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Bioactive Constituents, Metabolites, and Functions
Novel Flavoalkaloids from White Tea with Inhibitory Activity Against Formation of Advanced Glycation End Products Xiao Li, Guang-Jin Liu, Wei Zhang, Yv-Long Zhou, Tie-Jun Ling, Xiao-Chun Wan, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00650 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018
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Journal of Agricultural and Food Chemistry
Novel Flavoalkaloids from White Tea with Inhibitory Activity against Formation of Advanced Glycation End Products Xiao Li†,§, Guang-Jing Liu†,§, Wei Zhang†, Yv-Long Zhou†, Tie-Jun Ling†, Xiao-Chun Wan†, Guan-Hu Bao*, † †
Natural Products Laboratory, International Joint Lab of Tea Chemistry and Health effects, State
Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 230036, Hefei, People’s Republic of China *
Phone: +86-551-65786401. Fax: +86-551-65786765. E-mail:
[email protected].
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Abstract: Two novel flavoalkaloids, (−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-epigallocatechin
2
-O-gallate
(ester-type
catechins
pyrrolidinone
3
(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-O-gallate (etc-pyrrolidinone B,2) and
4
new
5
(−)-8-N-ethyl-2-pyrrolidinone-epigallocatechin-O-gallate (etc-pyrrolidinone C,3a and etc-
6
pyrrolidinone D,3b), were isolated from white tea (Camellia sinensis). Their structures were
7
identified by extensive NMR spectra. Absolute configuration of 1 and 2 was decided by
8
comprehensive CD spectroscopic analyses. The isolated flavoalkaloids together with
9
(−)-epigallocatechin-O-gallate (EGCG) were evaluated for their inhibition against the
10
formation of advanced glycation end products (AGEs) with IC50 values ranging from 10.3 to
11
25.3 µM. UPLC−DAD−ESI/MS detected these flavoalkaloids in both white tea and fresh tea
12
leaves, which demonstrated the existence of a corresponding biosynthetic pathway in tea
13
plant. Therefore, a possible pathway was proposed to involve deamination, decarboxylation,
14
and spontaneously cyclization of l-theanine, and then attachment of the product to EGCG to
15
form the flavoalkaloids.
naturally
A,
occurring
etc-pyrrolidinone
A,
1),
flavoalkaloids
16
Keywords: pyrrolidinone, epigallocatechin-O-gallate (EGCG), Camellia sinensis, absolute
17
configuration, advanced glycation end products (AGEs), l-theanine
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Introduction
19
White tea is one of the six traditional Chinese tea categories (green, white, yellow,
20
oolong, black, and dark tea), which is mostly produced in Fujian province, China. It is
21
a lightly fermented tea since its manufacture process has only two simple steps called
22
withering and drying.1 White tea can be ranked into four classes according to its
23
quality as the following sequence: Silver Needle (or Baihao-Yinzhen), White Peony
24
(or Bai-Mudan), Tribute Eyebrow (or Gong-Mei), and Longevity Eyebrow (Shou
25
-Mei).2 Among them, Bai-Mudan is a popular one which was prepared by one bud
26
with one or two fresh tea leaves (Camellia sinensis var. sinensis, C. sinensis var.
27
sinensis). Recent studies have indicated that white tea shows anti-oxidant,
28
anti-obesity, anti-cancer, and preventive effect on cardiovascular diseases and so
29
on.3-6 One study found that white tea was the richest in catechin derivatives associated
30
with the best intestinal bioaccessibility and bioavailability among all of the tea
31
samples tested (green, white, black tea).7 Another research suggested that white tea
32
had better antimutagenic activity than green tea in the Salmonella assay, which might
33
be related to the different levels of the nine major constituents, and these major
34
components may act synergistically with other minor constituents.8
35
The diverse functional properties of white tea can be attributed to its abundant active
36
components, whereas few studies have been conducted on systematic purification and
37
structural identification of chemical constituents from white tea. Recently, we have
38
found several minor catechin derivatives from both dark and green tea.9,10 One minor
39
catechin derivative (−)-epicatechin 3-O-caffeoate (ECC) showed the strongest
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inhibitory effect against acetylcolinesterase and neutrophil gelatinase-associated
41
lipocalin (NGAL) among different catechins,10,11 which suggested minor new
42
catechins in tea materials are worthy of study. UPLC-MS analysis showed that
43
flavoakaloids may exist in the white tea Bai-Mudan (big white tea originated in
44
Fuding, Fujian province, cultivar: Fuding-Dabai, class: Bai-Mudan) (Figure S1),
45
which encouraged us to study the chemical constituents from this type of tea.
46
Flavoalkaloids are an unusual group of secondary metabolites from plant, which have
47
a unique molecular framework possessing a nitrogen ring system linked to a flavonoid
48
skeleton, as the name imply.12 (+)−Ficine and (+)−isoficine were the first two
49
flavoalkaloids isolated from the wild fig, Ficuspantoniana, Moraceae in 1965.13 Since
50
then, more than 100 flavoalkaloids have been found from different plants,
51
successively.14 Ethylpyrrolidinonyl theasinensin A was the first flavoalkaloid isolated
52
from black tea in 2005.15 In 2014, eight more flavoalkaloids, puerins I-VIII with
53
significant antioxidative activity, were reported in the Chinese dark tea.16 Recently,
54
due to their potential possibility of multiple biosynthetic pathways and pronounced
55
bioactivities, flavoalkaloids have gathered much more attention.17-19 Flavopiridol, a
56
semi-synthetic flavoalkaloid also known as alvocidib, was performed effectively for
57
the treatment of acute myeloid leukaemia in phase II clinical trials by alteration of
58
tyrosine phosphorylation of cyclin-dependent kinase (CDK) 1/2 and competitive
59
inhibition with adenosine triphosphate (ATP).20 Furthermore, flavoalkaloids were also
60
demonstrated as inhibitors against the formation of advanced glycation end products
61
(AGEs), which are the pathogenic factor of some chronic degenerative diseases such
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as diabetic and neurodegenerative diseases.21 As it could be anticipated,
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flavoalkaloids indicate promising potential for the discovery of new therapeutic
64
agents and deserve more research.
65
In spite of studies on the flavoalkaloids from black and dark tea, there is less attention
66
to flavoalkaloids from other tea categories. Therefore, in this research, we
67
systematically studied flavoalkaloids from white tea and successfully obtained two
68
novel together with new naturally occurring flavoalkaloids from white tea. The
69
isolation, structural elucidation, AGEs inhibition, and possible biosynthetic pathway
70
of the flavoalkaloids were presented in this study.
71
Materials and Methods
72
Chemicals. HPLC grade acetonitrile, methanol, and formic acid were purchased from
73
Duksan pure chemicals Co., Ltd (Ulsan, Korea). Bovine serum albumin was
74
purchased from Nanjing Duly Biotech Co., Ltd (Nanjing, China). Penicillin was
75
obtained from Harbin Pharmaceutical Group Co., Ltd (Harbin, China). Phosphate
76
buffered
77
Aminoguanidine hydrochloride was bought from Shanghai TCI Development Co.,
78
Ltd (Shanghai, China). (−)-Epigallocatechin-3-O-gallate (EGCG) was isolated from
79
tea plants in our laboratory and the purity was ≥ 98% confirmed by HPLC analysis.
80
The purification materials filled in column chromatography in this study included
81
MCI-Gel CHP20P (Mitsubishi Ltd., Tokyo, Japan), Sephadex LH-20 (GE Healthcare
82
Bio-Sciences AB, Stockholm, Sweden), ODS C-18 (ODS, Fuji Silysia Chemical Ltd.,
83
Kasugai, Japan), Toyopearl HW-40F (Tosoh Bioscience Shanghai Co., Ltd., Shanghai,
saline
(PBS)
was
purchased
from
Solar-bio
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China).
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China), DIAION HP20SS gel (Mitsubishi Ltd., Tokyo, Japan), and silica gel (Yantai
85
jiangyou silicon development co., Ltd., Shandong, China).
86
IR spectrum was measured on an FTIR-650 spectrometer purchased from Tianjin
87
GangDong Sci. & Tech. Development Co., Ltd (Tianjin, China). Optical rotation was
88
measured on MCP 100 Modular Circular Polarimeter (Anton Paar GmbH, Graz,
89
Austria). 1H and 13C NMR, 1H-1H COSY, ROESY, HSQC, and HMBC spectra were
90
recorded with a DD2 (600 MHz) spectrometer in methanol-d4 or dimethylsulfoxide
91
(DMSO)-d6 (Agilent Inc., Santa Clara, CA, USA). CD spectra were obtained with a
92
Jasco-810-CD apparatus (Jasco, Tokyo, Japan). Mass spectra were performed on
93
Agilent 1290 UPLC with a photodiode detector array (PDA) coupled to a 6545
94
time-of-flight (TOF) mass spectrometer with electrospray ionization (ESI) source in
95
negative mode (Agilent Inc., Santa Clara, CA, USA). HPLC semi-preparation was
96
performed on a Waters e2695 combined with a Waters 2998 PDA detector (Waters,
97
Milford, Massachusetts, USA). The semi-preparative column was X Bridge Prep C18
98
(10 × 250 mm i.d., 5 µm) (Waters, Wexford, Ireland). The fluorescence of the
99
samples was measured on a SpectraMax M2e ELIASA (Molecular Devices, Santa
100
Clara, CA, USA). The melting point (mp) was measured on SGWX-4 Micromelting
101
point apparatus purchased from Beijing century science instruments Co., Ltd (Beijing,
102
China).
103
Tea Materials. The commercial white tea Bai-Mudan was purchased from Fujian
104
Pinpinxiang Tea Co., Ltd (Fujian, China) in 2014 and it belongs to Fuding-Dabai tea
105
cultivar.
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Extraction and Isolation. Bai-Mudan tea (4 kg) was ground and extracted with 80%
107
acetone/water for three times at room temperature and then concentrated under
108
reduced pressure to produce a water-soluble extract.22 The aqueous extract was then
109
mixed into dichloromethane (1:1, v/v) to provide dichloromethane-soluble fraction
110
(300 g) and an aqueous phase. The aqueous phase was further extracted with ethyl
111
acetate (1:1, v/v), and n-butanol, successively to provide ethyl acetate-soluble fraction
112
(680 g), n-butanol soluble fraction (200 g), and residue water-soluble fraction (630 g).
113
The n-butanol soluble fraction was subjected to Sephadex LH-20 column
114
chromatography (CC), eluting with water/methanol (1:0−0:1), to get fractions A1-A5,
115
and then the fraction A4 was subjected to MCI-Gel CHP20P gel CC (water/methanol
116
= 1:0−0:1) to obtain fractions B1-B20. Fraction B19 was subjected to Toyopearl CC
117
and eluted with methanol/water (1:1, v/v), yielding ten fractions (C1 to C10). Fraction
118
C1 was then performed on the semi-preparative HPLC eluted with gradient
119
acetonitrile/water (The gradient elution of acetonitrile was set as follows: 0−6 min,
120
18%; 6−8 min, from 18% to 20%; 8−13 min, 20%; 13−13.5 min, from 20% to 18%;
121
13.5−22 min, 18%) to get compound 1 (10 mg), 2 (5 mg), 3a and 3b (15 mg),
122
respectively (Figure 1). Fraction C5 was subjected to Toyopearl CC (water/methanol
123
= 8:2−1:0) to give compound 15 (74 mg). Fraction B4 was applied to Sephadex
124
LH-20 CC with H2O containing increasing proportion of methanol to get fractions
125
D1-D20. Fraction D7 was eluted with water/methanol (6:4) on Toyopearl CC to get
126
compound 4 (12 mg). Fraction D10 was eluted with methanol on Toyopearl CC to
127
obtain compound 5 (8 mg). Fraction D9 was separated by Toyopearl CC
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(methanol/water = 8:2) to give compound 6 (11 mg) and compound 9 (9 mg). Fraction
129
D12 was purified by Sephadex LH-20 CC with 95% ethanol, followed by Toyopearl
130
CC with methanol to afford compound 7 (7 mg) and compound 10 (15 mg). Use of
131
Sephadex LH-20 CC on fraction D15 with methanol and then Toyopearl CC with
132
methanol gave fractions E1-E6. Fraction E2 was subjected to Sephadex LH-20 CC
133
with methanol to obtain compound 11 (11 mg). Fraction D18 was eluted with
134
methanol on Sephadex LH-20 CC and Toyopearl CC to give compound 12 (15 mg).
135
Fraction A1 was subjected to Sephadex LH-20 CC eluted by methanol/water with
136
increasing polarity (1:9 to 10:0) to obtain fraction F1-F5. Fraction F4 was separated
137
by silica gel CC with ethyl acetate/methanol (40:1) and Toyopearl CC with methanol
138
to gain compound 8 (9 mg). Fraction A3 was separated into G1−G4 fractions by
139
Sephadex LH-20 CC with methanol. Fraction G2 was successively subjected to ODS
140
CC (water/methanol = 9:1) and Sephadex LH-20 CC (water/methanol = 1:0−0:1) to
141
obtain ten fractions (H1−H10). DIAION HP20SS gel CC eluting with water/methanol
142
(7:3) was used to separate fraction H4 to yield Fractions I1-I3. Fraction I3 was further
143
separated by Toyopearl CC (water/methanol = 6:4) to afford six subfractions (J1−J6).
144
Fraction H6 and J5 were merged and separated by Sephadex LH-20 CC, eluted with
145
water/methanol (1:0−0:1) in a gradient elution to afford fraction K6 which was further
146
purified by ODS CC (water/methanol = 1:9) to get compound 14 (79 mg). Fraction I2
147
was then purified by Toyopearl CC (water/methanol = 1:0−0:1), ODS CC
148
(water/methanol = 1:0−0:1), followed by Sephadex LH-20 CC (water/methanol =
149
1:0−0:1) to get compound 13 (10 mg).
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UPLC−DAD−ESI/MS Analysis. UPLC−MS analysis was performed on an Agilent
151
1290 UPLC instrument with a PDA coupled to a 6545 TOF mass spectrometer with
152
ESI source in negative mode. The analysis was carried out using an ACQUITY
153
UPLC® BEH Shield RP18 column (2.1 × 150 mm, i.d., 1.7 µm) and Agilent
154
qualitative analysis software for data acquisition. The mobile phase A was 0.1%
155
aqueous formic acid and mobile phase B was 0.1% formic acid acetonitrile. The
156
gradient elution of mobile phase B was set as follows: 0−1.5 min, 6%; 1.5−4 min,
157
from 6 to 12%; 4−8 min, from 12 to 25%; 8−10 min, from 25 to 35%; 10−14 min,
158
from 35 to 90%; then kept at 90% for 5 min and return to 6% in 1 min and kept at 6%
159
for 3 min. The flow rate was 0.22 mL/min under the wavelength of 280 nm and the
160
injection volume was 2 µL. The tea sample was prepared by ultrasonic extracting 0.25
161
g of ground tea powder in 10 mL of 80% aqueous acetone three times within 12 h (15
162
min each time) for UPLC−DAD−ESI/MS analysis. Mass spectra were acquired in
163
negative and full scan mode from m/z 100 to 1700.
164
Determination of AGEs Formation. Determination of the AGEs formation was
165
based on a former reported method with modification.23 The buffer with 0.02%
166
penicillin to prevent degradation. The reaction mixture contained bovine serum
167
albumin 10 mg/mL in PBS with penicillin to prevent bacterial growth, and added with
168
36 mg/mL fructose and glucose. The reaction mixture was then mixed with
169
compounds (EGCG, 1, 2 and 3a, 3b) or positive control aminoguanidine. After
170
incubating at 37 °C for 14 d, the fluorescent reaction products were assayed on
171
ELISA at the excitation and emission maximum of 350 nm and 450 nm. Then a series
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of gradient concentrations of the compounds were measured, all samples were
173
prepared in triplicate.
174
Statistical Analysis. All assay experiments were done in triplicate and the values
175
were presented as mean ± SD. We used GraphPad Prism software (version 6.0) for
176
statistical analysis and IC50 calculation.
177
Results and Discussion
178
Isolation and Identification of Compounds 1, 2 and 3a, 3b. For the phytochemical
179
investigation of white tea, the 80% aqueous acetone extract of white tea was
180
concentrated. The aqueous residue was successively extracted with dichloromethane,
181
ethyl acetate, and n-butanol. And the n-butanol soluble fraction was fractionated by
182
repeated column chromatography (CC) and semi-preparative HPLC to provide four
183
flavoalkaloids (Figure 2), two novel pure compounds (1, 2) and a mixture of two
184
isomers (3a, 3b). At the same time, 12 known compounds were obtained from the
185
Chinese commercial white tea including gallicin (4),24 (+)-catechin (5),25
186
(−)-epicatechin (6),25 gallic acid (7),26 kaempferol (8),27 procyanidin B2 (9),28
187
kaempferol-3-7-O-β-D-dirhamnoside (10),29 epigallocatechin-(4β-8)-epicatechin-3-O
188
-gallate (11),30 EGCG (12),31 1,6-di-O-galloyl-β-D-glucose (13),32 1-O-galloyl-4,6-
189
(−)-hexahydroxydiphenoyl-β-D-glucose (14),30 1,4,6-tri-O-galloyl-β-D-glucose (15).
190
30
191
Compounds 1, 2, 3a and 3b showed similar IR spectrum, which suggested the
192
presence of hydroxyl groups (broad peak around 3397 cm-1), carbonyl group (1695
193
cm-1), aromatic rings (1620, 1539 cm-1).33 Their ESI−HR−MS spectrum showed the
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same deprotonated molecular ion peak at m/z 568.1475 [M-H]− (calcd for 568.1455)
195
corresponding to the molecular formula C28H27NO12 with 16 degrees of unsaturation.
196
The odd number of the molecular weight (569) suggested the presence of a nitrogen
197
atom in the molecule. The UV λmax (MeOH) peaks of compounds 1, 2, 3a and 3b are
198
at 209, 276 nm. The 1H and 13C NMR data of the isomers 1, 2, 3a and 3b were nearly
199
the same (Table 1). Above experimental results suggest that the four flavoalkaloids
200
are regioisomers.
201
Compound 1 was observed as white amorphous powder, mp: 200 °C, [α]25D -36.94 (c
202
0.11, methanol). The 1H and
203
are shown in table 1. The existence of an EGCG skeleton in the molecule could be
204
easily deduced from the 1H NMR spectrum recorded in DMSO-d6 compared with that
205
of EGCG (Figure 3). The typical proton signals for rings A, B and C are similar to
206
those of EGCG,31 at δH 5.00 (1H, s, H-2), δH 5.56 (1H, s, H−3), 3.03 (1H, m, H−4β),
207
2.84 (1H, m, H−4α) (ring C), δH 6.51 (2H, s, H−2', 6') (ring B), δH 6.07 (s, 1H) (ring
208
A), δH 6.95 (2H, s) (galloyl−H), respectively (Figure 3).31 A single proton signal at
209
A-ring suggested a substitute at C−6 or C−8 of the A−ring. Besides signals from
210
flavan-3-ol and galloyl unit, the 1H and
211
attributable to a methine (C−5''' ), two methylenes (C−3''', C−4''' ), a carbonyl (C−2''' )
212
and an ethyl group. The 1H−1H COSY correlations of these methine and methylenes
213
indicated the presence of a partial structure of −CH2−CH2−CH−. In the HMBC
214
spectrum, the methylene protons of the N-ethyl group were correlated with the
215
carbonyl carbon (δC 178.4, C−2''') and the methine carbon (δC 55.6, C−5'''). The
13
C NMR data of compound 1 recorded in methanol-d4
13
C NMR spectra also showed signals
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216
methylene protons (δH 2.20, H−3''') were also correlated with carbonyl carbon. These
217
1
218
N-ethyl-2-pyrrolidinone ring (Figure 4). The weak ROESY correlations of singal at
219
A−ring (δH 6.07, CH-8) with that of B−ring (δH 6.51, CH−2', 6'), C−ring (δH 5.00,
220
CH−2) and 7-OH proton (δH 9.44) (Figure S12) allowed the attachment of the
221
N-ethyl-2-pyrrolidinone group at C−6 position and the proton signal (δH 6.07) belongs
222
to C−8 position (Figure 4),34 which are proven with the corresponding HMBC
223
correlations from H−5''' (δH 5.43) to C−5 (δC 156.4) and C−7 (δC 158.5).
224
The absolute configurations at C−2/3 of four flavoalkaloids were confirmed as 2R, 3R
225
by comparing CD curves with those of EGCG (Figure S32). The configuration at
226
C−5''' can be distinguished by subtracting one CD spectrum from the other
227
stereoisomer with the same configuration at C−2/3.16,35 In this research, compounds 1
228
and 2 had the same skeleton as EGCG. In order to determine the configuration at
229
C−5''' of 1 and 2, the CD spectra of 1 and 2 were compared after subtracting the CD
230
spectrum from each other (Figure 5).16 For compound 1, the arithmetically isolated
231
CD curves of C−5''' showed a strong positive cotton effect at 213 nm (∆ε+10.5) and
232
was determined to be of the 5''' S-configuration. Therefore, the structure of compound
233
1
234
epigallocatechin-3-O-gallate and named as etc-pyrrolidinone A.
235
Compound 2 were obtained as white amorphous powder, mp: 193 °C, [α]25D -143.21
236
(c 0.02, methanol). Comparing 1H and
237
(Table 1), compound 2 was determined to be an isomer of 1. The position of
H−1H
COSY
was
and
HMBC
determined
correlations
to
be
13
revealed
the
presence
of
an
(−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-
C NMR spectra between compound 1 and 2
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N-ethyl-2-pyrrolidinone in 2 was also determined to be at C−6 by the analysis of the
239
ROESY (Figure S18) and HMBC spectra. Meanwhile, compound 2 presented a
240
negative cotton effect at 213 nm (∆ε − 10.5) comparing with 1 by arithmetically CD
241
curves (Figure 5) and was determined to be 5''' R-configuration. Therefore, the
242
structure
243
(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate
244
etc-pyrrolidinone B.
245
For compounds 3a and 3b, we observed them as a mixture in the format of white
246
amorphous powder, [α]25D -78.37 (c 0.22, methanol). Their melting point is greater
247
than 300 °C. The 1H and
248
those of 1. But for 3a, the ROESY correlation of a proton (δH 6.06) with 5-OH and
249
7-OH proton (δH 9.38, δH 9.53) confirmed that this proton (δH 6.06) belongs to C-6
250
and the N-ethyl-2-pyrrolidinone group linked to the C-8 position (Figure S28). So,
251
compounds
252
(−)-8-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate. Furthermore, 3a and 3b
253
can be identified by 2D NMR spectroscopy. Compound 3a was determined as
254
(−)-8-(5'''S)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate
255
etc-pyrrolidinone
256
8-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate
257
etc-pyrrolidinone D. Although 3a and 3b had been synthesized and identified as a
258
mixture too,15 they were isolated as new natural products from tea in present study.
259
Naturally occurring flavoalkaloid tend to exist as isomers in the plant kingdom
of
compound
13
C.
determined and
to named
be as
C NMR spectra of 3a and 3b were also closely similar to
and
3a
was
2
were
3b
3b
was
determined
and
determined
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named as
and
named
be
as (−)as
Journal of Agricultural and Food Chemistry
260
including tea and other food materials,14,16,21 which results in the difficulty to purify
261
and identify these compounds from plants.
262
UPLC−MS Analysis. In this study, the four flavoalkaloids (1, 2, 3a and 3b) were
263
used as standards to analyze Bai-Mudan tea and fresh tea leaves of Fuding-Dabai by
264
UPLC−PDA−ESI/MS analysis. The result showed that these four flavoalkaloids (1, 2,
265
3a and 3b) could be found in white tea Bai-Mudan (Figure 6A), as well as the fresh
266
tea leaves (material of the former) (Figure S1), which implies the original presence of
267
flavoalkaloids in tea as pyrrolidinonated ester-type catechins (such as EGCG) with
268
epi-configuration at C-2 and -3 position (2R, 3R) and also suggests the existence of a
269
related biosynthetic pathway in Camellia sinensis. The MS/MS spectrum gave the
270
fragmental peaks at 416, 398, 236, 169, 125 (Figure 6B) corresponding to the
271
elucidated fragmental structures as shown in Figure 6C, which further confirmed the
272
identified flavoalkaloid structure.16,36 The retention time of compound 1, 2, 3a and
273
3b are 11.07, 11.88, 10.86 min, respectively. The content of these compounds is
274
around several ppm/dry weights on the base of the amount of the valualbe
275
flavoalkaloids we isolated, further accumulation of these flavoalkaloids and
276
quantification of them in different tea leaves is highly warranted.
277
Inhibition of Compounds 1, 2 and 3 (3a and 3b) on Formation of AGEs
278
The effect of the compounds on the formation of AGEs was evaluated with different
279
concentrations (0.1, 1, 5, 10, 25, 50, 100 µM). Figure 7 shows that the four
280
compounds (1, 2, 3a and 3b) and EGCG can effectively reduce the formation of
281
AGEs with a dose-response inhibition. Among them, the mixture compounds of 3a
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and 3b exhibited the most potential inhibitory activity against AGEs formation, with
283
the IC50 values of 10.3 µM. The new compounds (1, 2) and EGCG also showed a
284
promising activity with IC50 values at 25.3, 13.5, and 11.7 µM, respectively, better
285
than the well known glycation inhibitor, aminoguanidine with the IC50 value at 228.8
286
µM .
287
Flavoalkaloids represent a convergence of diverse array of structures resulted from
288
multiple biosynthetic pathways.14,37 To date, a dozen of flavoalkaloids including these
289
in the present study were detected and isolated from Camellia sinensis.15,16 The first
290
tea flavoalkaloid ethylpyrrolidinonyl theasinensin A was obtained from black tea. The
291
researchers posed that l-theanine was degraded to Strecker aldehyde and conjugated
292
with tea polyphenol A rings during the drying and enzyme deactivation stages of
293
black tea production.15 The later study demonstrated that puerins I-VIII were
294
biosynthesized from catechins and l-theanine through a kind of typical fungi
295
Aspergillus niger in the Chinese dark tea during the post-fermentation process.16 Both
296
above researches suggested that flavoalkaloids were formed through a Strecker
297
aldehyde during manufacture process of tea. In our study, the four flavoalkaloids (1, 2,
298
3a and 3b) were isolated from the Chinese white tea (Bai-Mudan), and identified as
299
etc-pyrrolidinones sharing the same EGCG skeleton (Figure 2), which are different
300
from those reported from dark tea.16 The flavoalkaloids from dark tea all lose the
301
galloyl group in the structure since galloyl group of EGCG tends to fall off during the
302
fermentation process.10,16 However, the four flavoalkaloids (Figure 2) isolated from
303
commercial white tea were also detected in fresh tea leaves (Figure S1), which
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suggested that manufacture process is not the only pathway for the formation of tea
305
flavoalkaloids. EGCG and epicatechin gallate (ECG) are the most abundant catechins
306
in tea. L-theanine is the characteristic amino acid accounting for about 50% of total
307
amino acids in C. sinensis. Flavoakaloids in tea may be the products from the
308
interaction between the above most abundant metabolites EGCG or ECG, and
309
l-theanine in tea plant. Most of the original biosynthetic products may be present in
310
tea plant as pyrrolidinonated EGCG or ECG. The flavoalkaloids in dark tea may be
311
derived from losing galloy group of these pyrrolidinonated EGCG or ECG and then
312
configuration changes during the process.16 Therefore, flavoalkaloids may also be
313
biosynthesized by the function of enzymes in the fresh leaves of tea plants, with
314
EGCG or ECG and l-theanine as the vital precursors. As such, a biosynthetic pathway
315
in tea plant for these flavoalkaloids was proposed in Figure 8. The biosynthesis may
316
be
317
N-ethyl-pyrrolidinone, followed by coupling of the product and EGCG. The key
318
enzymes such as glutamic acid dehydrogenase38 and glutamic acid decarboxylase39
319
may play important roles in the reaction.
320
Flavoalkaloids are not common but often rewarded with pronounced biological
321
activities.14,37 We evaluated the isolated flavoalkaloids for their effect on the
322
formation of AGEs and demonstrated them as effective inhibitor against the formation
323
of AGEs with IC50 values ranging from 10.3 to 25.3 µM compared with the positive
324
control aminoguanidine with the IC50 at 228.8 µM (Figure 7). This observation is
325
well consistent with a former report that two new flavoalkaloids isolated from the
completed
through
deamination,
decarboxylation
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l-theanine
into
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roots of Actinidia arguta, which also showed stronger activity than that of
327
aminoguanidine.21 The unsubstituted carbons at A ring of these flavoalkaloids are the
328
active sites for trapping reactive dicarbonyl species according to previous reports,
329
which suggest that these flavonoids may prevent the development of diabetic
330
complications.40 However, they showed no big difference in the assay from that of
331
EGCG, which implied that more assays are needed for determination of the special
332
bioactivity of these tea flavoalkaloids since they shared a pyrrolidinone ring and
333
should have different effect in some assays from EGCG.
334
In conclusion, fifteen compounds including two novel (1 and 2) and new naturally
335
occurring flavoalkaloids (3a and 3b) were isolated from the commercial Chinese
336
white tea. The absolute configuration at C−5''' of the pyrrolidinone ring of the novel
337
compounds were unambiguously assigned by CD analyses. These flavoalkaloids 1, 2,
338
and 3 showed inhibitory effect against the formation of AGEs with the IC50 values at
339
25.3, 13.5, and 10.3 µM, respectively.
340
Supplementary Data
341
1
342
and 3b (Figure S5-28). The optical rotation value, CD value, and infrared
343
spectrogram of compound 1, 2, 3a and 3b (Figure S29-32).
344
Author Contributions: X. Li and G. J. Liu contribute equally to the paper
345
Funding
346
Financial assistances were received with appreciation from Nutrition and Quality
347
& Safety of Agricultural Products, National Modern Agriculture Technology System
348
Grant CARS-23.
H,
13
C, COSY, HSQC, HMBC, ROESY and DEPT spectra of compounds 1, 2, 3a
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Notes
350
Guan-Hu Bao, Xiao Li, Xue-Shi Liu declare patent applications (CN201810201244.4)
351
with Anhui Agricultural University for the use of the two new flavoalkaloids 1 & 2.
352
All the other authors declare no competing financial interest. All the other authors
353
declare no competing financial interest.
354
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Figure Captions
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Figure 1. The semi-preparative chromatogram and ultraviolet absorption spectrogram
474
of four flavoalkaloids by HPLC.
475
Figure 2. The structure of four flavoalkaloids isolated from white tea.
476
Figure 3. 1H NMR spectra of (−)-epigallocatechin-O-gallate (EGCG) and compound
477
1 in dimethylsulfoxide (DMSO)-d6.
478
Figure 4. Selected two dimensional nuclear magnetic resonance (2D NMR)
479
correlations including the key 1H-1H COSY (heavy solid line),HMBC (solid single
480
arrowhead line), ROESY (dashed double arrowhead line) correlations of 1 and 3a, 3b.
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Figure 5. The configuration of compounds 1 and 2 was determined by arithmetically
482
CD curves subtracted each other for a couple of stereoisomers.
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Figure 6. A: LC-MS total (TIC) and extracted (EIC at 568) ion chromatograms of the
484
80% aqueous acetone extract of Bai-Mudan tea together with the TIC of compounds 1
485
2, and 3. B: fragmental peaks of compounds 1-3 from MS/MS spectrum. C: the
486
deduced structures of the fragment peaks of compounds 1-3.
487
Figure 7. Does-response inhibition curves and IC50 of compounds 1, 2, 3, and
488
(−)-epigallocatechin -O-gallate (EGCG) against AGEs, with the IC50 of the positive
489
control aminoguanidine (AG).
490
Figure 8. A possible biosynthetic route to flavoalkaloids in tea plant.
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Table 1. NMR Spectroscopic Data of Compound 1, 2, 3a, 3ba position 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 1'' 2'' 3'' 4'' 5'' 6'' 7'' 1''' 2''' 3''' 4''' 5''' 6''' 7''' a1
compound 1 δH (J, δc Hz) 5.00 s 79.3 5.56 s 70.4 3.03 m 28.2 2.84 m 156.4 109.0 158.5 6.07 s 98.2 157.3 100.7 131.4 6.51 s 107.7 147.5 134.7 147.5 6.51 s 107.7 122.2 6.95 s 111.1 147.1 140.7 147.1 6.95 s 111.1 168.4
compound 2 ∆H (J, δc Hz) 4.98 s 79.5 5.56 s 70.6 3.00 m 28.5 2.86 m 156.8 109.3 158.8 6.07 s 98.5 157.5 101.0 131.6 6.50 s 108.0 147.8 135.0 147.8 6.50 s 108.0 122.4 6.94 s 111.3 147.4 141.0 147.4 6.94 s 111.3 168.7
178.4 25.3 33.4
178.7 25.6 33.7
2.20 m 2.44 m 2.66 m 5.43 dd (5.2,9.2) 2.66 m 3.49 m 1.00 m
55.6 37.2 13.4
2.15 m 2.39 m 2.68 m 5.45 dd (4.8,9) 2.65 m 3.50 m 0.99 m
55.7 37.5 13.7
compound 3a δH (J, Hz) δc 5.00 s 5.53 s 2.99 m 2.88 m 6.06 s
6.52 s
6.52 s 6.95 s
6.95 s
2.20 m 2.39 m 2.65 m 5.55 m 2.90 m 3.56 m 1.14 m
79.8 70.5 27.6 157.3 97.0 158.3 106.4 155.9 99.9 131.6 107.7 147.6 134.8 147.6 107.7 122.5 111.2 147.2 140.8 147.2 111.2 168.4 178.4 25.3 33.5 55.1 37.8 14.0
compound 3b δH (J, Hz) δc 4.91 s 5.47 s 2.95 m 2.85 m 6.03 s
6.44 s
6.44 s 6.92 s
6.94 s
2.18 m 2.34 m 2.59 m 5.46 m 2.72 m 3.38 m 0.97 m
80.1 70.3 27.9 157.3 97.8 158.3 106.7 155.9 100.9 131.3 108.2 147.6 135.0 147.6 108.2 122.5 111.2 147.2 140.8 147.2 111.2 168.6 178.1 25.3 33.1 55.1 37.3 13.6
H at 600 MHz and 13 C NMR at 150 MHz in methanol-d4. s: single peak; d: double
peaks; m:multipeaks.
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