Hypohalous acid-mediated halogenation of resveratrol and its role in antioxidant and antimicrobial activities

Article abstract of DOI:10.1016/j.foodchem.2012.05.043

The reactions of resveratrol with proinflammatory oxidants including hypochlorous and hypobromous acids in phosphate-buffered saline/methanol solution were carried out and eight halogenated resveratrol derivatives differing in the number and position of halogen atoms, and the configuration of double bond were obtained. Halogenation of resveratrol took place only at the aromatic A ring, and interestingly, the halogenation increased antioxidant activity of this parent molecule in the 2,2′-azobis(2-amidinopropane) hydrochloride-induced RBC haemolysis model. Additionally, antimicrobial activity of the derivatives against Gram-positive bacteria, Gram-negative bacteria and fungi were tested, and toward Candida albicans, 2-chloro-resveratrol and 2-bromo-resveratrol were more active than the unmodified form and the reference compound fluconazole.

Full text of DOI:10.1016/j.foodchem.2012.05.043

Food Chemistry 135 (2012) 1239–1244
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Hypohalous acid-mediated halogenation of resveratrol and its role
in antioxidant and antimicrobial activities
a
a
Xiu-Zhuang Li a, Xia Wei a, Chun-Jiang Zhang b, Xiao-Ling Jin a, , Jiang-Jiang Tang , Gui-Juan Fan ,
⇑
Bo Zhou a,
⇑
a State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China
b School of Life Science, Lanzhou University, Lanzhou, Gansu 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The reactions of resveratrol with proinflammatory oxidants including hypochlorous and hypobromous
acids in phosphate-buffered saline/methanol solution were carried out and eight halogenated resveratrol
derivatives differing in the number and position of halogen atoms, and the configuration of double bond
were obtained. Halogenation of resveratrol took place only at the aromatic A ring, and interestingly, the
halogenation increased antioxidant activity of this parent molecule in the 2,20-azobis(2-amidinopropane)
hydrochloride-induced RBC haemolysis model. Additionally, antimicrobial activity of the derivatives
against Gram-positive bacteria, Gram-negative bacteria and fungi were tested, and toward Candida albi-
cans, 2-chloro-resveratrol and 2-bromo-resveratrol were more active than the unmodified form and the
reference compound fluconazole.
Received 29 February 2012
Received in revised form 19 April 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
Myeloperoxidase
Resveratrol
Hypohalous acids
Antioxidant
Ó 2012 Elsevier Ltd. All rights reserved.
Antimicrobial
Cytotoxicity
1. Introduction
Southwell-Keely, 2000; Nguyen & Southwell-Keely, 2007). Inter-
estingly, chlorination of quercetin and soy isoflavones (genistein
Myeloperoxidase (MPO), a haem enzyme released by activated
phagocytes during inflammation, catalyses the reaction of hydro-
gen peroxide with physiological concentrations of chloride and
bromide anions to produce hypochlorous acid (HOCl) and hypobro-
mous acid (HOBr), respectively (Chapman, Skaff, Senthilmohan,
Kettle, & Davies, 2009). HOCl and HOBr could function as potent
antibacterial agents and play a pivotal role in the immune response
(Thomas, 1979), but they are also oxidants and electrophiles which
react easily with biological molecules such as proteins, lipids and
DNA (Davies, Hawkins, Pattison, & Rees, 2008; Hawkins & Davies,
2002; Hawkins, Pattison, & Davies, 2003; Pattison & Davies,
2001, 2006). Thus, tissue damage resulted from excessive or
misplaced production of hypohalous acids has been implicated in
many inflammatory diseases including atherosclerosis, neurode-
and daidzein) mediated by HOCl and neutrophil MPO, respectively,
leads to the enhanced antioxidant activity in comparison with their
parent molecules. This suggests that inflammatory cell-specific
metabolism could be an important pathway to generate novel
pharmacophores for polyphenolics and modify their properties at
the local site of inflammation (Binsack et al., 2001; Boersma
et al., 2001, 2003).
Resveratrol (3,5,40-trihydroxy-trans-stilbene), a polyphenolic
phytoalexin present in grape and red wine, has attracted consider-
able attention because of its multiple pharmacological actions
ranging from antibacterial and antifungal effects to antioxidative,
anti-inflammatory, cardioprotective, neuroprotective and cancer
chemopreventive activities (Aggarwal & Shishodia, 2006; Baur &
Sinclair, 2006; Pezzuto, 2008). Studies have indicated that this
compound shows significant dose-dependent inhibitory effect on
the production of HOCl and nitric oxide by stimulated human neu-
trophils through preventing the release of MPO (Cavallaro, Ainis,
Bottari, & Fimiani, 2003), and on the chlorination, oxidation and
nitration activities of MPO by a direct interaction with the enzyme
(Kohnen et al., 2007). However, the reaction products of resveratrol
with hypohalous acids (HOCl and HOBr) and their biological activ-
ities, are yet to be determined. As part of our laboratory’s continu-
ous effort to find resveratrol-inspired antioxidants and cancer
chemopreventive agents (Cao et al., 2012; Fan et al., 2009; Shang
generation and cancer (Nicholls
& Hazen, 2005; Ohshima,
Tatemichi, & Sawa, 2003; Yap, Whiteman, & Cheung, 2007). This
has prompted interest in modulating hypohalous acid-mediated
damage by using antioxidants (Folkes, Candeias, & Wardman,
1995; Pattison, Hawkins, & Davies, 2003; Skaff, Pattison, & Davies,
2007), and investigating hypohalous acid-mediated fate of
antioxidants (Boersma et al., 1999; Ho, Soldevilla, Hook,
&
⇑
Corresponding authors. Fax: +86 931 8915557.
E-mail addresses: jinxling@lzu.edu.cn (X.-L. Jin), bozhou@lzu.edu.cn (B. Zhou).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.05.043

1240
X.-Z. Li et al. / Food Chemistry 135 (2012) 1239–1244
et al., 2009; Tang et al., 2011; Yang et al., 2010), we initiated the
current study on the reaction of resveratrol with hypohalous acids
under physiological conditions to mimic MPO-mediated fate of this
molecule and to investigate antioxidant, antimicrobial and anti-
proliferative activities of the related products.
C-2, C-6), 116.5 (1C, C-30), 116.6 (1C, C-50), 120.4 (1C, C-7), 129.0
(1C, C-10), 129.1 (2C, C-20,C-60), 134.9 (1C, C-1), 138.0 (1C, C-8),
149.9 (2C, C-3, C-5), 159.1 (1C, C-40); HR-ESI-MS: m/z calcd. for
C
14H9Cl3O3 + H: 330.9690; found: 330.9692, error = 0.6 ppm.
(Z)-2, 4, 6-trichloro-3, 5, 40-trihydroxystilbene (5): Yield: 0.14%;
1H NMR (400 MHz, (CD3)2CO): d (ppm) = 6.20 (d, J = 12.4 Hz, H-7),
6.66 (d, J = 8.8 Hz, H-30, H-50), 6.71 (d, J = 12.4 Hz, H-8), 6.93 (d,
J = 8.8 Hz, H-20, H-60); HR-ESI-MS: m/z calcd. for C14H9Cl3O3ꢀH:
328.9545; found: 328.9550, error = 1.5 ppm.
2. Materials and methods
2.1. General experimental procedures
To a solution of resveratrol (550 mg) in methanol (90 ml) was
slowly added PBS (200 ml). After being stirred for 10 min, a solu-
tion of 5% self-prepared NaOBr (50 ml, 22.1 mmol) was added
dropwise over 1 h and the mixture stirred for 7 h at room temper-
ature under conditions of darkness. The mixture was extracted
with ethyl acetate, and the organic layer was washed with water
and brine, and dried over MgSO4 followed by evaporation in vacuo.
The residue was separated by silica gel column chromatography
using chloroform/methanol from 40:1 to 80:1 as elution phase,
and the products were collected and recrystallized from light
petroleum/acetone to give pure compounds 6–8.
Resveratrol was purchased from Shanxi Sciphar Biotechnology
Company, China and further purified before use. NaOCl and N-bro-
mosuccinimide (NBS) were from Tianjin Guangfu Fine Chemical
Research Institute, China. 2,20-Azobis (2-amidinopropane hydro-
chloride) (AAPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) were obtained from Sigma–Aldrich
and used as received. RPMI 1640 medium were purchased from
GIBCO. All 1H, 13C and 2D NMR spectra were measured using a Bru-
ker Avance III 400 MHz NMR spectrometer with d6-CH3COCH3 as
solvent. High-resolution mass spectra (HR-MS) were obtained on
an APEX II FT-ICR MS spectrometer.
(E)-2-bromo-3, 5, 40-trihydroxystilbene (6): Yield: 3.72%; 1H
NMR (400 MHz, (CD3)2CO): d (ppm) = 6.48 (d, J = 2.8 Hz, H-4),
6.79 (d, J = 2.8 Hz, H-6), 6.87 (d, J = 8.4 Hz, H-30, H-50), 7.01 (d,
J = 16.4 Hz, H-8), 7.32 (d, J = 16.4 Hz, H-7), 7.47 (d, J = 8.4 Hz, H-
20, H-60), 8.43 (brs, 40-OH), 8.54 (brs, 3-OH, 5-OH); 13C NMR
(100 MHz, (CD3)2CO): d (ppm) = 102.6 (1C, C-2), 103.5 (1C, C-4),
105.7 (1C, C-6), 116.6 (2C, C-30, C-50), 125.6 (1C, C-7), 129.2 (2C,
C-20, C-60), 129.8 (1C, C-10), 132.1 (1C, C-8), 139.9 (1C, C-1), 155.9
(1C, C-3), 158.4 (1C, C-5), 158.7 (1C, C-40); HR-ESI-MS: m/z calcd.
for C14H11BrO3+H: 306.9964; found: 306.9957, error = 1.3 ppm.
(E)-2, 4-dibromo-3, 5, 40-trihydroxystilbene (7): Yield: 1%; 1H
NMR (400 MHz, (CD3)2CO): d (ppm) = 6.88 (d, J = 8.4 Hz, H-30,
H-50), 7.01(s, H-6), 7.03 (d, J = 16.0 Hz, H-8), 7.26 (d, J = 16.0 Hz,
H-7), 7.47 (d, J = 8.4 Hz, H-20, H-60), 8.22 (brs, 3-OH), 8.57 (brs, 40-
2.2. Reaction of resveratrol with HOCl or HOBr
Phosphate-buffered saline (PBS, pH 7.3, 200 ml) was added to a
solution of resveratrol (550 mg) in methanol (90 ml). The suspen-
sion was stirred vigorously for 10 min before the addition of 5%
NaOCl (50 ml, 36.1 mmol). Stirring was continued for 7 h at room
temperature under conditions of darkness. The mixture was ex-
tracted with ethyl acetate, and the organic phase was washed with
water and brine, dried, and concentrated. The residue was sepa-
rated by silica gel column chromatography using chloroform/
methanol from 40:1 to 120:1 as elution phase, and the products
were collected and recrystallized from light petroleum/acetone to
give pure compounds 1–5.
OH), 8.93 (brs, 5-OH); 13C NMR (100 MHz, (CD3)2CO):
d
(ppm) = 98.9 (1C, C-4), 103.0 (1C, C-2), 105.7 (1C, C-6), 116.7 (2C,
C-30, C-50), 124.8 (1C, C-7), 129.3 (2C, C-20, C-60), 129.5 (1C, C-10),
132.8 (1C, C-8), 138.5 (1C, C-1), 152.6 (1C, C-3), 155.1 (1C, C-5),
158.9 (1C, C-40); HR-ESI-MS: m/z calcd. for C14H10Br2O3 + H:
384.9069; found: 384.9073, error = 1.0 ppm.
(E)-2-chloro-3, 5, 40-trihydroxystilbene (1): Yield: 1%; 1H NMR
(400 MHz, (CD3)2CO): d (ppm) = 6.47 (d, J = 2.8 Hz, H-4), 6.79 (d,
J = 2.8 Hz, H-6), 6.87 (d, J = 8.4 Hz, H-30, H-50), 7.06 (d, J = 16.0 Hz,
H-8), 7.32 (d, J = 16.0 Hz, H-7), 7.47 (d, J = 8.4 Hz, H-20, H-60), 8.41
(brs, –OH), 8.49 (brs, –OH), 8.54 (brs, –OH); 13C NMR (100 MHz,
(CD3)2CO): d (ppm) = 103.7 (1C, C-4), 105.1 (1C, C-6), 111.6 (1C,
C-2), 116.6 (2C, C-30, C-50), 122.7 (1C, C-7), 129.2 (2C, C-20, C-60),
129.8 (1C, C-10), 132.0 (1C, C-8), 138.1 (1C, C-1), 154.9 (1C, C-3),
157.6 (1C, C-5), 158.7 (1C, C-40); HR-ESI-MS: m/z: calcd. for
(E)-2, 6-dibromo-3, 5, 40-trihydroxystilbene (8): Yield: 0.65%;
1H NMR (400 MHz, (CD3)2CO): d (ppm) = 6.71 (s, H-4), 6.80 (d,
J = 16.4 Hz, H-7), 6.89 (d, J = 16.4 Hz, H-8), 6.89 (d, J = 8.8 Hz, H-
30, H-50), 7.47 (d, J = 8.8 Hz, H-20, H-60), 8.56 (brs, 40-OH), 8.81
(brs, 3-OH, 5-OH); 13C NMR (100 MHz, (CD3)2CO):
d
C
14H11ClO3 + H: 263.0469; found: 263.0476, error = 2.7 ppm.
(ppm) = 102.7 (1C, C-4), 103.2 (2C, C-2, C-6), 116.6 (2C, C-30, C-
50), 125.7 (1C, C-7), 129.0 (2C, C-20, C-60), 129.3 (1C, C-10), 137.2
(1C, C-8), 140.6 (1C, C-1), 155.1 (2C, C-3, C-5), 158.9 (1C, C-40);
(E)-2, 4-dichloro-3, 5, 40-trihydroxystilbene (2): Yield: 1%; 1H
NMR (400 MHz, (CD3)2CO): d (ppm) = 6.86 (d, J = 8.8 Hz, H-30, H-
50), 6.99 (s, H-6), 7.06 (d, J = 16.0 Hz, H-8), 7.26 (d, J = 16.0 Hz, H-
7), 7.45 (d, J = 8.8 Hz, H-20, H-60), 8.74 (brs, –OH), 8.86 (brs, –OH),
9.29 (brs, –OH).
HR-ESI-MS: m/z calcd. for
382.8934, error = 2.6 ppm.
C14H10Br2O3ꢀH: 382.8924; found:
(E)-2, 6-dichloro-3, 5, 40-trihydroxystilbene (3): Yield: 1%; 1H
2.3. Synthesis of (E)-2, 4, 6-tribromo-3, 5, 40-trihydroxystilbene
NMR (400 MHz, (CD3)2CO):
d (ppm) = 6.67 (s, H-4), 6.88 (d,
J = 8.0 Hz, H-30, H-50), 6.96 (d, J = 16.4 Hz, H-7), 7.03 (d,
J = 16.4 Hz, H-8), 7.48 (d, J = 8.0 Hz, H-20, H-60), 8.59 (s, 40-OH),
Dissolved resveratrol (545 mg) in the mixture of anhydrous ace-
tonitrile (25 ml) and methanol (2 ml), and NBS (1.27 g) was slowly
added to the solution. After being stirred for 24 h at room temper-
ature, the solvent was removed in vacuo, giving a tan oil. The oil
was disolved in ethyl acetate, and washed with water and brine be-
fore dried over MgSO4. Removal of the solvent in vacuo again
yielded a brown oil, which was separated by silica gel column
chromatography using chloroform/methanol (120/1, v/v) as elut-
ing solvent. The crude product was collected and recrystallized
from light petroleum/acetone to give pure compound 9.
8.74 (s, 3-OH, 5-OH); 13C NMR (100 MHz, (CD3)2CO):
d
(ppm) = 103.7 (1C, C-4), 112.4 (2C, C-2, C-6), 116.6 (2C, C-30, C-
50), 121.2 (1C, C-7), 129.1 (2C, C-20, C-60), 129.5 (1C, C-10), 136.9
(1C, C-1), 137.5 (1C, C-8), 153.5 (2C, C-3, C-5), 158.9 (1C, C-40);
_
HR-ESI-MS: m/z: calcd. for C14H10Cl2O3
H: 294.9934; found:
294.9928, error = 2.0 ppm.
(E)-2, 4, 6-trichloro-3, 5, 40-trihydroxystilbene (4): Yield: 6.51%;
1H NMR (400 MHz, (CD3)2CO): d (ppm) = 6.89 (d, J = 8.4 Hz, H-30, H-
50), 6.94 (d, J = 16.4 Hz, H-7), 7.05 (d, J = 16.4 Hz, H-8), 7.49 (d,
J = 8.4 Hz, H-20, H-60), 8.57 (s, 40-OH), 8.81 (s, 3-OH, 5-OH); 13C
NMR (100 MHz, (CD3)2CO): d (ppm) = 109.7 (1C, C-4), 113.2 (2C,
(E)-2, 4, 6-tribromo-3, 5, 40-trihydroxystilbene (9): Yield: 29.3%;
1H NMR (400 MHz, (CD3)2CO): d (ppm) = 6.81 (d, J = 16.4 Hz, H-7),
6.87 (d, J = 16.4 Hz, H-8), 6.89 (d, J = 8.4 Hz, H-30, H-50), 7.48 (d,

X.-Z. Li et al. / Food Chemistry 135 (2012) 1239–1244
1241
J = 8.4 Hz, H-20, H-60), 8.50 (s, 3-OH, 5-OH), 8.55 (s, 40-OH); 13C NMR
(100 MHz, (CD3)2CO): d (ppm) = 99.0 (1C, C-4), 103.3 (2C, C-2, C-6),
116.6 (2C, C-30, C-50), 125.2 (1C, C-7), 128.9 (1C, C-10), 129.2 (2C, C-
20, C-60), 137.7 (1C, C-8), 139.4 (1C, C-1), 152.0 (2C, C-3, C-5), 159.0
(1C, C-40); HR-ESI-MS: m/z calcd. for C14H9Br3O3ꢀH: 460.8029;
found: 460.8023, error = 1.3 ppm.
genase of intact cells to a purple formazan product (Hussain, Nouri,
& Oliver, 1993). HL-60 cells (5 ꢁ 104/ml) were seeded in 96-well
microtiter plates and incubated with various concentrations of
compounds for 48 h at 37 °C, then 10 ll MTT solution (5 mg/ml
in PBS) was added to each well and the cells were incubated for
4 h at 37 °C in the CO2 (5%) incubator. One hundred ten micro litres
extraction buffer (10% SDS-5% isobutanol-0.1 M HCl) was added
and the incubation was continued overnight at 37 °C. A549 cells
(5 ꢁ 104/ml) were seeded in 96-well microtiter plates and cultured
for 24 h, then the medium was replaced by fresh medium before
the cells were treatment with various concentrations of com-
pounds. After 48 h, the medium was replaced by fresh medium
which contained 0.5 mg/ml MTT and the cells were incubated for
2.4. Assay for anti-haemolysis activity
Human red blood cells (RBCs) were obtained from the Red Cross
Centre for Blood (Gansu, China). The procedures for determining
the extent of haemolysis have been described previously (Qian
et al., 2011).
4 h. Afterwards, the medium was removed and 100 ll DMSO was
2.5. Assay for antimicrobial activity
added. The absorbance was measured at 570 nm using a Bio-Rad
M680 ELISA microplate reader.
Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922
and Candida albican were provided by the Clinic Microbial Test
Centre of Gansu Provincial People’s Hospital.
3. Results and discussion
A broth microdilution method (Carson, Hammer, & Riley, 1995)
was used to determine the minimal inhibitory concentration (MIC),
which is defined as the lowest concentration of a sample at which
the microorganism does not demonstrate visible growth. All tests
were performed in nutrient broth. Overnight broth cultures of each
strain were prepared and the final concentration of each well was
adjusted to 2 ꢁ 106 CFU/ml. Each tested compound was dissolved
in 5% dimethylsulphoxide (DMSO) and then diluted with the high-
est concentration (2 mg/ml) to prepare serial twofold dilutions in a
96-well microtiter plate with the range of 0.0039–2 mg/ml. Stan-
dards and controls were made as follows: wells containing nutrient
broth only; each type of bacteria but with no compounds; nutrient
broth containing compounds. Levofloxacin and fluconazole at the
concentration range of 0.0039ꢂ2 mg/ml were prepared in nutrient
broth, and served as the positive controls. The plate was covered
with a sterile plate sealer. Contents of each well were mixed on a
plate shaker for 20 s, and plates were incubated at 37 °C for 24 h
for bacteria and at 28 °C for 48 h for the yeast. Each test in this
study was repeated at least twice.
3.1. Halogenation of resveratrol
The reaction of resveratrol with HOCl or HOBr formed from
NThe reaction of resveratrol with HOCl or HOBr formed from NaO-
Cl or NaOBr (Nguyen & Southwell-Keely, 2007) was conducted in
PBS/methanol solution. Under physiological conditions, HOCl and
HOBr are in equilibrium with the corresponding conjugated base
ꢀOCl and ꢀOBr, respectively. Thus, the used terms of HOCl and
HOBr are representative of the mixture of these species (Morris,
1966; Skaff et al., 2007). Eight products (Fig. 1) were isolated and
identified as (E)-2-chloro-3, 5, 40-trihydroxystilbene (1), (E)-2,
4-dichloro-3, 5, 40-trihydroxystilbene (2), (E)-2, 6-dichloro-3, 5,
40-trihydroxystilbene (3), (E)-2, 4, 6-trichloro-3, 5, 40-trihydroxy-
stilbene (4), (Z)-2, 4, 6-trichloro-3, 5, 40-trihydroxystilbene (5),
(E)-2-bromo-3, 5, 40-trihydroxystilbene (6), (E)-2, 4-dibromo-3, 5,
40-trihydroxystilbene (7) and (E)-2, 6-dibromo-3, 5, 40-trihydroxy-
stilbene (8) by HR-ESI-MS and 1H, 13C and 2D NMR spectrum (see
the Supporting Information). It should be pointed out that the
yields of halogenation of resveratrol in the presence of HOCl or
HOBr are quite low and the synthesis is not valuable as a prepara-
tive method, but the reaction research helps to clarify the MPO-
mediated the fate of resveratrol and understand the reaction
details. Additionally, the products could be more efficiently
obtained by MPO in neutrophils than in vitro. To facilitate the
structure–activity study in the following experiments, (E)-2, 4, 6-
tribromo-3, 5, 40-trihydroxystilbene (9) was also synthesized by
the reaction of resveratrol (1 eq.) with N-bromosuccinimide (3 eq.).
Only the chlorinated and brominated resveratrol derivatives
were obtained in the reactions and the halogenation took place only
at the aromatic A ring, in support of an electrophilic mechanism of
hypohalous acids. In contrast to the aromatic B ring, A ring bears
two electron-donating hydroxyl groups at positions 3 and 5 and
has a relatively high electron density, hence facilitating occurrence
of the electrophilic reactions. By analysing the products obtained, it
is clear that position 2 on the aromatic A ring of resveratrol is the
preferred site of electrophilic attack. It has been revealed that Cl+ it-
self is not the actual species involved in chlorination by HOCl
(Swain & Crist, 1972). However, the actual chlorinating species de-
rived from NaOCl is unclear (Ho et al., 2000; Nguyen & Southwell-
Keely, 2007). On the basis of the reference deals with the reaction
2.6. Calculation of ClogP
The octanol–water partition coefficient P, a descriptor of lipo-
philicity, is defined as the ratio of the concentrations of a com-
pound in two immiscible phases (octanol and water) under
equilibrium conditions, and is used usually in its logarithmic form,
logP. The calculated logP (ClogP) values of the halogenated resvera-
trol derivatives were obtained by Bio-Loom software (Biobyte
Corp. version 5) (Hansch & Leo, 1995; Selassie, Kapur, Verma, &
Rosario, 2005).
2.7. Cell culture
Human promyelocytic leukemia (HL-60) and human lung ade-
nocarcinoma epithelial (A549) cell lines were purchased from
Shanghai Institute of Biochemistry and Cell Biology, Chinese Acad-
emy of Sciences. Cells were cultured in RPMI 1640 medium supple-
mented with 10% (v/v) heat-inactivated foetal bovine serum,
penicillin (100 kU/L) and streptomycin (100 kU/L) at 37 °C in a
CO2 (5%) incubator. Exponentially growing cells were used
throughout these experiments.
of c-tocopherol model compound and HOCl (Ho et al., 2000), the
2.8. Assessment of antiproliferative activity
probable mechanism for formation of 1 are showed in Fig. 2.
Noticeably, a cis-configuration product 5 was obtained in the
reaction mediated by HOCl. This can be rationalised by isomeriza-
tion of product 4 under the condition of light or usage of solvents
with low dielectric constant during the post-processing stage of
Antiproliferative activity of the halogenated resveratrol deriva-
tives was assessed by the MTT colourimetric assay which is based
on the reduction of MTT by the mitochondrial succinate dehydro-

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X.-Z. Li et al. / Food Chemistry 135 (2012) 1239–1244
Fig. 1. The products formed in the reaction of resveratrol with HOCl, HOBr or NBS.
Fig. 2. Probable mechanism for formation of 1 by the reaction of resveratrol and HOCl.
reaction. It has been previously reported that the cis-resveratrol
can be easily transformed from the trans form under intense UV
light (Trela & Waterhouse, 1996), and the formation rate of cis-
diethylstilbestrol from its trans form appears to be facilitated by
solvents with low dielectric constant such as ethylacetate, chloro-
form, heptane and benzene (Winkler, Nyman, & Egan, 1971).
3.2. Anti-haemolysis activity of the halogenated resveratrol derivatives
An initiating radical produced by thermal decomposition of
AAPH in the aqueous dispersion of human red blood cells (RBCs)
could induce membrane lipid peroxidation, resulting in the occur-
rence of haemolysis, which is easily detected by measuring the
absorbance of the haemolysate at 540 nm (Qian et al., 2011). Thus,
the AAPH-induced RBC haemolysis model was used to evaluate
antioxidant activity of the six selected compounds representative
of mono-, di- and tri-chlorinated and brominated resveratrol
derivatives.
Fig. 3. Effects of resveratrol and its halogenated derivatives (20 lM) on 50 mM
AAPH-induced haemolysis of 5% human RBCs in 0.15 M PBS (pH 7.4) under air
atmosphere at 37 °C. (a) AAPH; (b) AAPH + resveratrol; (c) AAPH + 1; (d) AAPH + 3;
(e) AAPH + 4; (f) AAPH + 6; (g) AAPH + 7; (h) AAPH + 9. Data are expressed as the
mean of three determinations.
Fig. 3 shows time courses for AAPH-dependent RBC haemolysis
in the absence and presence of resveratrol and its halogenated
derivatives. A typical time course for haemolysis consists of an ini-
tial inhibition phase followed by a propagation phase and finally
termination. Addition of 50 mM AAPH caused fast haemolysis after
an inhibition time (tinh) of 95 min depending on the used sample
(line a), which stems from the presence of the endogenous antiox-
idant such as vitamin E and/or ubiquinol-10 in the RBC membrane
(Esterbauer & Ramos, 1995). Twenty micro molar resveratrol and
its halogenated derivatives inhibited effectively AAPH-dependent
RBC haemolysis, as judged from an increase in the intrinsic inhibi-
tion time of haemolysis (lines b–h). The inhibition times of resve-
ratrol and compounds (1, 3, 4, 6, 7 and 9) were 120, 127, 145, 150,
135, 160 and 136 min, respectively, which correspond to the addi-
tional, or effective, inhibition time, teff, being 25, 32, 50, 55, 40, 65
and 41 min, respectively (Table 1). Anti-haemolysis activity of res-
veratrol and its halogenated derivatives decreased in the order of
7 > 4 > 3 > 9 ꢂ 6 > 1 > resveratrol by comparing the teff values.
Interestingly, all of the used halogenated derivatives were more
effective at inhibiting RBC haemolysis than this parent molecule
(resveratrol). Given that RBCs are heterogeneous media, such a
change in anti-haemolysis activity could be related to the lipophil-
icity differences among the compounds. The lipophilicity of com-
pounds can be characterised by the octanol–water partition
coefficient, P. Consequently, the calculation of the logP values for
resveratrol and its halogenated derivatives was performed with
Bio-Loom software (Hansch & Leo, 1995; Selassie et al., 2005),
and the values are listed in Table 1. It can be seen from Table 1 that
lipophilicity of compounds augments with the number of chlorine
or bromine atoms attached to the aromatic A ring, and their
anti-haemolysis activity first increases and then reduces with a
maximum for compound 7 (teff = 65 min; ClogP = 4.240) as the
lipophilicity is augmented. In contrast to the situation in homoge-
neous solutions, the antioxidant efficacy in heterogeneous media

X.-Z. Li et al. / Food Chemistry 135 (2012) 1239–1244
1243
Table 1
Anti-haemolysis, antimicrobial and antiproliferative activities of resveratrol and its halogenated derivatives.
a
a
Compounds
tinh (min)
teff (min)
ClogP
MICs (
l
g/ml)
IC50
(lM)
E. coli
S. aureus
C. albican
HL-60
A549
Resveratrol
1
3
4
6
7
9
120
127
145
150
135
160
136
4
2
4
2
4
7
4
25
32
50
55
40
65
41
2.833
3.602
4.120
4.550
3.802
4.240
5.150
250
31.3
125
3.90
31.3
62.5
125
3.90
250
36.3 1.8b
77.7 0.5
41.6 1.9
71.3 0.7
39.7 0.4
>150
104
118
95.2 1.4
90.9 3.7
135
159
164
2
6
c
c
c
–
–
–
62.5
15.6
31.3
31.3
3.90
7.81
3.90
62.5
125
1
2
2
>150
Fluconazole
Levofloxacin
12.5
0.156
0.156
a
b
c
Data are expressed as the mean SD for three determinations.
Cited from Ref. Fan et al. (2009).
No determination.
depends more on the lipophilicity, localisation and mobility than
the chemical reactivity (Niki & Noguchi, 2004). On the one hand,
halogenation of resveratrol improves its lipophilicity and facilitates
its penetration into the membrane and subsequent reaction with
the propagating lipid peroxyl radicals (LOOÅ) within the mem-
branes. On the other hand, the excessive increase of lipophilicity
could lead to the improper localisation of antioxidants in the mem-
branes and thus decreases their LOOÅ-scavenging efficiency, as
exemplified by compound 9, which possesses the highest ClogP va-
lue (5.150) among the compounds investigated but is not the most
effective in the anti-haemolysis experiment.
4. Conclusion
This study describes the products formed by the reaction of res-
veratrol with HOCl or HOBr under physiological conditions and
their antioxidant and antimicrobial activities, with the aim to
investigate the fate of this bioactive stilbene in inflammatory
cell-specific metabolism, and to find this phytoalexin-inspired
antioxidants and antimicrobials. Eight chlorinated and brominated
resveratrol derivatives were formed in the reactions. Interestingly,
all of the investigated derivatives displayed the increased anti-
haemolysis activity as compared to this parent molecule. Addition-
ally, 2-chloro-resveratrol and 2-bromo-resveratrol surfaced as the
important lead compounds, exhibiting more effective activity
against C. albican than the unmodified form and the reference com-
pound fluconazole.
3.3. Antimicrobial activity of the halogenated resveratrol derivatives
In vitro antimicrobial activity of resveratrol and its halogenated
derivatives were assessed against Gram-positive bacteria
(S. aureus), Gram-negative bacteria (E. coli) and yeast (C. albicans).
The minimum inhibitory concentrations (MICs) of the compounds
are displayed in Table 1. Among the compounds tested, resveratrol
and di-brominated compound 7 turned out to be the most potent
against S. aureus. However, resveratrol was less active against
E. coli and C. albicans. Noticeably, 2-chloro-resveratrol (1) and
2-bromo-resveratrol (6) were most active against C. albicans
among the compounds tested, and exhibited roughly 30- and 3-
fold lower MIC values against C. albicans than this parent molecule
(resveratrol) and the reference compound (fluconazole), respec-
tively. Therefore, they could be employed for further development
of effective antifungal drugs. The above results demonstrate that
the introduction of chlorine or bromine atoms on the aromatic A
ring of resveratrol can modify its antimicrobial profile. Similar re-
sults have been also found in the case of the halogenated
bis(hydroxyphenyl)methanes (Oh et al., 2009).
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 20972063), the 111 Project, Program for
New Century Excellent Talents in University (NCET-06-0906) and
the Fundamental Research Funds for the Central Universities.
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.2012.
05.043.
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