Synthesis, molecular characterization and preliminary antioxidant activity evaluation of quercetin fatty esters

Article abstract of DOI:10.1007/s11746-013-2314-0

Quercetin shows interesting pharmacological effects, but its use in topical applications is limited by its low skin permeability and solubility. In this work, the synthesis of highly lipophilic quercetin esters with oleic, linoleic and linolenic acid useful as topical quercetin prodrugs is reported. Partial OH esterification is advisable to maintain the antioxidant activity of these compounds; tetraesters and triesters can be achieved by modulating the reaction conditions utilized for the total esterification of quercetin. The chemical structures of the esters were proven by spectroscopic techniques; quantum chemical NMR calculation were mandatory to unequivocally assign the free position in triesters. Finally, the antioxidant activity of all the synthesized compounds was determined by the 2,2-diphenyl-1-picryl-hydrazyl method and by 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) assay.

Full text of DOI:10.1007/s11746-013-2314-0

J Am Oil Chem Soc (2013) 90:1751–1759
DOI 10.1007/s11746-013-2314-0
ORIGINAL PAPER
Synthesis, Molecular Characterization and Preliminary
Antioxidant Activity Evaluation of Quercetin Fatty Esters
•
Francesca Mainini Alessandro Contini Donatella Nava
•
•
•
Paola Antonia Corsetto Angela Maria Rizzo
•
•
Elisabetta Agradi Elena Pini
Received: 18 January 2013 / Revised: 20 June 2013 / Accepted: 12 July 2013 / Published online: 6 September 2013
ꢀ AOCS 2013
Abstract Quercetin shows interesting pharmacological
effects, but its use in topical applications is limited by its
low skin permeability and solubility. In this work, the
synthesis of highly lipophilic quercetin esters with oleic,
linoleic and linolenic acid useful as topical quercetin pro-
drugs is reported. Partial OH esterification is advisable to
maintain the antioxidant activity of these compounds; tet-
raesters and triesters can be achieved by modulating the
reaction conditions utilized for the total esterification of
quercetin. The chemical structures of the esters were pro-
ven by spectroscopic techniques; quantum chemical NMR
calculation were mandatory to unequivocally assign the
free position in triesters. Finally, the antioxidant activity of
all the synthesized compounds was determined by the 2,2-
diphenyl-1-picryl-hydrazyl method and by 2,2-azinobis(3-
ethyl-benzothiazoline-6-sulfonic acid) assay.
Introduction
In recent years the interest in natural substances useful to
counteract aging has risen sharply. In fact, there is a sig-
nificant increase in skin cosmetics, nutraceuticals and even
pharmaceutical products based on the use of natural com-
pounds or on their semi-synthetic derivatives.
Besides, a strong interest in natural substances with high
antioxidant activity has been particularly observed. Indeed
oxidative stress induced by multiple factors is the main
cause of many pathological conditions such as inflamma-
tion, cancer, coronary heart disease and even skin aging.
Quercetin, 3,30,40,5,7-pentahydroxyflavone, is a natural
polyphenol abundant in plant food (apples, onions, grapes,
red wine and green tea) which exerts many pharmacolog-
ical effects, such as antioxidant activity [1–3], anti-
inflammatory effects [4–6], antitumor [7], pro-apoptotic [8,
9] and cardiovascular activity [10] inhibiting platelet
aggregation and thrombus formation. Moreover quercetin
shows in-vitro antiviral [11] and antibacterial activity [12–
14]. Recently it has been reported that the topical appli-
cation of quercetin inhibits oxidative skin damage [15] and
the inflammatory processes induced by solar UV radiation
[16]. However quercetin topical use is limited by its low
skin permeability and solubility making the development
of dermocosmetic and nutraceutical applications difficult.
In this work, the synthesis of new quercetin pentaesters
with x-3, x-6 and x-9 fatty acids is presented with the aim
of obtaining topical prodrugs with two moieties both of
them pharmacologically active. These molecules could
lead to an increase in quercetin lipophilicity which could
positively affect its bioavailability, skin permeability and
overall pharmacological activity. Since in the literature
[17–19] is reported that the different hydroxyl groups of
quercetin are responsible for the various pharmacological
Keywords Quercetin pentaesters ꢀ Quercetin
tetraesters ꢀ Quercetin trimesters ꢀ Fatty acids ꢀ
Bi-dimensional NMR techniques ꢀ Quantum chemical
NMR calculation ꢀ DPPH ꢀ ABTS
Electronic supplementary material The online version of this
article (doi:10.1007/s11746-013-2314-0) contains supplementary
material, which is available to authorized users.
F. Mainini ꢀ A. Contini ꢀ D. Nava ꢀ E. Pini (&)
Sezione di Chimica Generale e Organica ‘‘Alessandro
Marchesini’’, DISFARM, Via Venezian 21, 20133 Milan, Italy
e-mail: elena.pini@unimi.it
P. A. Corsetto ꢀ A. M. Rizzo
DISFEB, Via Trentacoste 2, 20134 Milan, Italy
E. Agradi
DISFEB, Via Balzaretti 9, 20133 Milan, Italy
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J Am Oil Chem Soc (2013) 90:1751–1759
activities, we also report herewith on the synthesis and
analytical characterization of tetra and triesters of quercetin
with oleic, linoleic and linolenic acids. Furthermore, a
preliminary evaluation of the antioxidant activity by the
2,2-diphenyl-1-picryl-hydrazyl (DPPH) method and by
2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid)
assay (ABTS) is reported.
was silica Ascentis 15 9 4.6 mm (3 lm particle size); the
mobile phase was a mixture of hexane/isopropanol 99.9:0.2
(v/v) eluted at 1.5 ml min-1 at room temperature.
Low resolution mass analyses were recorded with a
Thermo-Finnigan LCQ advantage AP electrospray/ion trap
equipped instrument by using a syringe pump device to
directly inject sample solutions.
The UV–Vis spectra were collected using a Jasco V530
instrument.
Experimental
General Procedure for the Preparation of Acyl Chloride
Materials
(1b–d)
Quercetin, oleic, linolenic and linoleic acid, solvents,
reagents and deuterated solvents were purchased from
Sigma Aldrich (Milan, Italy). Reaction were monitored by
thin layer chromatography on silica gel plates F254, visu-
alized with UV light or iodine vapor. Silica gel 230–400
mesh/60A was employed for column chromatography.
Antioxidant Assay Kits were from Sigma-Aldrich, St.
Louis, USA.
Under nitrogen atmosphere, oxalyl chloride (16 mmol,
1.31 ml) was added dropwise to an ice cold stirred solution
of the unsaturated fatty acid (2.47 mmol, 0.77 ml) in
anhydrous toluene (10 ml). The reaction mixture was then
allowed to reach room temperature and magnetically stir-
red for 3 h. The reaction was monitored by FT-IR. At the
end of the reaction (3 h) the solution was evaporated under
a vacuum (77 mbar, 40 ꢁC) in an inert atmosphere and the
acyl chloride was employed without further purification for
the following reaction.
Characterization Methods
Oleoyl chloride. IR (NaCl) cm-1 3005, 2926, 2855,
FT-IR spectra were collected using a Perkin Elmer (Wal-
tham, MA, USA) FT-IR Spectrometer ‘‘Spectrum One’’ in
a spectral region between 4,000 and 600 or 450 cm-1 for
solid or liquid compounds respectively and analysed by
transmittance technique through 32 scanning and 4 cm-1
resolution. Solid samples were mixed in a mortar with KBr
(1:100) and pressed in a hydraulic press (14 tons) to small
tablets, while for liquid samples one drop was placed
between two widows of sodium chloride. The 1H- and 13C-
NMR and Bi-dimensional analyses, COSY, heteronuclear
multiple-bond correlation spectroscopy (HSQC) and het-
eronuclear multiple-bond correlation spectroscopy
(HMBC) were carried out on a Bruker Avance 500
1801, 1464, 723.
Linoleoyl chloride. IR (NaCl) cm-1 3010, 2928, 2857,
1801, 1464, 729.
Linolenoyl chloride. IR (NaCl) cm-1 3011, 2961, 2930,
2857, 1801, 1463, 727.
General Procedure for the Preparation of Quercetin Esters
(2, 3, 4 a–d)
Under nitrogen flow, the opportune acyl chloride (for molar
ratio see Table 1) was added dropwise to a magnetically
stirred ice cold solution of quercetin (0.3 mmol, 0.10 g)
and TEA (2.47 mmol, 0.35 ml) in anhydrous dioxane
1
(Billerica, MA, USA) operating at 500 MHz for H and
125.75 MHz for 13C or with a Varian Mercury Plus 200,
operating at 200 MHz for 1H and 50.3 MHz for 13C.
Chemical shifts were expressed as ppm (d) using the cen-
tral peak of chloroform as the internal reference
(dH = 7.23 ppm; dC = 77.3 ppm). The APT sequence was
used to distinguish methine and methyl carbon signals from
those due to methylene and quaternary carbons.
Table 1 Polyesters obtained by the modulation of quercetin and fatty
acid molar ratio
Quercetin/fatty acid molar
ratio
Stearoyl
ester
Unsaturated fatty
ester
1/10
1/7.5
1/6
Pentaester
Pentaester
Pentaester
Pentaester
Pentaestera
Pentaestera
Pentaestera
Pentaestera
Pentaester
Pentaester
HPLC/DAD analyses were performed using an HPLC
system equipped with a quaternary pump Merck Hitachi
L-7100, an injector Rheodyne 7125 with a 20 ll loop, a
thermostat Column Block Heater (Mod. 7940 Hichrom
Ltd.), a detector DAD Hewlett Packard HP 1050. The
acquired data were processed by software HP CHEM. The
detector was a Diode Array (Waters mod. 2996, Milford,
MA, USA) operating at 254 nm. The column employed
Penta and tetraester
Tetraester
1/5
1/4
Tetra and triester
Tetra and triestera
Tetra and triestera
Tetra and triestera
1/3
1/2
1/1
a
And unreacted quercetin
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J Am Oil Chem Soc (2013) 90:1751–1759
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(10 ml). The reaction mixture obtained was then magnet-
ically stirred while being protected from daylight at room
temperature for 20 h. After solvent evaporation under a
vacuum (107 mbar, 40 ꢁC), the residue was dissolved in
chloroform (20 ml), washed with a saturated aqueous
NaHCO3 solution (20 ml) and then with water
(3 9 20 ml). The organic layer was dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure.
2-(3,4-Distearoylossiphenyl)-4-oxo-4H-chromene-3,5,
7-tristearate (2a). Recrystallization from ethanol gave a
white solid with 57 % yield. IR (KBr) cm-1 2957, 2918,
2850, 1769, 1641, 1618, 1468, 1438, 1266, 1233, 1208,
2.61–2.57 (m, 6 H), 2.05 (m, 16 H), 1.81–1.74 (m, 8 H),
1.44–1.29 (m, 80 H), 0.90 (t, 12 H, J = 6.6 Hz). 13C NMR
(CDCl3) d 175.6, 170.4, 169.9, 169.9, 169.8, 161.0, 155.8,
155.3, 154.8, 144.1, 141.7, 131.6, 129.4, 129.3, 129.2,
129.0, 128.9 (2 9 C), 126.8, 125.8, 123.3, 123.2, 108.1,
104.8, 100.4, 33.7, 33.4, 33.3, 33.0, 31.2, 29.1, 29.0
(2 9 C), 28.9, 28.8, 28.6 (2 9 C), 28.5 (2 9 C), 28.4
(2 9 C), 28.3 (3 9 C), 26.6, 26.5, 26.4, 24.2, 24.1 24.0,
23.9, 21.9, 13.4. ESI–MS: C87H138O11, calc 1360.0, found
m/z = 1383 [M ? Na]?. Rt = 2.49 min. UV/Vis (CHCl3)
269, 337 nm.
2-(3,4-Dioleoylossiphenyl)-4-oxo-4H-chromene-3,5-
dihydroxy-7-oleate (4b). Separation from compound 3b
by column chromatography was performed using a mixture
hexane/acetone 9:1 giving a light yellow liquid in 15 %
yield. IR (NaCl) cm-1 3322, 3099, 3005, 2959, 2854,
1765, 1647, 1603, 1563, 1489, 1466, 1308, 1263, 1179,
1
1178, 1112, 990, 721. H NMR (200 MHz, CDCl3) d 7.70
(dd, 1 H, J = 8.3, 1.8 Hz), 7.66 (d, 1 H, J = 1.8 Hz), 7.32
(d, 1 H, J = 8.3 Hz), 7.30 (d, 1 H, J = 2.2 Hz), 6.83 (d, 1
H, J = 2.2 Hz), 2.73 (t, 2 H, J = 7.6 Hz), 2.62–2.52 (m, 8
H), 1.78–1.71 (m, 10 H), 1.55–1.26 (m, 100 H), 0.88 (t, 15
H, J = 5.6 Hz). 13C NMR (CDCl3) d 172.2, 171.0, 170.9,
170.8, 170.2, 157.1, 154.5, 153.8, 150.8, 144.7, 142.5,
134.3, 128.0, 126.6, 124.0, 115.0, 114.1, 109.1, 34.6, 34.3,
34.3, 34.0, 32.2, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.4,
29.3, 25.1, 24.9, 24.8, 24.7, 22.9, 14.3. ESI–MS:
1
1111, 722. H NMR (500 MHz, CDCl3) d 11.65 (s, 1 H,
exch. D2O), 8.14 (dd, 1 H, J = 8.6, 2.1), 8.08 (d,1 H,
J = 2.1 Hz), 7.38 (d, 1 H, J = 8.6 Hz), 6.90 (d, 1 H,
J = 2.0 Hz), 6.8 (s, 1 H, exch. D2O), 6.59 (d, 1H,
J = 2.0 Hz), 5.40–5.31 (m, 8 H), 2.06–2.57 (m, 6 H,),
2.06–2.02 (m, 12 H,), 1.82–1.78 (m, 6 H), 1.48–1.28 (m, 60
H,), 0.92 (t, 9 H, J = 5.2 Hz).13C NMR (CDCl3) d 175.1,
170.5, 170.2, 169.9, 159.9, 155.8, 154.9, 143.8, 143.2,
141.7, 136.4, 129.4, 129.0, 128.9, 128.1, 125.3, 123.1,
122.4, 106.3, 103.9, 100.6, 33.7, 33.4, 33.3, 31.2, 29.1,
29.0, 28.8, 28.6, 28.5, 28.4, 28.3 (2 9 C), 26.5, 26.4, 24.2,
24.1, 21.9, 13.4. ESI–MS: C69H106O10, calc 1095.6, found
m/z = 1094 [M - H]?. Rt = 3.58 min. UV/Vis (CHCl3)
250, 271, 316, 366 nm.
C
105H180O12, calc 1634.5, found m/z (%) = 1635
[M ? H]?. UV/Vis (CHCl3) 261, 298 nm.
2-(3,4-Dioleoylossiphenyl)-4-oxo-4H-chromene-3,5,7-
trioleate (2b). A light yellow liquid, obtained in 60 %
yield. IR (NaCl) cm-1 3005, 2925, 2854, 1770, 1653,
1
1632, 1466, 1434, 1266, 1234, 1205, 1178, 1114, 723. H
NMR(500 MHz, CDCl3) d 7.73 (dd, 1 H, J = 8.5, 2.0 Hz),
7.69 (d, 1 H, J = 2.0 Hz), 7.34 (d, 1 H J = 8.5 Hz), 7.33
(d, 1 H J = 2.2 Hz), 6.86 (d, 1 H, J = 2.2 Hz), 5.4–5.3 (m,
10 H), 2.75 (t, 2 H, J = 7.6), 2.64–2.58 (m, 8 H), 2.05 (m,
20 H), 1.82–1.70 (m, 10 H), 1.48–1.25 (m, 100 H), 0.91 (t,
J = 6.6 Hz, 15 H). 13C NMR d 171.3, 170.1, 170.0, 169.9,
169.8, 169.3, 156.2, 153.6, 152.9, 149.9, 143.8, 141.6,
133.4, 129.4, 129.3, 129.1 (2 9 C), 129.0, 128.9, 127.1,
125.7, 123.1 (2 9 C), 114.1, 113.2, 108.1, 33.7, 33.4
(2 9 C), 33.3, 33.1, 31.3, 29.1 (2 9 C), 29.0(3 9 C), 28.8,
28.7, 28.6 (3 9 C), 28.5 (4 9 C), 28.4 (4 9 C), 28.3, 26.5
(2 9 C), 26.4 (2 9 C), 24.2, 24.1, 24.0, 23.9, 23.7, 22.0
(3 9 C), 13.4. ESI–MS: C105H170O12, calc 1624.5, found
m/z = 1647 [M ? Na]?. Rt = 2.93 min. UV/Vis (CHCl3)
250, 296 nm.
2-(3,4-Dilinoleoylossiphenyl)-4-oxo-4H-chromene-3,5,
7-trilinoleate (2c). A light yellow liquid obtained in 85 %
yield. IR (NaCl) cm-1 3009, 2926, 2855, 1771, 1653,
1
1622, 1464, 1433, 1177, 1115, 724. H NMR (200 MHz,
CDCl3) d 7.71 (dd, 1 H, J = 8.2, 1.8 Hz), 7.57 (d, 1 H,
J = 1.8 Hz), 7.33 (d, 1 H, J = 8.2 Hz,), 7.32 (d,1 H,
J = 2.4 Hz), 6.84 (d, 1 H, J = 2.4 Hz), 5.46–5.26 (m, 20
H), 2.77 (t, 10 H, J = 5.5 Hz), 2.69–2.41 (m, 10 H), 2.10
(m, 20 H), 1.78–1.71 (m, 10 H), 1.36–1.07 (m, 70 H), 0.89
(t, 15 H, J = 6.1 Hz). 13C NMR d 172.2, 171.0, 170.8,
170.7, 170.2, 169.8, 157.1, 154.5, 153.8, 150.81, 144.7,
142.5, 134.3, 130.5-130.4, 130.4, 130.3, 130.3, 130.2,
130.2, 130.1, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1,
128.1, 128.0, 128.0, 126.6, 124.0, 115.0, 114.1, 109.0,
35.5, 34.6, 34.2, 34.1, 34.0, 31.7, 29.9, 29.8,29.6, 29.5,
29.4, 29.2, 29.1, 27.4, 25.9, 25.11, 24.9, 24.8, 24.7, 24.4,
22.8, 13.3. ESI–MS: C105H160O12, calc 1614.2, found m/
z (%) = 1615 [M ? H]?. Rt = 3.92 min. UV/Vis (CHCl3)
281 nm.
2-(3,4-Dioleoylossiphenyl)-4-oxo-4H-chromene-5-hydro
xy-3,7-dioleate (3b). Separation from compound 4b by
column chromatography was performed using a mixture
hexane/acetone 9:1 giving a light yellow liquid in 65 %
yield. IR (NaCl) cm-1 3008, 2922, 2853, 1767, 1651,
1
1605, 1488, 1468, 1297, 1224, 1203, 1187, 1114, 723. H
NMR (500 MHz, CDCl3) d 12.15 (s, 1 H, exch. D2O), 7.75
(dd, 1 H, J = 2.1, 8.5 Hz), 7.73 (d, 1 H, J = 2.1 Hz), 7.37
(d, 1 H, J = 8.5 Hz), 6.87 (d, 1 H, J = 2.0 Hz), 6.61 (d, 1
H, J = 2.0 Hz), 5.41-5.35 (m, 8 H,), 2.65 (t, 2 H, J = 7.6),
2-(3,4-Dilinoleoylossiphenyl)-4-oxo-4H-chromene-5-
hydroxy-3,7-dilinoleate (3c). Separation from com-
pound 4c by column chromatography, eluent hexane/
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J Am Oil Chem Soc (2013) 90:1751–1759
?
acetone 9:1 gave a light yellow liquid in 56 % yield. IR
(NaCl) cm-1 3010, 2927, 2856, 1772, 1655, 1615, 1465,
1434, 1266, 1236, 1196, 1179, 1128, 724. 1H NMR
(200 MHz, CDCl3) d 12.12 (s, 1 H, exch. D2O), 7.72 (dd, 2
H, J = 2.1, 8.2 Hz), 7.34 (d, 1 H, J = 8.2 Hz), 6.84 (d, 1
H, J = 2.1 Hz), 6.58 (d, 1 H, J = 1.8 Hz), 5.45–5.29 (m,
16 H), 2.77 (t, 8 H, J = 5.5 Hz), 2.66–2.53 (m, 8 H), 2.00
(m, 16 H), 1.75–1.56 (m, 8 H), 1.29–1.25 (m, 56H), 0.88 (t,
C105H150O12, calc 1604.3, found m/z = 1605 [M ? H]
Rt = 3.54 min. UV/Vis (CHCl3) 260, 284 nm.
.
2-(3,4-Dilinolenylossiphenyl)-4-oxo-4H-chromene-5-
hydroxy-3,7-dilinolenate (3d). Separation from compound
4d by column chromatography, eluent hexane/acetone 9:1
gave a light yellow liquid in 48 % yield. IR (NaCl) cm-1
3011, 2960, 2929, 2855, 1772, 1655, 1615, 1488, 1455,
1266, 1236, 1197, 1127, 722. 1H NMR (200 MHz, CDCl3)
d 12.12 (s, 1 H, exch. D2O), 7.73 (dd, 2 H, J = 2.1,
8.2 Hz), 7.34 (d, 1 H, J = 8.2 Hz), 6.84 (d, 1 H,
J = 2.1 Hz), 6.58 (d, 1 H, J = 2.1 Hz), 5.46–5.24 (m, 24
H), 2.81 (t, 16 H, J = 5.5 Hz), 2.66–2.52 (m, 8 H),
2.15–2.00 (m, 16 H), 1.78–1.56 (m, 8 H), 1.27–1.21 (m, 32
H), 0.94 (t, 12 H, J = 7.6 Hz). 13C NMR (CDCl3) d 176.5,
171.3, 170.9, 170.8, 170.7, 161.9, 156.7, 156.2, 155.7,
145.0, 142.6, 132.5, 132.2, 130,5, 130.4, 130.3, 130.2,
130.1, 128.6, 128.5, 128.5, 128.4, 128.23, 128.1, 128.0,
127.9, 127.7, 127.3, 126.7, 124.2, 108.9, 105.7, 101.4,
34.6, 34.3, 34.3, 33.9, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2,
12 H, J = 6.8 Hz). 13C NMR (CDCl3)
d 176.5,
171.3,170.8, 170.8, 170.7, 161.9, 156.7, 156.2, 155.7,
145.0, 142.6, 132.5, 130.5, 130.4, 130.3, 130.2, 130.1,
128.4, 128.3, 128.2, 128.1, 128.1, 127.7, 126.7, 124.2,
108.9, 105.7, 101.4, 34.6, 34.3, 34.3, 33.9, 31.7, 29.8, 29.6,
29.5, 29.4, 29.3, 29.2, 27.4, 25.9, 25.1, 24.9, 24.8, 22.8,
14.3. ESI–MS: C87H130O11, calc 1351.9, found m/z = 1374
[M ? Na] ?. Rt = 2.57 min. UV/Vis (CHCl3) 273 nm.
2-(3,4-Dilinoleoylossiphenyl)-4-oxo-4H-chromene-3,5-
dihydroxy-7-linoleate (4c). Separation from compound 3c
by column chromatography, eluent hexane/acetone 9:1
gave a light yellow liquid in 20 % yield. IR (NaCl) cm-1
3351, 3009, 3005, 2927, 2856, 1771, 1652, 1606, 1492,
27.4, 25.9, 25.8, 25.1, 24.9, 24.8, 22.8, 14.5, 14.5. ESI–MS:
?
C87H122O11, calc 1343.9, found: m/z 1346 [M ? H]
Rt = 2.46 min. UV/Vis (CHCl3) 269, 301, 340 nm.
.
1
1466, 1307, 1268, 1194, 1133, 724. H NMR (200 MHz,
2-(3,4-Dilinolenylossiphenyl)-4-oxo-4H-chromene-
3,5-dihydroxy-7-linolenate (4d). Separation from com-
pound 3d by column chromatography, eluent hexane/ace-
tone 9:1 gave a yellow liquid in 15 % yield. IR
(NaCl) cm-1 3369, 3011, 2960, 2928, 2855, 1771, 1652,
1606, 1491, 1464, 1194, 1128, 723. NMR (200 MHz,
CDCl3) d 11.64 (s, 1 H, exch. D2O), 8.12 (dd, 1 H, J = 8.5,
1.8 Hz’), 8.05 (d, 1 H, J = 1.8 Hz), 7.36 (d, 1 H,
J = 8.5 Hz), 6.89 (d, 1 H, J = 1.8 Hz), 6.84–6.69 (s, 1 H,
exch. D2O), 6.57 (d, 1H, J = 1.8 Hz), 5.46–5.31 (m, 18 H),
2.81 (t, 12 H, J = 5.5 Hz), 2.62–2.53 (m, 6 H), 2.15–2.01
(m, 12 H), 1.76–1.56 (m, 6 H), 1.26–1.17 (m, 24 H), 0.98
(t, 9 H, J = 7.6 Hz).13C NMR (CDCl3) d 176.1, 171.4,
171.1, 170.9, 160.9, 156.7, 155.9, 144.7, 144.1, 142.6,
137.3, 132.2, 130,4, 129.0, 128.5, 128.4, 128.1, 127.3,
126.2, 124.0, 123.3, 107.2, 104.8, 101.5, 34.6, 34.3, 34.2,
29.8, 29.6, 29.4, 29.3, 27.4, 25.9, 25.8, 25.1, 25.0, 20.9,
14.5. ESI–MS: C69H94O10, calc 1083.5, found m/z = 1085
[M ? H]?. Rt = 3.92 min. UV/Vis (CHCl3) 270, 313,
362 nm.
CDCl3) d 11.64 (s, 1 H, exch. D2O), 8.12 (dd, 1 H, J = 8.5,
2.1 Hz,), 8.05 (d, 1 H, J = 2.1 Hz), 7.36 (d, 1 H,
J = 8.5 Hz), 6.88 (d, 1 H, J = 2.1 Hz), 6.70 (s, 1 H, exch.
D2O), 6.57 (d, 1 H, J = 2.1 Hz), 5.45–5.26 (m, 12 H), 2.77
(t, 6 H, J = 5.5 Hz), 2.66–2.53 (m, 6 H), 2.07–2.00 (m, 12
H), 1.75–1.59 (m, 6 H,), 1.30–1.21 (m, 42 H), 0.88 (t, 9 H,
J = 6.7 Hz). 13C NMR (CDCl3) d 176.5, 171.4, 171.1,
170.9, 160.9, 156.7, 155.9, 144.7, 144.1, 142.6, 137.4,
130.5, 130.3, 130.2, 130.1, 129.0, 128.4, 128.3, 128.1,
126.8, 124.0, 123.3, 107.2, 104.8, 101.5, 34.6, 34.3, 34.2,
31.7, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2, 27.4, 25.9, 25.2,
25.0, 22.8, 14.3. ESI–MS: C69H100O10, calc 1088.7, found
?
m/z = 1090 [M ? H]
.
Rt = 4.13 min. UV/Vis
(CHCl3) = 269, 311, 359 nm.
2-(3,4-Dilinolenylossiphenyl)-4-oxo-4H-chromene-3,5,7-
trilinolenate (2d). A light yellow liquid obtained in 74 %
yield. IR (NaCl) cm-1 3011, 2960, 2929, 2855, 1772,
1655, 1621, 1463, 1434, 1177.68, 1123, 758, 723. 1H NMR
(200 MHz, CDCl3) d 7.70 (dd, 1 H, J = 8.5, 1.8 Hz), 7.65
(d, 1 H, J = 1.8 Hz), 7.32 (d, 1 H, J = 8.5 Hz), 7.31 (d, 1
H, J = 2.1 Hz), 6.84 (d, 1 H, J = 2.1 Hz), 5.58–5.24 (m,
30 H), 2.84 (t, 20 H, J = 5.8 Hz), 2.65–2.55 (m, 10 H),
2.26–2.00 (m, 20 H), 1.77–1.59 (m, 10 H), 1.36–1.21 (m,
40 H), 0.94 (t, 15 H, J = 7.6 Hz). 13C NMR (CDCl3) d
172.2, 171.0, 170.8, 170.7, 170.1, 157.1, 154.5, 153.9,
150.8, 144.7, 142.5, 134.3, 130.5, 130.4, 130.3, 130.2,
130.1, 128.5, 128.4, 128.4, 128.2, 128.1, 127.9, 127.3,
126.6,124.1, 115.0, 114.1, 109.1, 35.5, 34.6, 34.3, 34.3,
33.9, 29.8, 29.7, 29.6, 29.5, 29.3, 29.4, 29.1, 27.4, 25.1,
24.9, 24.8, 24.7, 24.4, 22.8, 20.8, 14.5, 14.5. ESI–MS:
Synthesis of 2-(3,4-Distearolylossiphenyl)-4-oxo-4H-
chromene-5-hydroxy-3,7-distearate (3a)
Under nitrogen flow, stearoyl chloride (1.32 9 10-3 mol,
0.45 ml) was added dropwise to a magnetically stirred ice
cold solution of quercetin (0.3 mmol, 0.10 g) and TEA
(2.47 mmol, 0.35 ml) in anhydrous dioxane (10 ml). The
solution was magnetically stirred at 5 ꢁC for 3 h and then
evaporated to dryness under vacuum (107 mbar, 40 ꢁC).
The obtained residue was dissolved in chloroform (20 ml)
123

J Am Oil Chem Soc (2013) 90:1751–1759
1755
and washed with a saturated aqueous NaHCO3 solution
(20 ml) and then with water (3 9 20 ml). The organic
layer was dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure, leaving a light yellow
solid in 53 % yield. IR (KBr) cm-1 3436, 2957, 2918,
2850, 1766, 1655, 1616, 1468, 1438, 1266, 1234, 1195,
provided the best results in terms of accuracy/computation
time [26, 27]. The absolute shieldings of penta, tetra and
triesters were calculated using the GIAO method [28, 29].
All calculations were performed in the gas phase. As in
previous works the inclusion of the solvent provided no
improvements over the general quality of results, but
resulted in severely more expensive computation time [26].
1
1178, 1145. H NMR (200 MHz, CDCl3) d 12.12 (s, 1 H,
exch. D2O), 7.72 (dd, 2 H, J = 2.1, 8.4 Hz), 7.35 (d, 1 H,
J = 8.4 Hz), 6.84 (d, 1 H, J = 2.1 Hz), 6.58 (d, 1 H,
J = 2.1 Hz), 2.67–2.52 (m, 8 H), 1.75–1.71 (m, 8 H),
1.26–1.05 (m, 112 H), 0.88 (t, 12 H, J = 6.1 Hz). 13C
NMR (CDCl3) d 176.5, 171.3, 170.9, 170.8, 170.2, 161.9,
156.7, 156.2, 155.7, 145.0, 142.6, 132.5, 127.7, 126.6,
124.2, 108.9, 105.7, 101.3, 34.6, 34.3, 34.2, 33.9, 32.1,
29.9, 29.74 29.6, 29.5, 29.4, 29.4, 29.3, 25.1, 24.9, 24.7,
22.9, 14.3. ESI–MS: C87H146O11, calc 1368.1, found
m/z = 1369 [M ? H]?. UV/Vis (CHCl3) 260, 300 nm.
Antioxidant Activity
The Trolox equivalent antioxidant capacity (TEAC) mea-
sures the ability of a compound to scavenge the ABTS
(2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as
radical cation, ABTS•1, a soluble chromogen that can be
determined spectrophotometrically at 734 nm.
In the presence of antioxidants the radical cation is
suppressed to an extent dependent on the activity of the
antioxidant and the color intensity is decreased
proportionally.
Theoretical Calculations
The assay is performed with the Antioxidant Assay Kit,
as suggested by the producer. Trolox, a water-soluble
vitamin E analogue, serves as a standard.
Results are expressed as TEAC g-1 of dry powder
(TEAC, lmol g-1) and are the means SD of three dif-
ferent experiments performed in triple.
DFT calculations were performed using the Gaussian 09
package [20]. Starting geometries were initially obtained
through a conformational search conducted in vacuo using
the Low Mode molecular dynamics method and the
MMFF949 force field implemented in MOE [21]. The
eight most stable conformers, included in an energy range
of 2 kcal mol-1, were completely optimized in vacuo at
the mPW1B95/6-31?G (d,p) level [22], a level of theory
that provided excellent results in previous works from our
group [23–25]. Moreover the frequencies were calculated
at the same level to confirm the minimized structures as a
minimum (no imaginary frequencies). NMR computations
were performed on the most stable conformations at the
mPW1B95/TZVP level. As in other works this basis set
The DPPH assay utilizes the stable DPPH free radical
that produces a purple color with strong absorbance at
517 nm [31]. When DPPH is placed in an assay system
containing free radical scavengers, the color vanishes:
therefore the change in absorbance is a measurement of
antioxidant scavenging capacity of test samples.
Results are expressed as the sample concentration that
quenches the 50 % of DPPH radical (IC50, lg ml-1), cal-
culated by linear regression, plotting together data obtained
OH
OH
COOH
b
HO
+
O
O
toluene
+
-
R COCl
COCl2
COOH
COOH
c
OH
-
1 a d
COCl
:
1a
OH
d
TEA
Dioxane
O
O
R
R
O
O
O
O
R
O
O
O
R"
O
R'
O
2 a-d: R=R'=R"
O
3 a-d: R=R" R'=H
4b-d: R'=R"=H
Scheme 1 Synthesis of total and partial quercetin esters with stearoyl chloride (a), oleic (b), linoleic (c) and linolenic (d) acids
123

1756
J Am Oil Chem Soc (2013) 90:1751–1759
by at least two different experiments performed in
hydrolyzed –OCO group is in the five position. This
hypothesis, in agreement with the literature data [31] for
quercetin tetracetyl esters, was confirmed by HMCQ and
HSQC analyses.
duplicate
Statistical analysis was performed between the TEAC
activity of the different derivatives by unpaired Student’s
t test.
The identification of free hydroxyl groups in the tri-
oleoyl ester 4b led to ambiguous results. The H6, H8 and
4-C shifts confirmed that the OH on 5-C was free, while the
0
0
0
Results and Discussion
comparison between the H6 , H2 , and H5 shifts in penta
and tetraester (7.73, 7.69 and 7.32 ppm) with that in 4b 1H
spectrum (8.14, 8.08 and 7.38 ppm) allow us to hypothe-
size that the second free –OH group could be in either the
30 or 40 position; not even 13C- and 2D-NMR techniques
were able to distinguish between them.
Synthesis of Quercetin Fatty Esters
As reported in Scheme 1 the quercetin pentaesters were
easily synthesized following the well-established esterifi-
cation procedure by the reaction between quercetin and the
proper acyl chloride, obtained from the corresponding
carboxylic acid with an excess of oxalyl chloride in
anhydrous toluene. Furthermore, stearoyl pentaester was
synthetized from stearoyl chloride commercially available.
As summarized in Table 1, quercetin and unsaturated
acyl chloride molar ratio modulation allowed us to obtain a
mixture of tetra and triesters which were then purified by
column chromatography. As reported in the literature [31]
for quercetin pivaloyl esters, the tetrastearoyl derivative
was obtained modulating the reaction temperature at 0 ꢁC
instead of room temperature and not in the reagents’ molar
ratio.
Quantum chemical NMR calculation were then per-
formed on 3,5,7,30,40-pentaester, 3,7,30,40-tetraester, 3,7,30
triester, 3,7,40-triester and 7,3,40-triester to assist the exper-
imental structural assignment. Indeed, this technique was
successfully used by our group to solve similar questions [26,
27]. Calculations on long and highly flexible fatty acid
quercetin esters could not be performed, as the huge con-
formational flexibility would have made both of the calcu-
lations too expensive, in terms of CPU time, as well as the
interpretation of results more difficult, due to the large noise
introduced by the side-chain signals. Concerned that dis-
criminating signals for the mentioned esters are principally
those of the quercetin nucleus, for the above reported reason,
we adopted the acetyl esters as a model for NMR calcula-
tions. Beside the triesters whose structure was equivocal, the
penta and tetraacetyl esters were also included in the study to
test the general reliability of the method and to eventually
rule out systematical errors. A conformational analysis (see
‘‘Experimental’’ for details) was performed and the eight
most stable conformation were selected for further geometry
optimization by density functional theory (DFT) at the
mPW1B95/6-31?G(d,p) level of theory [22]. Preliminary
NMR calculations were conducted on tetra and pentaesters
by computing 13C absolute shieldings for the most stable
conformation, as resulted from DFT calculation, or for
selected conformations (from 2 to 8), where average 13C
absolute shieldings were obtained. Theoretical results were
then compared to the experimental results, although no sig-
nificant variation in the computed NMR values was observed
within the ‘‘multi conformation’’ or the ‘‘single conforma-
tion’’ methods, so the less computationally expensive latter
one was chosen for our analysis.
Molecular Characterization
In FT-IR spectra, the C=O stretching of the carboxylic
acids, acyl chlorides and quercetin fatty esters allowed us
to verify the esterification; in fact, this band shifted from
1,710 to 1,810 to 1,770 cm-1 respectively. In addiction in
all the synthetized compounds at about 1,654 cm-1 is
shown the quercetin C=O stretching. Quercetin shows
intense absorption broad bands between 3,445 and
3,000 cm-1 due to O–H stretching, which disappear in the
pentaesters; while in the tetraester and mainly in the tri-
ester, a broad band between 3,445 and 3,000 cm-1 is
evident.
The full mass spectra of compounds 3a–d and 4a–d
showed a molecular ion peak in agreement with the tetra
and the triester respectively.
In order to identify the unesterified –OH groups, quer-
cetin pentaoleyl ester 2b, tetraoleyl ester 3b and trioleyl
ester 4b were analyzed by bi-dimensional NMR tech-
Both 13C absolute shieldings sets calculated for
3,5,7,30,40-pentaacetyl quercetin and 3,7,30,40-tetraacetyl
quercetin were compared with the corresponding experi-
mental chemical shifts through a linear regression analysis.
As shown in Fig. 1, the calculated regression coefficient
(R2) is quite good (0.9925), proving that the chosen com-
putational method performs well in predicting NMR values
for the systems reported herein.
1
niques, such as COSY, HMBC and HSQC. The H-NMR
shifts of the tetraoleyl ester 3b showed for H6 (6.61 ppm)
and H8 (6.87 ppm) a downfield shift compared with that of
the corresponding pentaester (6.86 and 7.31 ppm respec-
tively); furthermore, the 13C-NMR analysis showed that
4-C (174.60 ppm) had a higher frequency in comparison
with that in the pentaester (169.25 ppm) suggesting that the
123

J Am Oil Chem Soc (2013) 90:1751–1759
1757
Fig. 1 Correlation between
experimental 13C chemical
shifts (ppm) and absolute
shieldings calculated at the
mPW1B95/TZVP//mPW1B95/
6-31?G(d,p) level for
3,5,7,30,40-pentaacetyl quercetin
and 3,7,30,40-tetraacetyl
quercetin
Fig. 2 Comparison between
experimental 13C chemical
shifts (ppm) of 4b and
theoretical 13C chemical shifts
computed for the hypothetical
quercetin triesters (a), (b) and
(c). Correlation coefficients (R2)
between experimental and
theoretical chemical shifts are
also reported
The linear equation obtained was then used to convert the
absolute shieldings, computed for the hypothesized triesters
3,7,40-triacetyl quercetin (a), 3,7,30-triacetyl quercetin
(b) and 7,30,40-triacetyl quercetin (c), into chemical shifts
directly comparable with those experimentally obtained. As
can be clearly observed in Fig. 2, the only theoretical set that
fitted perfectly with experimental 13C-NMR chemical shifts
is the one obtained from the triester (c) (R2 = 0.9940).
Indeed, compounds (a) and (b) (R2 = 0.9767 and 0.9632,
respectively) could be safely ruled out due to the severely
discordant values computed for 60-C, 10-C, 30-C, 40-C and
most of all 2-C. Indeed, the chemical shift for this last carbon
atom is particularly low if compared to the corresponding
values computed for (a) and (b), suggesting that 2-C is more
severely shielded by the –OCO-hydrolysis at 3-C than that at
either 30-C or 40-C.
The purity of all the synthetized compounds was eval-
uated by HPLC/DAD analysis.
Antioxidant Activity
In order to evaluate the antioxidant power of different
quercetin fatty polyesters, we performed two common and
reliable in-vitro assays, i.e. ABTS, expressed as TEAC, and
the stable DPPH radical, expressed as IC50. Results are
reported in Table 3; with both assays the quercetin esters
display a lower antioxidant activity respect to free quer-
cetin; in fact they display a high IC50 and a low TEAC both
indicating a lower antioxidant capacity.
On the other hand, quercetin triesters with oleic, linoleic
and linolenic fatty acids displayed a higher antioxidant
activity with respect to their corresponding pentaester
compounds. The trilinolenoyl quercetin esters display the
highest antioxidant power even if still 20 times lower than
In Table 2 the 1H and 13C chemical shifts assignment for
quercetin oleic esters are summarized.
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J Am Oil Chem Soc (2013) 90:1751–1759
Table 2 Chemical shifts (d) of the aromatic protons and carbons of pentaoleoyl quercetin (2b), 3,30,40,7-tetraoleoyl quercetin (3b) and 30,40,7
trioleoyl quercetin (4b) in CDCl3
OR
3'
OR
2'
4'
5'
8
RO
O
9
2
1'
7
6
6'
4
10
3
OR (H)
5
O
OR (H)
Compound
d (H-6)
d (H-8)
d (H-20)
d (H-50)
d (H-60)
2b
3b
4b
6.86
6.61
6.59
7.31
6.87
6.90
7.69
7.73
8.08
7.32
7.37
7.38
7.73
7.75
8.14
Compound
d (4-C)
d (3-C)
d (2-C)
d (10-C)
d (30-C)
d (40-C)
2b
3b
4b
169.25
175.60
175.15
133.42
131.57
136.39
152.9
127.09
126.78
128.09
141.61
141.67
141.69
143.79
144.09
143.22
154.83
143.79
acidic and reactive due to intramolecular H-bonding with
4-C carbonyl; whereas the triesters, in which also OH at
3-C is free, showed a better antioxidant activity.
Table 3 Antioxidant capacity of the different fatty esters
Compounds
IC50 (lg ml-1
)
TEAC mean SD (mg g-1
)
Quercetin
2a
4.72
1101.90
990.20
2693.00
2101.6
808.00
1853.70
1071.70
224.42
607.26
480.27
107.26
1930.6 127.5
4.64 0.84
1.82 0.08*
1.26 0.30
0.7 0.28
3a
Conclusion
2b
3b
In conclusion, following a well-known procedure, we
successfully prepared a series of new quercetin fatty pen-
taesters and a series of esters bearing a free OH group on
5-C or on 5-C and 3-C. Quantum chemical calculations
allowed the unequivocal assignment of the free position in
triesters through the comparison of theoretically predicted
and experimental chemical shifts. The antioxidant activity
of the new compounds was also evaluated, highlighting the
relevance of the free OH on 3-C for biological activity.
4b
1.37 0.10
0.55 0.17
1.56 0.15b
1.26 0.40a
1.33 0.21
1.67 0.60
2.92 0.16c
2c
3c
4c
2d
3d
4d
n = 3
* p \ 0.05 vs 2A
a
p \ 0.05
b
p \ 0.01 vs 2C
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