1
38
Y. Zhao et al. / Food Chemistry 171 (2015) 137–143
reagents used were of HPLC grade (Jinmei Biotech Corporation,
Tianjin, China). The water was prepared by an EASYPure II UV Ultra
Pure Water System (Barnstead International, Dubuque, IA, USA).
energy was 22 eV. Full scan ranging from 100 to 400 atomic mass
unit (amu) and daughter ion scan with the mass of parent ion at
227 m/z, 225 m/z and 239 m/z were recorded. UV–visible spectrum
of each compound was recorded in the range 190–400 nm by a
photodiode detector.
2.2. Assessment of photostabilities of trans-resveratrol in different
mediums
2
.5.2.2. Fourier transforms infrared (FTIR) spectroscopic analysis. A
An ethanol solution of trans-resveratrol (0.25 mM, 100% etha-
Nicolet AVATAR 370 FTIR Spectrometer (Thermo Fisher, Massachu-
setts, USA) was used to determine the FTIR spectrum of the com-
pound B in KBr pellets in the wavelength ranging from 400 to
nol) and an aqueous solution of trans-resveratrol (0.038 mM,
water) were prepared for irradiation tests. The trans-resveratrol
solutions and trans-resveratrol powder were respectively exposed
to sunlight (75000 lux), laboratory light (35000 lux) and UV B for
ꢀ1
4
000 cm according to method described by Yang, Li, He, Ren,
and Wang (2009).
ꢀ2
4
h. The intensity of UV B was achieved at 100 lW cm by using
six UV lamps (SPECTRONICS BLE-1T158 Tube 15 watt, main output
at312 nm) according to the UV B intensity in the sunlight. The
trans-resveratrol solutions with and without irradiation treat-
ments were sampled for HPLC analysis. The trans-resveratrol pow-
der was dissolved by 100% ethanol for HPLC analysis.
1
13
2
.5.2.3. H and C nuclear magnetic resonance (NMR) measure-
1
13
ment. H and C NMR spectra were obtained from ꢂ20 mg of the
freeze dried compound B suspended in 0.8 mL acetone-d 6. The
spectra were recorded on a Bruker AVANCE III spectrometer at
6
00 MHz with a 5 mm DCH cryoprobe (Bruker, Wissembourg,
France). Integration of the spectra was performed with Bruker
Topsin 3.2 software.
2
.3. Study on the transformation of trans-resveratrol under UV B
radiation
3
. Results and discussion
Based on the HPLC profiles of the reaction mixture of trans-
resveratrol in liquids, Peaks 1–4 are assigned to trans-resveratrol,
cis-resveratrol, compound A and compound B respectively. A
kinetic study was carried out to investigate the evolution of
trans-resveratrol under UV B radiation. UV lamps were used to
achieve a constant UV B radiation at 100 lW cm . 60 mL ethanol
solution of trans-resveratrol (0.25 mM, 100% ethanol) was UV B
3
.1. Photostability of trans-resveratrol
Table 1 shows the HPLC profiles of trans-resveratrol in different
ꢀ2
mediums with and without irradiation treatments. For liquid state,
the trace levels of cis-resveratrol in the aqueous and ethanol solu-
tion of trans-resveratrol without irradiation indicated that trans to
cis conversion of resveratrol naturally occurred in liquid state, and
laboratory light exerted no obvious effect on the isomerization of
trans-resveratrol according to the HPLC profiles in Table 1. Two
remarkable new peaks were observed in the HPLC profiles of sun-
light/UV B irradiated trans-resveratrol in aqueous and ethanol
solutions (Table 1), suggesting that two new compounds are abun-
dantly generated. For solid state, no new compounds were
detected in all the irradiated trans-resveratrol in solids (Table 1).
The level of cis-resveratrol slightly increased after exposure of
trans-resveratrol powder to sunlight/UV B for 4 h, and accordingly
the trans-resveratrol powder turned reddish under 4 h sunlight/UV
B radiation while remained white in the laboratory light.
Sunlight basically consists of visible, UV and infrared light
whereas laboratory light mainly consists of visible light. From
above results, UV B spectrum in the sunlight may play an impor-
tant role in generating new photoproducts of trans-resveratrol,
which is in agreement with the conclusion demonstrated by
Nour, Trandafir, and Muntean (2012). Besides, the occurrence of
photo-induced chemical reaction of trans-resveratrol requires a
liquid medium. In our study, two principal photoproducts were
observed, which is inconsistent with the previous studies that only
one photoproduct of trans-resveratrol formed under UV B radiation
irradiated and sampled every 30 min for HPLC analysis.
2.4. HPLC analysis
The HPLC conditions were: Injection volume 10 lL, Agilent TC-
C18 column (4.6 mm ꢁ 250 mm, 5
lm), column temperature 28 °C,
mobile phase A = acetonitrile/acetic acid/water (6:1:193, v), mobile
phase B = acetonitrile, linear gradient elution: from 75% (v) A and
2
7
5% (v) B to 55% (v) A and 45% (v) B during early 30 min and then
5% (v) A and 25% (v) B till 35 min, flow rate 1 mL min , Shimadzu
ꢀ
1
SPD ultraviolet detector at 285 nm.
2
2
.5. Structural identification of the photoproducts
.5.1. Preparation of the compound B
The reaction mixture of trans-resveratrol (0.25 mM, 100%
ethanol) under UV B radiation was submitted to semi-preparative
HPLC (Varian Proster 218, USA). The fractions of compound B were
collected and combined. The compound B was obtained by freeze
drying.
2
2
.5.2. Structural identification
.5.2.1. UPLC–UV-MS/MS analysis. UPLC–UV-MS/MS (Waters Cor-
(López-Hernández et al., 2007; Wang et al., 2002; Yang et al.,
poration, Milford, USA) was employed to provide the molecular
mass information of compounds A and B. The UPLC conditions
2
012). This may be attributed to different HPLC separation systems
and irradiation treatments employed. Exceptionally, ethanol solu-
tions of trans-resveratrol with high concentrations ranging from
2
were: acquity UPLC HSST3 column (2.1 mm ꢁ 150 mm, 1.8
lm),
column temperature 35°C, injection volume 10 L, mobile phase
l
0 mM to 100 mM were also irradiated by UV B/sunlight for 4 h
A = 0.1% formic acid + 99.9% water (v/v), mobile phase B = 0.1% for-
in our study, but no new compounds were found (data not shown),
indicating that the concentration of trans-resveratrol significantly
affects the photochemical reaction of trans-resveratrol.
mic acid + 99.9% acetonitrile (v/v), linear gradient elution: from
9
9.9% (v) A/0.1% (v) B to 10% (v) A/90% (v) B during 38 min, flow
ꢀ1
rate 1 mL min . An electrospray ionisation (ESI) technique in a
negative ion mode was employed for MS/MS analysis. The ion
source conditions were set as follows: capillary voltage 3000 V,
cone voltage 30 V, extractor 3.0 V and RF lens 0.2 V, ion source
temperature 150 °C, desolvation gas nitrogen at a flow rate of
3.2. Photo-induced transformation of trans-resveratrol
Fig. 1 shows the chemical transformation of trans-resveratrol
in 100% ethanol as UV B exposure time increased. At 30 min of
UV B radiation, most of trans-resveratrol (Peak 1) converted to
ꢀ
1
6
00 L h
and temperature at 300 °C. Argon was used as the
ꢀ1
collision gas at a flow rate of 0.13 mL min
and the collision