Journal of Agricultural and Food Chemistry
Article
yield 86%; mp 213−215 °C; 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J
= 7.6 Hz, 1H), 7.48−7.59 (m, 3H), 7.40 (t, J = 7.6 Hz, 1H), 7.21 (s,
1H), 4.01 (s, 3H), 4.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
158.7, 158.3, 152.9, 150.2, 149.5, 148.1, 131.0, 124.5, 121.2, 117.3,
115.2, 112.8, 106.1, 102.1, 95.4, 56.4, 56.3.
9-Chloro-6H-benzofuro[3,2-c]chromen-6-one (4): Rf = 0.32 (ethyl
acetate/petroleum ether = 1:19, v/v), 0.59 g of white product, yield
87%; mp 242−244 °C; 1H NMR (400 MHz, CDCl3) δ 8.01−8.06 (m,
2H), 7.69 (s, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.50−7.53 (m, 1H), 7.41−
7.47 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.5, 157.7, 155.5,
153.8, 132.7, 132.3, 130.7, 128.8, 126.1, 124.8, 122.4, 121.9, 117.6,
112.5, 112.4.
9-Bromo-6H-benzofuro[3,2-c]chromen-6-one (5): Rf = 0.39 (ethyl
acetate/petroleum ether = 1:9, v/v), 0.42 g of white product, yield
53%; mp 213−215 °C; 1H NMR (400 MHz, CDCl3) δ 8.03 (t, J = 8.0
Hz, 2H), 7.87 (d, J = 1.2 Hz, 1H), 7.61−7.68 (m, 2H), 7.53−7.55 (m,
1H), 7.45 (t, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 160.3,
157.7, 155.6, 153.8, 132.3, 128.8, 124.8, 122.7, 122.6, 121.9, 120.1,
117.6, 115.4, 112.3, 105.6.
9-Hydroxy-6H-benzofuro[3,2-c]chromen-6-one (6): Rf = 0.29
(ethyl acetate/petroleum ether = 1:2, v/v), 0.24 g of white product,
yield 96%; mp 291−293 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.16
(s, 1H), 8.03 (s, 1H), 7.78 (s, 1H), 7.69 (s, 1H), 7.60 (m, 1H), 7.49 (s,
1H), 7.22 (s, 1H), 7.00 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
158.1, 157.7, 157.1, 156.4, 152.5, 131.5, 124.9, 121.2, 121.1, 117.0,
114.4, 114.3, 112.1, 105.3, 98.6.
presented herein is a study on the antioxidant effects of seven
coumestans with electron-withdrawing groups (−Cl, −Br) and
electron-donating groups (−OCH3 and −OH). The abilities to
quench ABTS•+, DPPH, and galvinoxyl radical, respectively,
•
and to inhibit the oxidations of DNA caused by OH, Cu2+/
GSH, and AAPH, respectively, are estimated to reveal the role
of the coumestan scaffold in the generation of antioxidant
effectiveness.
MATERIALS AND METHODS
■
Materials and Instrumentation. ABTS, DPPH, and galvinoxyl
radicals were purchased from Fluka Chemie GmbH, Switzerland, and
AAPH and naked DNA sodium salt were purchased from Acros
Organics, Belgium. Other agents were of analytical grade and used
directly. The structures of products were identified by H and 13C
1
NMR (Bruker Avance III 400 MHz sprctrometer), and the spectra are
included in the Supporting Information.
Synthesis and Identification of Coumestan Structure.
Esterification of Methyl Salicylate. To a mixture of methyl
salicylate (1.52 g, 10 mmol) and the corresponding phenylacetic acid
(11 mmol) in 10 mL of pyridine at 0−10 °C was added dropwise
POCl3 (1.1 mL, 12 mmol) within 0.5 h under stirring. After the above
mixture was stirred for 3 h at 0−10 °C, the concentrated HCl was
added to adjust pH 5−6 at the same temperature. The mixture was
extracted by ethyl acetate at room temperature, and the organic phase
was washed by brine and dried over Na2SO4. After the solvent was
evaporated under vacuum, the obtained crude product was purified by
silica chromatography with ethyl acetate and petroleum ether being
eluents to afford a white product with a yield >70%.
Ester Condensation for the Formation of Coumarins. The
phenylacetic ester of methyl salicylate (5 mmol) was mixed with
anhydrous pyridine (5 mL), followed by the addition of KOH powder
(0.7 g, 12.5 mmol) at room temperature. After the mixture was stirred
for 6 h in a nitrogen atmosphere, pyridine was evaporated under
vacuum, and 15 mL of NaOH (2.0 M) was added. The water phase
was washed with ethyl acetate and then acidized by concentrated HCl
to adjust the pH to 4−5. The aqueous solution was cooled to afford
the crude product, a white solid with a yield >55%.
Oxidative Annulation for the Formation of Coumestans. To
30 mL of dried 1,2-dichloroethane solution of the ester condensation
products (2.5 mmol) were added anhydrous FeCl3 (1.625 g, 10 mmol)
and silica powder (1.625 g), and the mixture was refluxed for 10 h.
The solvent was then evaporated under vacuum, and the residue was
purified by silica chromatography with ethyl acetate and petroleum
ether being eluents to afford white coumestans with yields >60%.
To obtain hydroxyl-substituted coumestan, the methoxyl group
should be converted into a hydroxyl group by demethylation. In brief,
methoxyl-substituted coumestan (1.0 mmol, dissolved in 10 mL of
anhydrous CH2Cl2) was added dropwise to 10 mL of CH2Cl2 solution
of BBr3 (1.0 M) within 30 min at 0 °C and stirred overnight at room
temperature. The reaction mixture was hydrolyzed at 0 °C. The
obtained precipitate was purified by silica chromatography with ethyl
acetate and petroleum ether (1:2, v/v) being eluent to afford a white
product with a yield >90%.
8,9-Dihydroxy-6H-benzofuro[3,2-c]chromen-6-one (7): Rf = 0.18
(ethyl acetate/petroleum ether = 1:2, v/v), 0.24 g of white product,
1
yield 91%; mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 9.65 (s,
1H), 9.53 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.64−7.68 (m, 1H), 7.57−
7.59 (m, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.30 (s, 1H), 7.24 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) δ 158.2, 157.9, 152.8, 149.9, 147.0,
145.2, 131.7, 125.4, 121.6, 117.5, 114.3, 112.9, 106.0, 105.4, 99.4.
Scavenging ABTS•+, DPPH, and Galvinoxyl Radicals. A 2.0 mL
of aqueous solution containing 4.0 mM ABTS and 1.41 mM K2S2O8
was kept for 20 h to form ABTS•+ and then diluted by 100 mL of
ethanol. The absorbance of ABTS•+ was around 1.00 at 734 nm
(εABTS = 1.6 × 104 M−1 cm−1). DPPH and galvinoxyl radicals were
•+
dissolved in ethanol directly. The absorbances of DPPH and galvinoxyl
radicals were around 1.00 at 517 nm (εDPPH = 4.09 × 103 M−1 cm−1)
and 428 nm (εgalvinoxyl = 1.4 × 105 M−1 cm−1), respectively. A certain
concentration of DMSO solution of a coumestan (0.1 mL) was added
to 1.9 mL of ABTS•+, DPPH, and galvinoxyl radical solutions,
respectively. The decreases of the absorbance of these radicals were
recorded at 25 °C at a certain time interval.
•
Cu2+/GSH- and OH-Induced Oxidations of DNA. Cu2+/GSH-
induced oxidation of DNA was carried out according to the following
description. Briefly, DNA, CuSO4, and GSH were dissolved in
phosphate-buffered solution (PBS1: 6.1 mM Na2HPO4, 3.9 mM
NaH2PO4), and coumestans were dissolved in dimethyl sulfoxide
(DMSO) as the stock solution. Then, a solution of 2.0 mg/mL DNA,
5.0 mM Cu2+, 3.0 mM GSH, and 0.2 mM coumestan was poured into
test tubes with each containing 2.0 mL. The test tubes were incubated
at 37 °C for initiating the oxidation of DNA. Three test tubes were
taken out at 90 min and cooled immediately. PBS1 solution of EDTA
(1.0 mL, 30.0 mM) was added to chelate Cu2+, followed by adding 1.0
mL of thiobarbituric acid (TBA) solution (1.00 g of TBA and 0.40 g of
NaOH dissolved in 100 mL of PBS1) and 1.0 mL of 3.0%
trichloroacetic acid aqueous solution. The test tubes were heated in
boiling water for 30 min and cooled to room temperature, and 1.5 mL
of n-butanol was added and shaken vigorously to extract thiobarbituric
acid reactive substance (TBARS) for measuring the absorbance at 535
nm.
6H-Benzofuro[3,2-c]chromen-6-one (1): Rf = 0.40 (ethyl acetate/
petroleum ether = 1:9, v/v), 0.38 g of white product, yield 64%; mp
177−179 °C; 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 4.8 Hz, 1H),
8.04 (d, J = 7.2 Hz, 1H), 7.60−7.68 (m, 2H), 7.42−7.52 (m, 4H); 13C
NMR (100 MHz, CDCl3) δ 159.9, 157.9, 155.4, 153.6, 131.9, 126.7,
125.2, 124.6, 123.4, 121.8, 121.8, 117.4, 112.5, 111.7, 105.8.
9-Methoxy-6H-benzofuro[3,2-c]chromen-6-one (2): Rf = 0.31
(ethyl acetate/petroleum ether = 1:9, v/v), 0.59 g of white product,
•OH can be generated in the mixture of H2O2 and tetrachlorohy-
droquinone (TCHQ, dissolved in DMSO as the stock solution). DNA
and H2O2 were dissolved in phosphate-buffered solution (PBS2: 8.1
mM Na2HPO4, 1.9 mM NaH2PO4, 10.0 μM EDTA). A solution of 2.0
mg/mL DNA, 4.0 mM TCHQ (dissolved in DMSO as the stock
solution), 2.0 mM H2O2, and 0.2 mM coumestan (dissolved in DMSO
as the stock solution) was poured into test tubes with each containing
1
yield 89%; mp 217−219 °C; H NMR (400 MHz, CDCl3) δ 7.98 (s,
2H), 7.57 (s, 1H), 7.50 (s, 1H), 7.40 (s, 1H), 7.19 (s, 1H), 7.07 (s,
1H), 3.91 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.7, 159.3,
158.2, 156.8, 153.2, 131.3, 124.6, 122.0, 121.5, 117.4, 116.5, 113.6,
112.9, 106.1, 96.8, 55.9.
8,9-Dimethoxy-6H-benzofuro[3,2-c]chromen-6-one (3): Rf = 0.46
(ethyl acetate/petroleum ether = 3:7, v/v), 0.64 g of white product,
B
dx.doi.org/10.1021/jf500013v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX