCG,
PubChem CID: 65064)
Epicatechin-3-gallate (ECG, PubChem CID:
65056)
Epigallocatechin (EGC, PubChem CID: 72277)
Xanthocerin (PubChem CID: 5315332)
Gallicin (PubChem CID: 7428)
()-Epiafzelechin (PubChem CID: 443639)
Phloroglucinol (PubChem CID: 359)
Pyrogallol (PubChem CID: 1057)
2,5-Dihydroxy benzoic acid (PubChem CID:
3469)
Quercetin (PubChem CID: 5280343)
Kaempferol (PubChem CID: 5280863)
Myricetin (PubChem CID: 5281672)
Canophyllol (PubChem CID: 623591)
a-Spinasterol (PubChem CID: 5281331)
Theobromine (PubChem CID: 2519)
Epicatechin-3-O-(40 -O-methyl) gallate
(PubChem CID: 21146794)
Astragalin (PubChem CID: 5282102)
Nicotiflorin (PubChem CID: 5318767)
Rutin (PubChem CID: 5280805)
a b s t r a c t
HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed that
the level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) dropped
increasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discovery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C–F (1–4) together with three
known BRFCs (5–7), six known catechins (8–13), five simple phenols (14–18), seven flavones and flavone
glycosides (19–25), two alkaloids (26, 27), three triterpenoids (28–30) and one steroid (31). The structures were elucidated by spectroscopic methods including 1D and 2D NMR, LC–HR-ESI-MS, IR, and CD
spectra. Five compounds (16–18, 28, 29) were reported for the first time in tea. Possible pathways for
the degradation of major tea catechins and the generation of BRFCs were also provided.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Fuzhuan brick tea (FBT)
B-ring fission metabolites of catechins (BRFCs)
Fuzhuanin C–F
Camellia sinensis
Eurotium spp.
Aspergillus spp.
⇑ Corresponding authors. Tel.: +86 551 5786401; fax: +86 551 5786765 (G.-H. Bao). Tel.: +86 551 5786002; fax: +86 551 5786765 (X.-C. Wan).
E-mail addresses: [email protected] (G.-H. Bao), [email protected] (X.-C. Wan).
http://dx.doi.org/10.1016/j.foodchem.2014.08.075
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
1. Introduction
The manufacturing process substantially determines the types
of tea and also relevant chemical constituents of tea, such as the
content and type of tea polyphenols (Harbowy, Balentine, Davies,
& Cai, 1997; Wang et al., 2014). Accordingly, tea could be categorised into four major types: unfermented (green tea belongs to this
type), semi-fermented (such as Oolong tea), fully fermented (black
tea), and post-fermented (dark tea, such as Fu brick tea, FBT) based
on the increasing degree of fermentation of tea (Ho, Lin, & Shahidi,
2008; Jiang et al., 2011; Kim, Goodner, Park, Choi, & Talcott, 2011).
Dark tea is of great interest due to the upsurge in popularity of
Puer tea. Brick dark tea is a kind of unique post-fermented tea with
brick form compressed from the older, coarse and rough leaves,
and small branches of Camellia sinensis var. sinensis and C. sinensis
var. assamica, mainly in Hunan, Sichuan, Shaanxi, and Yunnan
provinces in China. It has been reported that brick dark tea has special health benefits, such as antihyperlipidaemic (Fu et al., 2011),
anti-obesity (Li, Liu, Huang, Luo, et al., 2012), antibacterial (Amy,
Tiffany, Corey, & Elizabeth, 2013), antioxidant (Cheng et al.,
2013), inhibiting fat deposition (Peng et al., 2014) and so on. Its
typical fungal aroma has also been studied (Xu, Mo, Yan, & Zhu,
2007). According to the material and manufacturing process, brick
dark tea products can be categorised into several types: Heizhuan
brick tea, Huazhuan brick tea, Fu brick tea (FBT), Qingzhuan brick
tea, Kangzhuan brick tea, and Pu-erh brick tea (Wan, 2010).
The changes of major tea polyphenols, especially EGCG, were
reported in partially fermented Oolong tea, fully fermented black
tea, as well as post fermented dark tea. Tea catechins contained
in green tea are higher than other types of tea because polyphenol
oxidase and native microflora are inactivated in freshly plucked tea
leaves after being immediately steamed or pan-fired (Toschi et al.,
2000). However, during fermentation of black tea, polyphenol oxidase in the tea leaves catalyses the oxidation of the major catechins into theaflavin, hence reducing the catechins content
(Friedman, Levin, Choi, Lee, & Kozukue, 2009). The characteristic
reddish-black colour, reduced bitterness and astringency, and
removal of leafy and grassy flavour are derived from this oxidation
process, giving black tea a marked distinction from green tea
(Wang & Helliwell, 2000). As for puer tea, it has also been suggested that the levels of major catechins are reduced by oxidation
and polymerisation through thermal and enzymatic reactions (Xie
et al., 2009; Zuo, Chen, & Deng, 2002). For FBT, during the flowering
procedure, the level of tea catechins especially EGCG and ECG
greatly decreased, mainly due to enzymatic oxidation when the
large population of microorganisms appear and release extracellular enzymes. It was reported that the major reaction in the processing of FBT is oxidative polymerisation which lead to reduction of
coarse astringency and increase of alcoholic taste and thus
improves the quality and taste of FBT (Fu et al., 2008). Several B
ring fission metabolites of catechins (BRFCs) were also reported
(Jiang et al., 2011; Kanegae et al., 2013; Luo et al., 2013;
Wulandari et al., 2011), which are obviously distinct from the polymerised products and may also contribute to the unique flavour
and quality of FBT.
A number of studies have reported on FBT, including chemical
analysis (Amy et al., 2013) and components purification (Ling
et al., 2010; Luo et al., 2012, 2013). However, to date, we are not
aware of any HPLC analysis of the major tea polyphenolic changes
during the processing of FBT. Additionally, there is no systematic
chemical purification study on Jing-Wei FBT, a kind of FBT traditionally consumed in the northwestern area of China. In this paper,
we studied the chemical constituents of Jing-Wei Fu brick tea.
Through extensive liquid chromatography, 31 compounds were
isolated. Four new BRFCs, Fuzhuanin C–F, together with 27 known
111
compounds, were identified by IR, NMR, HR-ESI-MS, and CD spectroscopy. A dynamic HPLC analysis of the degree of fermentation
on the levels of major tea catechins during processing was conducted and a possible pathway for the generation of BRFCs was
also provided.
2. Materials and methods
2.1. Instrumentation
IR was measured on a Thermo Nicolet 8700 FT-IR spectrophotometer. 1H NMR and 13C NMR, HSQC and HMBC spectra were
recorded in dimethyl-d6 sulfoxide (DMSO-d6) with Bruker AM400 spectrometers operating at 400 MHz for 1H NMR and
100 MHz for 13C NMR, respectively. The Agilent 6210 HPLC/timeof-flight MS system with a binary high-pressure mixing pump, an
auto sampler, a column oven, a photodiode array detector (PAD),
a high resolution (HR) time-of-flight (TOF) MS, an ESI source (negative mode) and an Agilent workstation was from Agilent Technologies (Santa Clara, CA). HPLC was performed on a Waters 2695
separation module combined with a Waters 2489 UV detector. Circular dichroism (CD) spectra were detected with a JASCO J-815
spectropolarimeter (JASCO, Tokyo, Japan).
2.2. Chemicals, tea materials, and extraction for HPLC analysis
Gallic acid (GA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG) standards
were purchased from Chengdu Ultrapure Technology Co. Ltd.
(Chengdu, China) and identified in our laboratory for analysis. All
of these standards were of a purity higher than 98%.
Jing-Wei FBT for phytochemical research (produced in 2011)
was provided by Cangshan tea Company (Xianyang, China). The
processing samples of FBT were also supplied by the same company. Briefly, the manufacture of FBT is summarised as steaming,
rolling, microbial fermentation and drying (Wan, 2010). The
detailed processing procedure has been described in several articles (Jin, Chen, & Ji, 2003; Mo, Zhu, & Chen, 2008; Xu et al.,
2011). The fungal growth stage (known as flowering stage) is the
key procedure for FBT, during which the chemical constituents
change a lot. HPLC analysis of the major tea catechins was therefore carried out at the pre-growing stage of the fungi (pre-flowering, PF), the sixth day of fungal growth (six days of flowering, SDF),
the ninth day of fungal growth (nine days of flowering, NDF), and
the final FBT products (FBTP).
Extracts of different degree of fermentation during processing
of Jing-Wei FBT were prepared by ultrasonic extracting 2.5 g of
ground tea powder in 100 mL of 70% aqueous methanol twice in
12 h (15 min each time). A 2-mL aliquot of the liquid extracts
was centrifuged at 10,000 rpm for 10 min and then the supernatant was passed through a 0.22-lm filter. This filtrate was used
for HPLC analysis.
2.3. HPLC analysis of major tea polyphenols of Jing-Wei Fu brick tea
HPLC analysis was carried out using a XP ODS-A C18 column
(250 mm 4.6 mm i.d., 5 lm, H&E Co. Ltd., PR China). Column
temperature was set at 30 °C. The eluant was composed of mobile
phase A (water containing 0.17% acetic acid) and mobile phase B
(acetonitrile). The gradient of mobile phase B was as follows:
0–4 min, 6%; 4–16 min, from 6% to 14%; 16–22 min. from 14% to
15%; 22–32 min, from 15% to 18%; 32–37 min, from 18% to 29%;
37–45 min, from 29% to 45%; 45–50 min, 45%; 50–51 min, from
45% to 6%; then kept at 6% for 10 min. Solvent flow rate was
112
Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
1.0 mL/min and injection volume was 5 lL. The UV detection
wavelength was 280 nm.
The linear calibration curves contained five different concentrations of each reference compound diluted with methanol. Each
concentration was measured three times. All calibration curves
were constructed by plotting the peak areas of the standard substances versus the corresponding concentration of the injected
standard solutions for quantitative analysis: EGCG (r2 = 0.9994,
37.5–1000 lg/mL, RT = 29.15 min), EGC (r2 = 0.9995, 37.5–
1000 lg/mL, RT = 20.80 min), ECG (r2 = 0.9997, 12.5–625 lg/mL,
RT = 40.56 min), EC (r2 = 0.9998, 12.5–630 lg/mL, RT = 27.92 min);
GA (r2 = 0.9996, 11.25–300 lg/mL, RT = 7.06 min).
eluant, yielding four fractions (D1–D4). Fraction D1 (26 g) was subjected to a silica gel CC eluted with dichloromethane:methanol
(30:1), yielding four subfractions (D1-1 to D1-4). Compound 26
(1.0 g, caffeine) was obtained from subfraction D1-1 by recrystallisation. Subfraction D1-2 (2.0 g) was subjected to Sephadex LH-20,
polyamide CC, and HPLC to yield 2 (3.5 mg, Fuzhuanin D), 3
(1.2 mg, Fuzhuanin E), 4 (1.0 mg, Fuzhuanin F). Subfraction D1-3
(4.2 g) was subjected to Sephadex LH-20, polyamide, and silica
gel CC to yield 1 (4.2 mg, Fuzhuanin C), 5 (22 mg, planchol A), 6
(3.0 mg, xanthocerin), 18 (32 mg, gallicin). Subfraction D1-4
(3.3 g) was subjected to Sephadex LH-20 and MCI gel CC to yield
13 (23 mg, epiafzelechin), 16 (56 mg, phloroglucinol), 17 (62 mg,
pyrogallol). Fraction D2 (16 g) was subjected to Sephadex LH-20
and polyamide CC to yield 7 (38 mg, teadenol A), 25 (12 mg, taxifolin), 27 (250 mg, theobromine). Fraction D3 (23 g) was subjected
to Sephadex LH-20, polyamide and MCI gel CC to yield 10 (119 mg,
epicatechin gallate), 11 (31 mg, epigallocatechin gallate), 12
(12 mg, epicatechin-3-O-(40 -O-methyl)gallate). Also fraction D4
(33 g) was subjected to Sephadex LH-20, polyamide and MCI gel
CC to yield 22 (22 mg, astragalin), 23 (21 mg, nicotiflorin) and 24
(52 mg, rutin).
HPLC separation of compounds 3 and 4 was carried out using an
XP ODS-A C18 column (250 mm 4.6 mm i.d., 5 lm). Column
temperature was set at 30 °C. The eluant was composed of mobile
phase A (methanol) and mobile phase B (water). The gradient of
mobile phase A was as follows: 0–15 min, 50%; 15–17 min, from
50% to 100%; 17–20 min, 100%; 20–22 min, from 100% to 50%; then
kept at 50% for 10 min. Eluant was performed at a solvent flow rate
of 1.0 mL/min. The injection volume was 20 lL. The UV detection
wavelength was monitored at 280 nm and 210 nm.
The detailed purification procedure can be found in Supplementary Fig. 1 and the structures of all these compounds are given in
Supplementary Fig. 2.
Fuzhuanin C (1): white powder (CH3OH). IR (KBr) mmaz 3181,
1731, 1605, 1518, 1467, 1380, 1139, 996, 822 cm1. CD De (nm):
+10.8 (240), 3.6 (280) (c 0.10, CH3OH). 1H and 13C NMR data
(DMSO-d6) d see Table 1. HR-ESI-MS: m/z 323.12384 [MH],
(Calc. 323.11363 for C16H19O7). IR, 1D and 2D NMR, HRMS, UV
spectra are arranged in the Supplementary material.
Fuzhuanin D (2): white powder (CH3OH). IR (KBr) mmaz 3157,
1785, 1732, 1607, 1521, 1469, 1373, 1150, 995, 823 cm1. CD De
(nm): +12.7 (240), 3.4 (280) (c 0.10, CH3OH). 1H and 13C NMR
2.4. Isolation of phytochemicals from Jing-Wei Fu brick tea
Jing-Wei FBT (15 kg) was ground and extracted with petroleum
ether, ethyl acetate, and methanol three times (3 20 L) at room
temperature. The extracts were concentrated under reduced pressure to afford three residues: fraction A (the petroleum ether fraction, 165 g), fraction B (the ethyl acetate fraction, 200 g), and
fraction C (the methanol fraction, 2 kg). Fraction C was extracted
with dichloromethane and n-butyl alcohol, respectively. The nbutyl alcohol fraction (fraction D) was concentrated under reduced
pressure to afford residue (450 g).
Fraction A was separated by silica gel column chromatography
(CC), eluting with a mixture of petroleum ether:ethyl acetate with
increasing polarity (1:0 to 0:1), yielding thirteen fractions (A1 to
A13). Fractions A10 (6 g) and A12 (5 g) were subjected to ODS CC
eluted with methanol to yield 28 (122 mg, 2-hydroxydiplopterol),
29 (32 mg, canophyllol), 30 (12 mg, 3b, 6a, 13b-trihydroxyolean7-one), 31 (1.2 g, a-spinasterol). Fraction B was applied to a
polyamide CC, eluting with a mixture of dichloromethane:ethyl
acetate:methanol with increasing polarity (2:1:0–0:1:2), yielding
eight fractions (B1–B8). Fraction B5 (11 g) was subjected to a silica
gel CC using dichloromethane:methanol:formic acid (20:1:0.5) as
the eluant, yielding 15 (260 mg, 2,5-dihydroxybenzoic acid), 19
(136 mg, quercetin), 20 (154 mg, kaempferol). Fraction B7 (4.2 g)
was subjected to a Sephadex LH-20 CC, yielding 21 (112 mg,
myricetin). Fraction B8 (1.1 g) was subjected to a Sephadex LH20 CC, yielding 8 (110 mg, epicatechin), 9 (396 mg, epigallocatechin), 14 (80 mg, gallic acid). Fraction D was applied to a silica
gel CC using dichloromethane:methanol:water (6:1:0.1) as the
Table 1
H (d ppm, J Hz, s: single peak; d: double peaks; br s: broad single peak; m: multipeaks) and
1
Pos.
2
3
4b
4a
5
6
7
8
9
10
10
20 b
20 a
30
40
50
60
70
5-OH
7-OH
1
H
82.4d
73.2d
21.2t
4.37(1H,
4.33(1H,
2.53(1H,
2.64(1H,
172.1s
105.4s
19.8q
48.0q
51.3q
C NMR (d ppm, d: CH; t: CH2; q: CH3) data for compounds 1, 2 and 5.
2
C + DEPT
155.7s
96.6d
156.3s
96.2d
154.8s
100.9s
51.3d
32.1t
13
br d, J = 10.6 Hz)
m)
dd, J = 15.2, 6.0 Hz)
dd, J = 15.2, 3.6 Hz)
5.94(1H, d, J = 2.0 Hz)
5.71(1H, d, J = 2.0 Hz)
2.30(1H, dd, J = 11.6, 5.2 Hz)
2.47(1H, m)
2.53(1H, m)
1.20(3H,
3.10(3H,
3.62(3H,
9.19(1H,
8.99(1H,
s, CH3)
s, OCH3)
s, OCH3)
s)
s)
5 (planchol A)
C + DEPT
H
C + DEPT
H
77.3d
71.6d
20.1t
4.47 (1H, br s)
4.43 (1H, br s)
2.67 (2H, m)
79.1d
73.0d
19.9t
4.27
4.53
2.72
2.65
156.1s
95.9d
156.4s
94.3d
153.1s
97.0s
79.2d
36.1t
175.4s
93.1s
35.5t
169.9s
51.6q
5.92 (1H, d, J = 1.6 Hz)
5.71 (1H, d, J = 1.6 Hz)
4.68 (1H, d, J = 7.0 Hz)
2.56 (1H, d, J = 18.0 Hz)
3.13 (1H, dd, J = 18.0, 7.0 Hz)
3.16 (2H, s)
3.65 (3H, s, OCH3)
9.28 (1H, s)
9.00 (1H, s)
156.2s
95.6d
156.4s
94.0d
153.9s
96.7s
50.5d
31.2t
174.7s
115.6s
24.7q
(1H,
(1H,
(1H,
(1H,
d, J = 2.0 Hz)
dd, J = 5.0, 2.0 Hz)
m)
m)
5.91 (1H, d, J = 2.0 Hz)
5.70 (1H, d, J = 2.0 Hz)
3.02 (1H, s)
3.00 (1H, m)
2.68 (1H, m)
1.51 (3H, s)
9.29 (1H, s)
8.98 (1H, s)
113
Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
Table 2
H (d ppm, J Hz, s: single peak; d: double peaks; brs: broad single peak; m: multipeaks) and
1
Pos.
2
3
4b
4a
5
6
7
8
9
10
10
20
30
40
5-OH
7-OH
3
H
71.9d
73.6d
26.0t
4.58
4.52
2.58
2.95
158.4s
17.0q
C NMR (d ppm, d: CH; t: CH2; q: CH3) data for compounds 3, 4 and 6.
4
C + DEPT
157.1s
96.4d
157.4s
94.2d
154.2s
98.0s
162.4s
117.2d
13
C + DEPT
(1H,
(1H,
(1H,
(1H,
br d, J = 10.8 Hz)
ddd, J = 10.8, 10.4, 6.0 Hz)
dd, J = 15.2, 10.4 Hz)
dd, J = 15.2, 6.0 Hz)
5.99 (1H, d, J = 2.4 Hz)
5.79 (1H, d, J = 2.4 Hz)
5.90 (1H, q, J = 1.6 Hz)
2.07 (3H, brs)
9.52 (1H, s)
9.17 (1H, s)
138.4s
71.0d
25.9t
156.5s
96.6d
157.9s
93.5d
152.6s
96.4s
167.2s
33.8t
103.2s
13.2q
data (DMSO-d6) d see Table 1. HR-ESI-MS: m/z 335.07678 [MH]
(Calc. 335.07724 for C16H15O8). IR, 1D and 2D NMR, HRMS, UV
spectra are arranged in the Supplementary material.
Fuzhuanin E (3): white powder (CH3OH). IR (KBr) mmaz 3334,
1685, 1622, 1524, 1478, 1394, 1183, 1066, 809 cm1. CD De
(nm): +12.5 (210), 2.3 (250) (c 0.10, CH3OH). 1H and 13C NMR
data (DMSO-d6) d see Table 2. HR-ESI-MS: m/z 247.06156 [MH]
(Calc. 247.0612 for C13H11O5). IR, 1D and 2D NMR, HRMS, UV spectra are arranged in the Supplementary material.
Fuzhuanin F (4): white powder (CH3OH). IR (KBr) mmaz 3209,
1705, 1605, 1518, 1444, 1392, 1164, 1046, 824 cm1. CD De
(nm): +7.1 (192.5), 9.3 (250) (c 0.10, CH3OH). 1H and 13C NMR
data (DMSO-d6) d see Table 2. HR-ESI-MS: m/z 247.06007 [MH]
(Calc. 247.0612 for C13H11O5). IR, 1D and 2D NMR, HRMS, UV spectra are arranged in the Supplementary material.
3. Results and discussion
3.1. HPLC analysis of changes in major tea polyphenols during the
post-fermentation
The results are given in Fig. 1 and the HPLC chromatograms of
the four extracts during fermentation are given in Supplementary
Fig. 3. During the period of the fungal post-fermentation, the levels
of galloyl catechins dropped, especially those of EGCG and ECG,
which dropped from the beginning of the fermentation to the final
6 (xanthocerin)
H
5.18 (1H, br s)
2.37 (1H, dd, J = 16.0, 10.0 Hz)
3.20 (1H, dd, J = 16.0, 6.5 Hz)
6.00 (1H, d, J = 2.4 Hz)
5.85 (1H, d, J = 2.4 Hz)
3.05 (1H, m)
3.24 (1H, m)
1.69 (3H, s)
9.58 (1H, s)
9.27 (1H, s)
C + DEPT
H
68.3d
70.8d
23.4t
4.43 (1H, br s)
4.87 (1H, m)
2.78 (2H, dd, J = 6 Hz)
156.5s
95.9d
156.6s
94.0d
154.8s
96.3s
163.7s
118.6d
154.9s
20.4q
5.95 (1H, d, J = 2.4 Hz)
5.70 (1H, d, J = 2.4 Hz)
5.99 (1H, q, J = 1.6 Hz)
2.10 (3H, d, J = 1.6 Hz)
9.41 (1H, s)
9.07 (1H, s)
product. Both levels of EGCG and ECG of the FBT products dropped
to about one-third level of PF while the level of EGC increased. The
level of GA increased till the sixth day of fermentation because of
production of GA from degradation of galloyl catechins and/or gallotannins by microbial tannase (Bhat, Singh, & Sharma, 1998).
However, the level of GA and EC in the final product decreased,
suggesting that GA and EC continued to degrade from the ninth
day of flowering to form more simple phenols (Tanaka et al.,
2011). Interestingly, simple phenols such as 2,5-dihydroxybenzoic
acid (15), phloroglucinol (16), and pyrogallol (17) were purified
from the FBT product. In addition, EC and EGC also encountered
changes, especially on the B-ring, to form B-ring fission metabolites of catechin derivatives (BRFCs, 1-7). EGCG is the most abundant and active catechin and it is often used as a quality
indicator (Lakenbrink, Lapczynski, Maiwald, & Engelhardt, 2000;
Wang & Helliwell, 2000; Wang, Zhou, & Jiang, 2008). As there are
obvious changes in the levels of EGCG, ECG, and GA during processing, these three chemicals could be used as indices for quality control and real-time study of the processing of Jing-Wei Fu Brick tea
after further analysis of more samples.
To better understand the above changes of major tea polyphenols in final Jing-Wei FBT and what the catechins were transformed into, a system purification of the chemical constituents
was conducted. Thirty-one compounds were isolated and identified including simple phenols and seven BRFCs. Detailed structural
elucidation of the new BRFCs (Fuzhuanin C–F, 1–4) is given in the
following section.
3.2. Fuzhuanin C and D
Fig. 1. HPLC analysis of major tea catechins of Jing-Wei FBT during postfermentation; samples were PF (preflowering), SDF (sixth day of flowering), NDF
(ninth day of flowering), and FBTP (final Fuzhuan brick tea product); the data were
presented as means ± SD in triplicate.
Compound 1 was isolated as a colourless powder. Its molecular
weight was decided as C16H20O7 by HR-ESI-MS, with 7 degrees of
unsaturation. The IR spectrum (cm1) indicated the presence of
hydroxyl group (broad peak around 3181), ester carbonyl group
(1731), and aromatic ring (1605, 1518). The 1H-NMR spectrum is
similar to that of epicatechin (8) and exhibits the signals attributable to the A-ring (d 5.71 and 5.94, d, J = 2.0, H-8 and H-6; d 8.99
and 9.19, each s, OH-7 and OH-5) and the C-ring (d 4.37 and
4.33, H-2 and H-3; 2.53 and 2.64, H2-4). However, it has no aromatic B ring signals compared with epicatechin, which indicates
that it may be a B-ring fission metabolite of catechin (Jiang et al.,
2011; Luo et al., 2013). The TLC showed very weak colouration
with the FeCl3 reagent, which also confirmed it as a BRFC (Jiang
et al., 2011). Compared with known BRFC planchol A (5), compound 1 has two methoxyl groups (dH 3.62 and 3.10; dC 51.3q,
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Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
48.0q). The 13C-NMR and DEPT spectra of compound 1 also indicated the presence of A- and C-ring moieties of flavan-3-ol: one
phloroglucinol type aromatic A-ring (dC 155.7s, 96.6d, 156.3s,
96.2d, 154.8s, 100.9s), two oxygenated methine (dC 82.4d, 73.2d),
and one methylene (dC 21.2t). The remaining signals observed in
1
H-NMR, 13C-NMR, and DEPT spectra were assigned to one
methine (dC 51.3d, dH 2.30), one methylene (dC 32.1t, dH 2.47 and
2.53), one ester carbonyl (dC 172.1s), one oxygenated quaternary
carbon in very low field (dC 105.4s), and two methoxyl groups (dC
51.3q, 48.0q and dH 3.62s, 3.10s).
The HMBC correlations [dC 172.1 (C-30 )/dH 3.62 (OCH3); C-30 /dH
2.47 and 2.53 (H2-20 ); H-20 /dC 105.4 (C-40 ), 82.4 (C-2) and 51.3
(C-10 ); dH 1.20 (H3-50 )/C-10 and C-40 ; C-40 /dH 3.10 (OCH3)] can easily
decide the fragment CH3OCO-CH2-CH-C(-O-) (OCH3)-CH3 which is
attached to positions C-2 and C-3, to form a saturated furan ring
(THF). Thus, the proposed structure is shown in Fig. 2. The NOESY
correlation dH 3.10 for (OCH3)/H-3 suggest that they have the same
orientation while the H-50 /H-10 correlation suggests these two protons also have the same orientation (Fig. 3). Thus, the stereochemistry of the compound can be decided. The similar CD spectrum to
that of planchol A (5) further confirmed the structure of compound
1 as shown in Fig. 2. Compound 1 was named as Fuzhuanin C.
Compound 2 was isolated as a colourless powder. Its molecular
weight was decided as C16H16O8 by HR-ESI-MS, with 9 degrees of
unsaturation. The IR spectrum (cm1) suggested the presence of
hydroxyl group (broad peak around 3157), ester carbonyl group
(1732), five-membered lactone (1785), and aromatic ring (1607,
1521). Just like Fuzhuanin C (1), the 1H NMR spectrum showed that
compound 2 has no aromatic B-ring signals either. An ABX system
at d 4.68 (d, J = 7.0 Hz, H-10 ), 3.13 (dd, J = 18.0, 7.0 Hz, H-20 a), 2.56
(d, J = 18.0 Hz, H-20 b) indicated the presence of an O-CHCH2-fragment. These units (fragments) can also be confirmed by the
1
H-1H COSY spectrum. In addition, it has a unique singlet methylene unit at d 3.16 (s, H-50 ) and a methoxyl group (3.65, s, OCH3).
Fig. 2. The structures of compounds 1–8.
Fig. 3. The key HMBC (solid single arrowhead line), NOESY (dashed double arrowhead line) correlations of 1–4 (up), and CD spectra of 1–6 and 8 (down).
Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
The 13C NMR and DEPT spectrum indicated the presence of two
carbonyl groups (d 175.4 (s, C-30 ) and 169.9 (s, C-60 ), one quaternary carbon at d 93.1 (s, C-40 ). The HMBC correlations at dH 3.65
(OCH3)/C-60 , C-60 /H-50 , H-50 /C-40 and 79.2(C-10 ), dH 4.68 (H-10 )/C30 and 36.1 (C-20 ) indicated the presence of a CH3OCO-CH2-C-CHCH2-CO-unit. The HMBC signals at H-10 /71.6 (C-3) and H-50 /dC
77.3 (C-2) deduced that the above CH3OCO-CH2-C-CH-CH2-CO-unit
was connected with C-2 and C-3. Therefore, the structure can be
deduced as shown in Fig. 2. The stereochemistry can be deduced
by coupling constants (J-values) and NOESY spectrum. The small
coupling constants between H-2 and H-3 (both broad singlet) indicated a cis-orientation for these two protons. The NOESY correlations H-10 /H-50 indicated that these two protons were at the aorientation (Fig. 3). The similar CD spectrum to that of planchol
A (5) further confirmed the structure of compound 2, as shown
in Fig. 2. Compound 2 was named as Fuzhuanin D.
Biosynthetically, Fuzhuanin C (1), Fuzhuanin D (2), and planchol
A (5) may derive from epicatechin (8) through enzyme-catalysed
oxidative cleavage of the B-ring and then a sequential or simultaneous cycloaddition procedure (Luo et al., 2013). These proposed
biosynthetic pathways and the similar CD spectra of the four compounds further confirmed the stereochemistry of the two new
compounds (Fig. 3).
115
3.3. Fuzhuanin E and F
Compound 3 was isolated as a colourless powder. Its molecular
weight was decided as C13H12O5 by HR-ESI-MS, with 8 degrees of
unsaturation. The IR spectrum (cm1) suggested the presence of
a hydroxyl group (broad peak around 3334), a conjugated carbonyl
group (1685), and aromatic ring (1622, 1524). The 1H-NMR spectrum suggested it could be a derivative of (+)-catechin, by analysis
of the characteristic signals at d 5.79, 5.99 (both d, J = 2.4 Hz, H-8
and H-6) at the A-ring and d 4.58 (brd, J = 10.8 Hz, H-2), 4.52
(ddd, J = 10.8, 6.0, 10.4 Hz, H-3), 2.58 (dd, J = 15.2, 10.4 Hz, H-4b),
2.95 (dd, J = 10.8, 6.0 Hz, H-4a). Again the signals of the B-ring
were not observed. In addition, TLC showed very weak colouration
with the FeCl3 reagent. Thus, compound 3 is another BRFC. Its NMR
data are very similar to those of xanthocerin (6) (Table 2) except
for signals at positions 2–4. The bigger coupling constant of H-2
and H-3 (J = 10.8) suggested a trans-orientation for the two protons, which is different from those of xanthocerin, which has a
cis-orientation for the corresponding two protons. In addition,
the NOESY correlation H-2/H-4b and the methyl group (d 2.07, s)
indicated that they had the same orientation while the NOESY correlation H-3/H-4a indicated that these two protons also had the
same orientation (Fig. 3). Its CD spectrum is very similar to that
Fig. 4. Possible pathways for degradation of tea catechins in brick dark tea and generation of BRFCs.
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Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
of xanthocerin, which suggested that they have the same stereochemistry at position C-2 (2R) (Korver & Wilkins, 1971). Based
on the coupling constant of H-2/H-3, the NOESY correlations and
CD spectrum, the stereochemistry of compound 3 was determined
as 2R, 3S. Based on the above evidence, the structure was established as shown in Fig. 2. Compound 3 was named as Fuzhuanin E.
Compound 4 was isolated as a colourless powder. Its molecular
weight was decided as C13H12O5 by HR-ESI-MS, with 8 degrees of
unsaturation. The IR spectrum (cm1) suggested the presence of
a hydroxyl group (broad peak around 3209), carbonyl group
(1705), and aromatic ring (1605, 1518). Compound 4 is very similar
to compound 3. Interestingly, compounds 3 and 4 were initially
purified as one compound. All the 2D-NMR was thus conducted
on the mixture of the two compounds in DMSO-d6 (Supplementary
material). The two compounds were finally separated by HPLC. The
IR, CD, HPLC-PDA–HR-ESI-MS, and 1H NMR spectra of compounds
3 and 4 were then measured again (Supplementary material).
Compounds 3 and 4 have the same molecular weight, confirming
they are a pair of isomers. NMR data of 4 indicated the presence
of a methyl group (dH 1.69 s, dC 13.2q), a lactone (dC 167.2s), one
totally substituted double bond (dC 103.2s, 138.4s), and one oxygenated methine (dH 5.18 brs, dC 71.0d). In addition, it has one
more methylene signal (dC 33.8t, C-20 ; dH 3.05 and 3.24, H2-20 ).
The above data are different from those of compound 3, suggested
that a double bond had moved from one side to the other side at
the position of C-30 . Thus, the structure of compound 4 was decided
as shown in Fig. 2 and it was named as Fuzhuanin F.
Fuzhuanin E (3) and Fuzhuanin F (4) are two isomers of xanthocerin (6). Their CD spectra are similar except that the positive
Cotton effect is at shorter and shorter wavelength (3 is at
210 nm, 4 at 192.5 nm) compared to that of xanthocerin
(220 nm) (Fig. 3). Biosynthetically, the small amount of compounds
3 and 4 may derive from (+) catechin, which is also present in small
amounts in tea infusion. It was reported that (+)-catechin can be
transformed into BRFCs by many microorganisms (Das, Lamm, &
Rosazza, 2011); in this paper two new B-ring fission lactone products of (+)-catechin were produced. It was proposed that the diacidic compound from the cleavage of the B-ring could be the
intermediate of the new BRFCs (Das et al., 2011; Luo et al., 2013).
The linear 6/6/6 tricyclic BRFCs 3 and 4 could be formed through
recyclisation from a diacidic intermediate (Luo et al., 2013; Fig. 4).
A Japanese fermented tea, which was selectively fermented
with Aspergillus spp., was reported to produce teadenol A (7) and
teadenol B (Wulandari et al., 2011), both of which are linear 6/6/
6 tricyclic BRFCs which share the same skeleton with compounds
3 and 4. In addition, Aspergillus spp. was also reported to exist in
the final products of FBT (Luo et al., 2013) and teadenol A (7)
was also found in this study, which suggested that compounds 3
and 4 may be transformed by Aspergillus spp.
Since very few BRFCs have been found, very little bioassay work
has been reported on them, such as their antiproliferative (Luo
et al., 2013) and cytotoxic effects (Chang & Case, 2005; Luo et al.,
2013). More thorough bioactivity research on BRFCs is needed for
comprehensive evaluation of the health benefits of brick dark tea.
3.4. Possible pathway for degradation of tea catechins and generation
of BRFCs in brick dark tea
Tanaka, Matsuo, & Kouno, 2010); such reactions occur mainly on
the A-ring of catechins. Interestingly, since the first BRFC was
found from FBT, seven BRFCs had been found only in brick dark
tea up to date (Jiang et al., 2011; Kanegae et al., 2013; Luo et al.,
2013; Wulandari et al., 2011), in which all reactions happened
on the B-ring. It was suggested that BRFCs originated from tea catechins through oxidation and subsequent recyclisation of the
B-ring by the micro-organisms in FBT (Jiang et al., 2011; Luo
et al., 2013). The biosynthetic pathway of BRFCs was firstly studied
by Das et al. (2011), based on 18O2 labelling. Possible pathways for
lactone formation of the two new BRFCs transformed from (+)-catechin involved initial dioxygenase-mediated meta-B-ring cleavage
followed either by aldehyde oxidation to a dicarboxylic acid that
lactonises, or by hemiacetal (lactal) formation followed by alcohol
oxidation. Possible pathways for the production of Fuzhuanin A
and B, planchol A (5), and xanthocerin (6) were proposed in a
recent paper (Luo et al., 2013). Both of the pathways suggested
the formation of BRFCs through a diacidic or dicarboxylic acid
intermediate. As shown in Fig. 4, galloyl catechins (EGCG, ECG,
GCG, CG) endured a gradual decomposition during microbial fermentation. At the beginning, they were degalloylated through
hydrolysis by the effect of heat treatment during the first stage
of processing of FBT, and related degalloyl catechins (EGC, EC, C,
GC) together with GA were produced. This step was confirmed
by the HPLC analysis of changes in the levels of major tea polyphenols during post-fermentation procedure (Fig. 2).
In conclusion, besides being degraded to form simple phenols,
the degalloyl catechins were also degraded to three major classes
of BRFCs (the B-ring lactone type, the linear 6/6/6 tricyclic type,
and the angular 6/6/5/5 tetracyclic type) through three different
recyclisation pathways of the oxidised dicarboxylic acid intermediate (Fig. 4).
4. Conclusion
In the present study, HPLC-based analysis of major tea polyphenolic profiles can easily provide fermentation behaviour of tea catechins and GA during processing of Jing-Wei FBT, and thus provide
a better understanding of unique changes in tea metabolites during
tea fermentation. A systematic study on the chemical components
of FBT was performed and four new BRFCs together with 27 other
compounds were isolated and identified from Jing-Wei FBT for the
first time. The possible mechanism of the changes of major tea
polyphenols and the possible pathway of the formation of BRFCs
were also provided. The galloyl catechins firstly decomposed into
non-galloyl catechins and GA. On the one hand, the subsequent
non-galloyl catechins and GA continued the decomposition
tendency to firm simple phenols. On the other hand, they were
transformed into three types of BRFCs through initial dioxygenase-mediated meta-B-ring cleavage followed by oxidation to a
dicarboxylic intermediate that was subsequently recyclised
through three different pathways.
Funding
The authors declare no competing financial interest.
Acknowledgements
The post-fermented tea is characterised by the microbial fermentation procedure. During the post-fermentation procedure,
complex biochemical reactions such as microbial enzyme and
autoxidation lead to the polymerisation or decomposition of tea
catechins, which produce catechin polymers or simple phenolic
compounds, respectively. The polymerisation of catechins occurs
through hydrogen-peroxide-dependent peroxidase oxidation, polyphenol oxidase oxidation and autoxidation (Das et al., 2011;
We thank Ming-Jie Chu, Department of Chemistry, Anhui
Agricultural University, for IR recording. Financial assistance was
received with appreciation from Anhui Agricultural University
Talents Foundation (YJ2011-06), the Earmarked Fund for Modern
Agro-industry Technology Research System in Tea Industry of
Chinese Ministry of Agriculture (nycytx-26), and Program for Changjiang Scholars and Innovative Research Team in University IRT1101.
Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
08.075.
References
Amy, C. K., Tiffany, L. W., Corey, D. B., & Elizabeth, P. R. (2013). Antibacterial activity
and phytochemical profile of fermented Camellia sinensis (fuzhuan tea). Food
Research International, 53(2), 945–949.
Bhat, T. K., Singh, B., & Sharma, O. P. (1998). Microbial degradation of tannins—A
current perspective. Biodegradation, 9, 343–357.
Chang, J., & Case, R. (2005). Cytotoxic phenolic constituents from the roots of
Actinidia chinensis. Planta Medica, 71, 955–959.
Cheng, Q., Cai, S. B., Ni, D. J., Wang, R. J., Zhou, F., Ji, B. P., et al. (2013). In vitro
antioxidant and pancreatic a-amylase inhibitory activity of isolated fractions
from water extract of Qingzhuan tea. Journal of Food Science and Technology.
http://dx.doi.org/10.1007/s13197-013-1059-y.
Das, S., Lamm, A. S., & Rosazza, J. P. N. (2011). Biotransformation of (+)-catechin to
novel B-ring fission lactones. Organic Process Research & Development, 15(1),
231–235.
Friedman, M., Levin, C. E., Choi, S. H., Lee, S. U., & Kozukue, N. (2009). Changes in the
composition of raw tea leaves from the Korean Yabukida plant during high
temperature processing to pan-fried Kamairi-Cha green tea. Journal of Food
Science, 74, C406–C412.
Fu, D., Liu, Z. H., Huang, J. A., Hao, F., Wang, F., & Lei, Y. G. (2008). Variations of
components of Fuzhuan tea during processing. Food Science, 29, 64–67 ((in
Chinese)).
Fu, D., Ryan, E. P., Huang, J., Liu, Z., Weir, T. L., Snook, R. L., et al. (2011). Fermented
Camellia sinensis, Fu Zhuan tea, regulates hyperlipidemia and transcription
factors involved in lipid catabolism. Food Research International, 44(9),
2999–3005.
Harbowy, M. E., Balentine, D. A., Davies, A. P., & Cai, Ya. (1997). Tea chemistry.
Critical Reviews in Plant Sciences, 16(5), 415–480.
Ho, C.-T., Lin, J.-K., & Shahidi, F. (2008). Tea and tea products. Chemistry and health
promoting properties. Boca Raton: CRC Press.
Jiang, H. Y., Shii, T., Matsuo, Y., Tanaka, T., Jiang, Z. H., & Kouno, I. (2011). A new
catechin oxidation product and polymeric polyphenols of post-fermented tea.
Food Chemistry, 129(3), 830–836.
Jin, X. Y., Chen, J. B., & Ji, K. W. (2003). Tea process engineering (1st ed.). Beijing: China
Agriculture Press (in Chinese, pp. 45–125).
Kanegae, A., Sakamoto, A., Nakayama, H., Nakazono, Y., Yakashiro, I., Matsuo, Y.,
et al. (2013). New phenolic compounds from Camellia sinensis L. fermented
leaves. Journal of Natural Medicines, 67(3), 652–656.
Kim, Y., Goodner, K. L., Park, J. D., Choi, J., & Talcott, S. T. (2011). Changes in
antioxidant phytochemicals and volatile composition of Camellia sinensis by
oxidation during tea fermentation. Food Chemistry, 129, 1331–1342.
Korver, O., & Wilkins, C. K. (1971). Circular dichroism spectra of flavanols.
Tetrahedron, 22, 5459–5465.
Lakenbrink, C., Lapczynski, S., Maiwald, B., & Engelhardt, U. H. (2000). Flavonoids
and other polyphenols in consumers brews of tea and other caffeinated
beverages. Journal of Agricultural and Food Chemistry, 48(7), 2848–2852.
117
Li, Q., Liu, Z. H., Huang, J. A., Luo, G. A., Liang, Q. L., Wang, D., et al. (2012). Antiobesity and hypolipidemic effects of Fuzhuan brick tea water extract in high-fat
diet-induced obese rats. Journal of the Science of Food and Agriculture, 93(6),
1310–1316.
Ling, T. J., Wan, X. C., Ling, W. W., Zhang, Z. Z., Xia, T., Li, D. X., et al. (2010). New
triterpenoids and other constituents from a special microbial-fermented tea—
Fuzhuan brick tea. Journal of Agriculture and Food Chemistry, 58(8), 4945–4950.
Luo, Z. M., Du, H. X., Li, L. X., An, M. Q., Zhang, Z. Z., Wan, X. C., et al. (2013).
Fuzhuanins A and B—The B-ring fission lactones of flavan-3-ols from Fuzhuan
brick-tea. Journal of Agriculture and Food Chemistry, 61(28), 6982–6990.
Luo, Z. M., Ling, T. J., Li, L. X., Zhang, Z. Z., Zhu, H. T., Zhang, Y. J., et al. (2012). A new
norisoprenoid and other compounds from Fuzhuan brick tea. Molecules, 17,
3539–3546.
Mo, H. Z., Zhu, Y., & Chen, Z. M. (2008). Microbial fermented tea—A potential source
of natural food preservatives. Trends in Food Science & Technology, 19, 124–130.
Peng, Y. X., Xiong, Z., Li, J., Huang, J. A., Teng, C. Q., Gong, Y. S., et al. (2014). Water
extract of the fungi from Fuzhuan brick tea improves the beneficial function on
inhibiting fat deposition. International Journal of Food Science and Nutrition.
http://dx.doi.org/10.3109/09637486.2014.898253.
Tanaka, T., Matsuo, Y., & Kouno, I. (2010). Chemistry of secondary polyphenols
produced during processing of tea and selected foods. International Journal of
Molecular Sciences, 11, 14–40.
Tanaka, T., Umeki, H., Nagai, S., Shii, T., Matsuo, Y., & Kouno, I. (2011).
Transformation of tea catechins and flavonoid glycosides by treatment with
Japanese post-fermented tea acetone powder. Food Chemistry, 134, 276–281.
Toschi, T. G., Bordoni, A., Hrelia, S., Bendini, A., Lercker, G., & Biagi, P. L. (2000). The
protective role of different green tea extracts after oxidative damage is related
to their catechin composition. Journal of Agricultural and Food Chemistry, 48,
3973–3978.
Wan, X. C. (2010). Chinese tea (2nd ed.). China Forestry Press (in Chinese, pp. 208–
222).
Wang, H., & Helliwell, K. (2000). Epimerization of catechins in green tea infusions.
Food Chemistry, 70, 337–344.
Wang, W. N., Zhang, L., Wang, S., Shi, S. P., Jiang, Y., Li, N., et al. (2014). 8-C N-ethyl2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese
dark teas formed in the post-fermentation process provide significant
antioxidative activity. Food Chemistry, 152, 539–545.
Wang, R., Zhou, W., & Jiang, X. (2008). Reaction kinetics of degradation and
epimerization of epigallocatechin gallate (EGCG) in aqueous system over a wide
temperature range. Journal of Agricultural and Food Chemistry, 56, 2694–2701.
Wulandari, R. A., Amano, M., Yanagita, T., Tanaka, T., Kouno, I., Kawamura, D., et al.
(2011). New phenolic compounds from Camellia sinensis L. leaves fermented
with Aspergillus sp.. Journal of Natural Medicines, 65(3–4), 594–597.
Xie, G. X., Ye, M., Wang, Y. G., Ni, Y., Su, M. M., Huang, H., et al. (2009).
Characterization of pu-erh tea using chemical and metabolic profiling
approaches. Journal of Agricultural and Food Chemistry, 57, 3046–3054.
Xu, X. Q., Mo, H. Z., Yan, M. C., & Zhu, Y. (2007). Analysis of characteristic aroma of
fungal fermented Fuzhuan brick-tea by gas chromatography/mass
spectrophotometry. Journal of the Science of Food and Agriculture, 87(8),
1502–1504.
Xu, A. Q., Wang, Y. L., Wen, J. Y., Liu, P., Liu, Z. Y., & Li, Z. J. (2011). Fungal community
associated with fermentation and storage of Fuzhuan brick-tea. International
Journal of Food Microbiology, 146, 14–22.
Zuo, Y. G., Chen, H., & Deng, Y. W. (2002). Simultaneous determination of catechins,
caffeine and gallic acids in green, oolong, black and pu-erh teas using HPLC with
a photodiode array detector. Talanta, 57, 307–316.