Influence of side chain structure changes on antioxidant potency of the [6]-gingerol related compounds

Article abstract of DOI:10.1016/j.foodchem.2014.05.077

[6]-Gingerol and [6]-shogaol are the major pungent components in ginger with a variety of biological activities including antioxidant activity. To explore their structure determinants for antioxidant activity, we synthesized eight compounds differentiated by their side chains which are characteristic of the C₁-C₂ double bond, the C₄-C₅ double bond or the 5-OH, and the six- or twelve-carbon unbranched alkyl chain. Our results show that their antioxidant activity depends significantly on the side chain structure, the reaction mediums and substrates. Noticeably, existence of the 5-OH decreases their formal hydrogen-transfer and electron-donating abilities, but increases their DNA damage- and lipid peroxidation-protecting abilities. Additionally, despite significantly reducing their DNA strand breakage-inhibiting activity, extension of the chain length from six to twelve carbons enhances their anti-haemolysis activity.

Full text of DOI:10.1016/j.foodchem.2014.05.077

Food Chemistry 165 (2014) 191–197
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Influence of side chain structure changes on antioxidant potency
of the [6]-gingerol related compounds
⇑
Dong-Liang Lu, Xiu-Zhuang Li, Fang Dai , Yan-Fei Kang, Yan Li, Meng-Meng Ma, Xiao-Rong Ren,
Gao-Wei Du, Xiao-Ling Jin, Bo Zhou
⇑
State Key Laboratory of Applied Organic Chemistry, 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:
[6]-Gingerol and [6]-shogaol are the major pungent components in ginger with a variety of biological
activities including antioxidant activity. To explore their structure determinants for antioxidant activity,
we synthesized eight compounds differentiated by their side chains which are characteristic of the C1–C2
double bond, the C4–C5 double bond or the 5-OH, and the six- or twelve-carbon unbranched alkyl chain.
Our results show that their antioxidant activity depends significantly on the side chain structure, the
reaction mediums and substrates. Noticeably, existence of the 5-OH decreases their formal hydrogen-
transfer and electron-donating abilities, but increases their DNA damage- and lipid peroxidation-protect-
ing abilities. Additionally, despite significantly reducing their DNA strand breakage-inhibiting activity,
extension of the chain length from six to twelve carbons enhances their anti-haemolysis activity.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 25 January 2014
Received in revised form 15 April 2014
Accepted 14 May 2014
Available online 22 May 2014
Keywords:
Gingerol
Shogaol
Antioxidant
DNA damage
Lipid peroxidation
1. Introduction
keto moiety, and the latter possesses a,b-unsaturated carbonyl
moiety. Such a slight difference significantly distinguishes their
biological activities, as reported by numerous studies showing that
[6]-shogaol is more effective than [6]-gingerol in antioxidant
(Dugasani et al., 2010), anti-inflammatory (Dugasani et al., 2010;
Pan, Hsieh, Hsu et al., 2008; Sang et al., 2009) and anti-tumorigenic
assays (Wu et al., 2010) as well as in inhibiting proliferation (Gan
et al., 2011; Pan, Hsieh, Kuo et al., 2008; Sang et al., 2009), inducing
apopotosis (Pan, Hsieh, Kuo et al., 2008) and restraining invasion of
cancer cells (Weng, Chou, Ho, & Yen, 2012; Weng, Wu, Huang, Ho,
& Yen, 2010). Additionally, [6]-shogaol can act as a Michael accep-
tor to react with sulphydryl groups of cysteine residues in tubulin,
and hence impair tubulin polymerisation, whereas [6]-gingerol is
inactive (Ishiguro, Ando, Watanabe, & Goto, 2008). The above
observations highlight the importance of the side chain structure
for biological activities of the gingerol related compounds.
Because oxidative stress imposed by reactive oxygen species
(ROS) or free radicals plays an important role in the development
of many chronic diseases including cancer (Hussain, Hofseth, &
Harris, 2003; Reuter, Gupta, Chaturvedi, & Aggarwal, 2010), antiox-
idant activity of the gingerol related compounds has also attracted
attention (Dugasani et al., 2010; Kikuzaki & Nakatani, 1993; Li
et al., 2012; Masuda, Kikuzaki, Hisamoto, & Nakatani, 2004; Yeh
et al., 2014). Interestingly, Halvorsen et al. (2006) observed that
ginger presented the highest antioxidant activity among the foods
tested based on the ferric reducing antioxidant power assay. Anti-
oxidant activity of gingerol related compounds should be mediated
Ginger (Zingiber officinale Roscoe), one of the most popular
spices, is widely used in various foods and beverages, and has a
long history of medicinal usages especially in China and India
(Baliga et al., 2011; Shukla & Singh, 2007). Numerous studies have
shown that ginger and its extracts possess a variety of biological
and pharmacological activities, including cancer preventive and
antioxidative effects (Baliga et al., 2011; Gundala et al., 2014;
Shukla & Singh, 2007). The active and major pungent components
of ginger consist of gingerols and shogaols, which are a series of
chemical homologs with varying alkyl side chain lengths (Baliga
et al., 2011; Jolad, Lantz, Chen, Bates, & Timmermann, 2005).
Generally, gingerols are thermally labile owing to the presence of
b-hydroxy keto moiety and readily undergo dehydration to yield
the corresponding shogaols, resulting in the increased concentra-
tions of shogaols in dry ginger compared with fresh ginger
(Baliga et al., 2011; Jolad et al., 2005).
Of all the gingerols and shogaols, [6]-gingerol and [6]-shogaol
(Fig. 1) bearing an unbranched alkyl chain of six carbon atoms
are the most representative (Baliga et al., 2011; Jolad et al.,
2005). [6]-Gingerol and [6]-shogaol share the same vanillyl moiety
and have very similar chemical structures. Their structural differ-
ences exist only in the side chains, the former contains b-hydroxy
⇑
Corresponding authors. Fax: +86 931 8915557.
E-mail addresses: daifang@lzu.edu.cn (F. Dai), bozhou@lzu.edu.cn (B. Zhou).
http://dx.doi.org/10.1016/j.foodchem.2014.05.077
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

192
D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
Fig. 1. Molecular structures of [6]-gingerol, [6]-shogaol and their derivatives.
by their o-methoxyphenolic moiety. It has been proved that hydro-
gen atom abstraction is surprisingly easy from intramolecularly
hydrogen bonded methoxyphenols, in contrast to intermolecularly
hydrogen bonded molecules (de Heer, Mulder, Korth, Ingold, &
Lusztyk, 2000). Besides, the small solvent kinetic effect of o-meth-
oxyphenols renders them good antioxidants, even in a polar envi-
ronment (de Heer et al., 2000). It has been firmly established that
in addition to its innately chemical reactivity towards radicals, the
effectiveness of an antioxidant in biological system is also affected
by its localisation, concentration, and mobility at the microenvi-
ronment, because the environment of biological system is quite
heterogeneous (Niki & Noguchi, 2004). Therefore, we believe that
antioxidant potency of the gingerol related compounds depends
on not only their o-methoxyphenolic moiety but also functional
groups, and the number of carbons in their side chains. Although
a few studies has been conducted to investigate antioxidant activ-
ity of gingerol related compounds (Dugasani et al., 2010; Kikuzaki
et al., 1993; Li et al., 2012; Masuda et al., 2004; Peng et al., 2012;
Yeh et al., 2014), the literature regarding the influence of side chain
structure on antioxidant potency of them are still limited. Thus, as
a part of our ongoing research project on bioantioxidants (Cao
et al., 2012; Li et al., 2014; Qian et al., 2011; Shang et al., 2009,
2010; Tang et al., 2011; Zhou, Miao, Yang, & Liu 2005), we synthe-
sized eight gingerol related compounds (Fig. 1) including [6]- and
[12]-gingerol, shogaol, dehydrogingerol and dehydroshogaol, and
used them to probe how the side chain structure, such as the
C1–C2 double bond, the C4–C5 double bond or the 5-OH, and the
unbranched alkyl chain length, affect their antioxidant potency.
Their antioxidant potency were characterised by four experimen-
tal models including the 2,2-diphenyl-1-picrylhydrazyl free radi-
cal (DPPHÅ)-scavenging, ferric reducing antioxidant power (FRAP),
DNA strand breakage-inhibiting and human red blood cell
haemolysis-protecting assays which are indicative of their formal
hydrogen-transfer, electron-donating, DNA damage and lipid
peroxidation-protecting abilities, respectively.
Esquire 6000 spectrometer (ESI-MS) or a VG ZAB-HS spectrometer
(EI-MS).
2.2. Materials
2,2-Diphenyl-1-picrylhydrazyl free radical (DPPHÅ), pBR322
DNA and 2,20-azobis (2-amidinopropane hydrochloride) (AAPH)
were purchased from Sigma-Aldrich. 2,4,6-Tri(2-pyridyl)-s-triazine
(TPTZ) was from Alfa Aesar. Other chemicals used were of analyt-
ical grade. All anhydrous solvents were dried and purified by
standard techniques.
2.3. Synthesis of (E)-4-(40-hydroxy-30-methoxyphenyl)-3-buten-2-one
(E)-4-(40-Hydroxy-30-methoxyphenyl)-3-buten-2-one was syn-
thesized
by
following
previous
published
procedures
(Ramachandra & Subbaraju, 2006). 25 ml 20% aq. sodium hydrox-
ide was added to the acetone (30 ml) solution of vanillin (5.0 g,
32.89 mmol) and then this was stirred overnight at room temper-
ature. The reaction mixture was diluted with ice water and 35 ml
conc. HCl was added, a brown precipitate formed which was fil-
tered, washed with cold water and dried to obtain (E)-4-(40-
hydroxy-30-methoxyphenyl)-3-buten-2-one.
2.4. General procedure for the synthesis of dehydroshogaols and
dehydrogingerols
Dehydroshogaols and dehydrogingerols were synthesized based
on the published procedures with some modifications (Fleming,
Dyer, & Eggington, 1999). 1.0 equivalent of (E)-4-(40-hydroxy-30-
methoxyphenyl)-3-buten-2-one in dry THF (0.7 M) was added to
a flame-dried flask under argon, and cooled to ꢀ78 °C. 1.0 equiva-
lent of n-BuLi in hexane (1.60 M) was then added. After the mix-
ture was stirred for 30 min, LDA prepared with 1.3 equivalent of
diisopropyl amine and 1.0 equivalent of n-BuLi in 0.7 M THF at
ꢀ78 °C was sequentially added dropwise. The reaction was stirred
at ꢀ78 °C for 3 h, 1.1 equivalent of the appropriate aldehyde
(hexanal or dodecanal) in THF (0.7 M) was then introduced into
the reaction system. After stirring for another 3 h at the same tem-
perature, the mixture was warmed to 0 °C for 1 h, then quenched
with 10% HCl and extracted using ether. The combined organic
layers were dried over sodium sulphate and concentrated. Silica
gel column purification of the crude product provided dehydrogin-
gerols or dehydroshogaols, respectively.
2. Materials and methods
2.1. General experimental procedures
1H NMR spectra and 13C NMR were recorded by a Bruker AV 400
(400 MHz) spectrometer with CDCl3 or CD3COCD3 as a solvent.
Chemical shifts (d) are reported in parts per million (ppm) using
the solvent peak. Mass spectra were recorded on a Bruker Daltonics

D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
193
2.4.1. (E)-5-Hydroxy-1-(40-hydroxy-30-methoxyphenyl)-1-decen-3-
one ([6]-dehydrogingerol)
145.7, 133.7, 121.5, 115.7, 112.8, 68.4, 56.3, 51.0, 46.0, 38.2, 32.7,
26.0, 23.4, 14.4; HRMS (ESI): m/z calcd. for C17H26O4 (MꢀH)ꢀ:
293.1747; found: 293.1751, error = 1.4 ppm.
Yield: 44.5%. 1H NMR (400 MHz, (CD3)2CO) d 8.18 (s, 1H), 7.57
(d, J = 16.0 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 7.18 (dd, J = 8.0,
1.6 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 16.0 Hz, 1H), 4.11–
4.04 (m, 1H), 3.91 (s, 3H), 3.72 (d, J = 4.4 Hz, 1H), 2.78–2.76
(m, 2H), 1.48–1.44 (m, 3H), 1.32–1.30 (m, 5H) 0.90–0.87 (m, 3H);
13C NMR (100 MHz, (CD3)2CO) d 200.2, 150.3, 148.9, 143.9, 127.8,
125.3, 124.3, 116.3, 111.6, 68.7, 56.4, 48.5, 38.2, 32.7, 26.1, 23.4,
14.4; HRMS (ESI): m/z calcd. for C17H24O4 (M+H)+: 293.1747;
found: 293.1744, error = 1.0 ppm.
2.5.2. 5-Hydroxy-1-(40-hydroxy-30-methoxyphenyl)hexadecan-3-one
([12]-gingerol)
Yield: 64.0%. 1H NMR (400 MHz, (CD3)2CO) d 7.29 (s, 1H), 6.82
(d, J = 2.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.64 (dd, J = 8.0,
2.0 Hz, 1H), 4.04–3.99 (m, 1H), 3.81 (s, 3H), 3.65 (d, J = 4.8 Hz,
1H), 2.79–2.74 (m, 4H), 2.53–2.52 (m, 2H), 1.42–1.38 (m, 3H),
1.29 (s, 17H), 0.89-0.86 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
(CD3)2CO) d 210.2, 148.3, 145.7, 133.8, 121.5, 115.7, 112.9, 68.4,
56.3, 51.0, 46.0, 38.3, 32.7, 30.5, 30.2, 26.4, 23.4, 14.4; HRMS
2.4.2. (E)-5-Hydroxy-1-(40-hydroxy-30-methoxyphenyl)-1-hexadecen-
3-one ([12]-dehydrogingerol)
(ESI): m/z calcd. for
396.3112, error = 1.0 ppm.
C
23H38O4 (M+NH4)+: 396.3108; found:
Yield: 37.5%. 1H NMR (400 MHz, (CD3)2CO) d 8.20 (s, 1H), 7.57
(d, J = 16.0 Hz, 1H), 7.34 (s, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.88 (d,
J = 8.4 Hz, 1H), 6.73 (d, J = 16.0 Hz, 1H), 4.12–4.05 (m, 1H), 3.91
(s, 3H), 3.75 (d, J = 4.8 Hz, 1H), 2.78-2.76 (d, 2H), 1.48-1.45
(m, 2H), 1.28 (brs, 18H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, (CD3)2CO) d 200.3, 150.3, 148.8, 144.0, 127.8, 125.2,
124.3, 116.2, 111.6, 68.7, 56.4, 48.5, 38.2, 32.7, 30.5 (3C), 30.2,
2.6. General procedure for the synthesis of shogaols
Shogaols were synthesized by following previously published
procedures (Fleming et al., 1999). An equal volume of 10% HCl
was added to the THF solution (20 ml) of [6]-gingerol ([12]-ginger-
ol) and the mixture was heated for 4 h. The cooled mixture was
extracted with ether and washed with water. The combined
organic parts were washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to give a crude oil. Purification
with column chromatography on silica gel gave shogaols.
29.4, 26.4, 23.4, 14.5; HRMS (ESI): m/z calcd. for
C23H36O4
(M+H)+: 377.2686; found: 377.2684, error = 1.1 ppm.
2.4.3. (1E,4E)-1-(40-Hydroxy-30-methoxyphenyl)-1,4-decadien-3-one
([6]-dehydroshogaol)
Yield: 17.5%. 1H NMR (400 MHz, CDCl3) d 7.59 (d, J = 16.0 Hz,
1H), 7.14 (dd, J = 8.4, 2.0 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 7.00 (dt,
J = 15.6, 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 16.0 Hz,
1H), 6.44 (dt, J = 15.6, 1.2 Hz, 1H), 5.98 (s, 1H), 3.94 (s, 3H), 2.27
(dq, J = 7.2, 1.2 Hz, 2H), 1.55–1.48 (m, 2H), 1.35–1.31 (m, 4H),
0.92–0.89 (m, 3H); 13C NMR (100 MHz, CDCl3) d 189.4, 148.3,
148.1, 146.9, 143.4, 129.2, 127.5, 123.4, 123.0, 114.9, 109.8, 56.1,
32.8, 31.5, 28.0, 22.5, 14.1; MS (EI) m/z: 275 (M+).
2.6.1. (E)-1-(40-Hydroxy-30-methoxyphenyl)-4-decen-3-one ([6]-
shogaol)
Yield: 70.8%. 1H NMR (400 MHz, CDCl3) d 6.86–6.78 (m, 2H),
6.71 (d, J = 2.0 Hz, 1H), 6.68 (dd, J = 8.0, 2.0 Hz, 1H), 6.09 (dd,
J = 16.0, 1.6 Hz, 1H), 5.55 (s, 1H), 3.87 (s, 3H), 2.86–2.84 (m, 4H),
2.20 (dq, J = 7.2, 1.6 Hz, 2H), 1.44 (quint, J = 6.8 Hz, 2H), 1.32–
1.25 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
199.8, 147.9, 146.4, 143.8, 133.2, 130.3, 120.7, 114.3, 111.1, 55.8,
41.9, 32.4, 31.3, 29.8, 27.7, 22.4, 13.9; MS (EI) m/z: 277 (M+).
2.4.4. (1E,4E)-1-(40-Hydroxy-30-methoxyphenyl)-1,4-hexadecadien-3-
one ([12]-dehydroshogaol)
Yield: 19.9%. 1H NMR (400 MHz, (CD3)2CO) d 8.18 (s, 1H), 7.60
(d, J = 16.0 Hz, 1H), 7.36 (d, J = 1.6 Hz, 1H), 7.20 (dd, J = 1.6,
8.4 Hz, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.99 (dt, J = 15.6, 7.2 Hz, 1H),
6.88 (d, J = 8.0 Hz, 1H), 6.48 (d, J = 15.6 Hz, 1H), 3.91 (s, 3H), 2.27
(q, J = 7.2 Hz, 2H), 1.52–1.47 (m, 2H), 1.33–1.28 (m, 16H), 0.87
(t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO) d188.9, 150.2,
148.8, 147.6, 143.7, 130.5, 128.1, 124.2, 123.3, 116.2, 111.7, 56.4,
33.3, 32.7, 30.5, 30.4 (2C), 30.2 (2C), 30.1, 29.1, 23.4, 14.5; HRMS
2.6.2. (E)-1-(40-Hydroxy-30-methoxyphenyl)-4-hexadecen-3-one
([12]-shogaol)
Yield: 45.5%. 1H NMR (400 MHz, (CD3)2CO) d7.29(s, 1H), 6.90-
6.81 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 6.65 (dd, J = 8.0, 2.0 Hz, 1H),
6.10 (dt, J = 16.0, 1.2 Hz, 1H), 3.81 (s, 3H), 2.87–2.83 (m, 2H),
2.80-2.74 (m, 2H), 2.21 (dq, J = 7.2, 1.2 Hz, 2H), 1.48–1.43 (m,
2H), 1.30-1.28 (bs, 16H), 0.89-0.86 (m, 3H); 13C NMR (100 MHz,
(CD3)2CO) d 199.5, 148.2, 147.8, 145.7, 133.8, 131.3, 121.6, 115.7,
112.9, 56.3, 42.5, 33.1, 32.7, 30.5 (2C), 30.4, 30.2, 30.1, 30.0, 29.0,
(ESI): m/z calcd. for
359.2588, error = 1.9 ppm.
C
23H34O3 (M+H)+: 359.2581; found:
23.4, 14.4; HRMS (ESI): m/z calcd. for
C
23H36O3 (M+H)+:
361.2737; found: 361.2730, error = 1.9 ppm.
2.5. General procedure for the synthesis of gingerols
Gingerols were synthesized by following previous published
procedures (Ramachandra & Subbaraju, 2006). A solutions of [6]-
dehydrogingerol ([12]-dehydrogingerol) (1 mmol) in anhydrous
ethyl acetate (10 ml) was hydrogenated over 10% Pd/C at room
temperature. The resulting mixture was allowed to stir at room
temperature for 4 h and then monitored by TLC. After filtering off
the catalyst, the filtrate was evaporated in vacuo and then purified
by column chromatography on silica gel to afford gingerols.
2.7. DPPHÅ-scavenging assay
The EC50 values of [6]-gingerol, [6]-shogaol and their deriva-
tives for DPPHÅ-scavenging were determined by monitoring the
absorbance of this radical (60 lM) at 517 nm in methanol accord-
ing to the previously published method (de Gaulejac, Provost, &
Vivas, 1999) with minor changes, and using a Beijing purkinje
TU-1901 UV/Vis spectrometer after the solution was allowed to
stand 60 min in the dark.
The reaction rates of [6]-gingerol, [6]-shogaol and their deriva-
tives with DPPHÅ in methanol were determined by monitoring the
absorbance change at 517 nm, using a Hitachi 557 spectrophotom-
eter equipped with a quartz cell (optical path length, 1 cm), and
using the second-order kinetics with the ratio of [compounds]/
[DPPHÅ] being 1/1. The temperature in the cell was kept at 25 °C
by means of a thermostated bath.
2.5.1. 5-Hydroxy-1-(40-hydroxy-3-methoxyphenyl)decan-3-one ([6]-
gingerol)
Yield: 80.4%. 1H NMR (400 MHz, (CD3)2CO) d 7.33 (m, 1H), 6.81
(d, J = 2.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.64 (dd, J = 8.0, 2.0 Hz,
1H), 4.03–3.99 (m, 1H), 3.81 (s, 3H), 3.72-3.68 (m, 1H), 2.77–2.76
(m, 4H), 2.54–2.52 (m, 2H), 1.42–1.25 (m, 8H), 0.89-0.86 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO) d 210.2, 148.3,

194
D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
2.8. Assay for ferric reducing/antioxidant power (FRAP)
ability of a phenolic antioxidant by monitoring the decrease in this
absorbance. It has been pointed out that the radical-scavenging
antioxidant activity should be assessed by the reactivity toward
radicals and stoichiometric factor (n), that is, the radical-scaveng-
ing rate and number of radicals that can be trapped by each anti-
oxidant molecule (Niki & Noguchi, 2004). Consequently, we first
determined the EC50 values (defined as the amount of antioxidant
required to eliminate the initial DPPHÅ concentration by 50%) of
[6]-gingerol, [6]-shogaol and their derivatives by plotting the
remaining DPPHÅ concentrations at the end of this reaction versus
the concentrations of the compounds tested in methanol. This plot-
ting gave excellent linear correlations for all the compounds and
their stoichiometric factors (nDPPHÅ) were obtained from the gradi-
ents of the straight lines (Fig. 2). According to the EC50 or nDPPHÅ val-
ues listed in Table 1, we can conclude that in the two series with
different chain lengths, shogaols are the most active followed by
gingerols, whereas dehydroshogaols and dehydrogingerols are rel-
atively less active than shogaols and gingerols. Introduction of the
C1-C2 double bond in the chain structure of shogaols and gingerols,
to generate dehydroshogaols and dehydrogingerols, respectively,
results in a reduction of the DPPHÅ-scavenging activity. This is line
with the previous finding that [6]-shogaol is more active than [6]-
dehydroshogaol in the DPPHÅ-scavenging assay (Li et al., 2012). A
comparison of shogaols with gingerols, or dehydroshogaols with
dehydrogingerols clearly indicates that the C4–C5 double bond is
superior to the 5-OH in increasing the activity, in agreement with
the previous results that [6]-shogaol can more effectively scavenge
the DPPHÅ than [6]-gingerol (Dugasani et al., 2010). Additionally,
extension of the unbranched alkyl chain length from six to twelve
carbons had no significant influence on the activity of gingerols
and dehydrogingerols as suggested by statistical analysis (Table 1).
Previously, Masuda et al. (2004) had also found that there is no sig-
nificant difference in the activity among the gingerol related com-
pounds. In contrast, Dugasani et al. (2010) suggested that the
activity increases as extension of the chain length ([10]-ginger-
ol > [8]-gingerol > [6]-gingerol). The discrepancy in the influence
of the chain length on the activity probably comes from the differ-
ent incubation time of the radical with the test compounds. Taken
together, our results highlight that the influence of functional
group change is greater than that of the chain length change on
the DPPHÅ-scavenging activity.
FRAP assay was used to evaluate the reducing capacity of [6]-
gingerol, [6]-shogaol and their derivatives according to the proce-
dure of Benzie and Strain (1996) and the related details were
described in our previous work (Tang et al., 2011).
2.9. Protective effects against AAPH-induced DNA damage
The experiment was carried out by incubation of plasmid
pBR322 DNA (100 ng) with AAPH (5 mM) and [6]-gingerol, [6]-sho-
gaol or their derivatives (40
lM) in 10 mM phosphate-buffered sal-
ine (PBS, pH 7.4) at 37 °C for 1 h with the final volume of 25
the related details were described in our previous work (Qian et al.,
2011).
ll, and
2.10. Assay for anti-haemolysis activity
Human red blood cells (RBCs) were provided by the Red Cross
Center for Blood (Gansu, China). Anti-haemolysis activity of [6]-
gingerol, [6]-shogaol or their derivatives was determined as previ-
ously described (Qian et al., 2011).
2.11. Statistical analysis
The IBM SPSS Statistics 19 was used for statistical analysis of
data. Data were subjected to analysis of variance (ANOVA) and
multiple comparisons of means were carried out using Duncan’s
multiple range test. Data were expressed as means SD of three
independent analyses. Significance was accepted at P < 0.05.
3. Results and discussion
3.1. Synthesis of [6]-gingerol, [6]-shogaol and their derivatives
Synthesis of dehydrogingerols and dehydroshogaols was well-
established by an Aldol condensation reaction of vanillin acetone
with the different alkyl aldehyde (Ramachandra & Subbaraju,
2006). Gingerols were prepared by hydrogenation of dehydrogin-
gerols over Pd/C catalysts, and shogaols were then obtained by
dehydration of gingerols under acid conditions (Scheme 1).
We further measured the rate constants (k2) for hydrogen atom
abstraction from the compounds by DPPHÅ in methanol at 25 °C by
following the second order decay of the absorbance at 517 nm due
to the radical. It can be judged from the k2 values (Table 1) that
similarly to that determined by the nDPPHÅ values, introduction of
the C1-C2 double bond extends the conjugated link between the
aromatic region and side chain, but reduces the formal hydrogen
3.2. DPPHÅ-scavenging activity of [6]-gingerol, [6]-shogaol and their
derivatives
DPPHÅ is a stable N-centered radical with a characteristic absor-
bance at 517 nm and is widely used as the prototypic model for
peroxyl radical to evaluate the formal hydrogen atom transfer
Scheme 1. Synthesis of [6]-gingerol, [6]-shogaol and their derivatives.

D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
195
3.4. Inhibitory activity of [6]-gingerol, [6]-shogaol and their
derivatives against AAPH-induced DNA strand breakages
DNA is one of the most biologically important targets of free
radicals, and oxidative damage of DNA including strand breakages
and base and nucleotide modifications could result in gene muta-
tion. For example, 8-hydroxy-20-deoxyguanosine (8-OHdG), an oxi-
dised nucleoside of DNA, induces transversion of G to T, which is
also strong evidence that oxidative stress is intimately associated
with carcinogenesis (Wu, Chiou, Chang, & Wu, 2004). In view of
implication of DNA damage and the above two models being sub-
strate-free, thus, we further selected the AAPH-induced DNA
strand breakage model to probe inhibitory activity of [6]-gingerol,
[6]-shogaol and their derivatives against the damage by using aga-
rose gel electrophoresis analysis.
As shown in lane 2 of Fig. 3A, supercoiled pBR322 plasmid DNA
was completely broken down to the open circular and linear forms
upon addition of 5 mM 2,20-azobis (2-amidinopropane hydrochlo-
ride) (AAPH), a water-soluble azo compound which is extensively
used as a free radical initiator for biologic studies. The open circu-
lar and linear forms are indicative of single- and double-strand
breakages, respectively. The compounds bearing a six-carbon
unbranched alkyl chain significantly inhibits the DNA strand
breakages with the activity sequence of [6]-dehydroginger-
ol > [6]-gingerol > [6]-dehydroshogaol > [6]-shogaol (lanes 3–6).
The activity sequence is completely different from that obtained
by the DPPHÅ-scavenging and FRAP assays, highlighting the impor-
tance of the 5-OH in inhibiting the DNA strand breakages. A possi-
ble reason is that this group would facilitate the binding affinity of
[6]-dehydrogingerol and [6]-gingerol with plasmid DNA, resulting
in the activity enhancement. Furthermore, the C1–C2 double bond
could be responsible for their interaction with DNA. The extension
from six- to twelve-carbon unbranched alkyl chains almost com-
pletely abolishes their inhibitory activity. Even in the case of
[12]-dehydrogingerol bearing the 5-OH, it only retains the weak
activity as evidenced by lane 4 of Fig. 3B where the linear form
was decreased compared to that in the APPH control lane, but no
the supercoiled form appeared. This indicates that the chain exten-
sion significantly disrupts and intervenes with their interaction
with DNA.
Fig. 2. Dependency of the remaining DPPHÅ concentrations on the concentrations of
[6]-gingerol, [6]-shogaol and their derivatives in methanol after 60 min of reaction.
(a) [12]-shogaol; (b) [6]-shogaol; (c) [12]-gingerol; (d) [6]-gingerol; (e) [12]-
dehydroshogaol; (f) [6]-dehydroshogaol; (g) [12]-dehydrogingerol; and (h) [6]-
dehydrogingerol.
Table 1
Antioxidant activity of [6]-gingerol, [6]-shogaol and their derivatives.
Compounds
DPPHÅ-Scavenging activity
EC50
M) nDPPHÅ k (Mꢀ1 sꢀ1
FRAP
teff
(l
)
ne
(min)
[6]-Gingerol
[6]-Shogaol
[6]-Dehydrogingerol
[6]-Dehydroshogaol
[12]-Gingerol
21.4 0.9a 1.29
16.3 0.4b 1.81
32.6 0.7c 0.88
29.2 0.6d 1.00
20.8 0.6a 1.40
11.6 0.8e 2.29
243 12ab
260 5b
2.39 0.04 46af
2.81 0.01 33b
1.88 0.02 52c
2.08 0.05 41a
1.90 0.02 76d
2.15 0.06 44af
1.19 0.02 69e
1.37 0.03 48cf
173 15c
230 11a
254 8b
[12]-Shogaol
292 11d
168 15c
226 16a
[12]-Dehydrogingerol 31.1 2.0c 0.93
[12]-Dehydroshogaol 26.6 0.9f 1.06
For the DPPHÅ-Scavenging activity and FRAP assays, data are expressed as the
mean SD for three determinations. For the anti-haemolysis assay, data are the
averages of three determinations which were reproducible with deviation less
than 10%. In the same column, mean values bearing different superscripts are
significantly different (P < 0.05).
atom transfer ability of the compounds. Moreover, extension of the
chain length has no significant influence on the reaction rate
constant of gingerols, dehydrogingerols and dehydroshogaols
with the DPPHÅ.
3.5. Inhibitory activity of [6]-gingerol, [6]-shogaol and their
derivatives against AAPH-induced haemolysis of human erythrocytes
3.3. FRAP of [6]-gingerol, [6]-shogaol and their derivatives
Biological membranes are the heterogeneous system where
antioxidant efficacy could be significantly different from that
determined in the homogeneous solutions as used in the DPPHÅ-
scavenging, FRAP and DNA strand breakage-inhibiting experiments
A phenolic antioxidant can neutralize free radicals not only by
transferring a hydrogen atom but also by donating an electron.
Therefore, we next tested the electron-donating ability of [6]-ging-
erol, [6]-shogaol and their derivatives by the FRAP method (Benzie
& Strain, 1996) based on the reduction of the Fe3+ complex of
tripyridyltriazine Fe(TPTZ)32+ to the intensely blue coloured Fe2+
complex Fe(TPTZ)22+ in acidic medium. The results are expressed
as the number (ne) of donated electrons per molecule and summa-
rised in Table 1. On the basis of the ne values, the electron-donating
ability decreases in the order of shogaols > gingerols > dehydros-
hogaols > dehydrogingerols for two series compounds, in agree-
ment with the result obtained form the DPPHÅ-scavenging assay.
Installation of the C1–C2 double bond attenuates the electron-
donating ability as exemplified by the ne values of shogaols versus
dehydroshogaols, and gingerols versus dehydrogingerols. Replace-
ment of the C4–C5 double bond by the 5-OH also reduces the ability
as suggested by comparing the ne values of shogaols with ginge-
rols, and dehydroshogaols with dehydrogingerols. In addition,
extension of the unbranched alkyl chain length from six to twelve
carbons mildly decreased the ability, a result different from that of
the DPPHÅ-scavenging assay.
Fig. 3. Agarose gel electrophoresis pattern showing protection of [6]-gingerol, [6]-
shogaol and their derivatives (40
lM) against the AAPH (5 mM)-induced strand
breakages of plasmid pBR322 DNA (100 ng/25
ll) in PBS (pH 7.4) at 37 °C
for 60 min. (A) Lane 1: control; Lane 2: AAPH alone; Lanes 3-6: AAPH + [6]-
dehydroshogaol, AAPH + [6]-dehydrogingerol, AAPH + [6]-shogaol and AAPH + [6]-
gingerol, respectively. (B) Lane 1: control; Lane 2: AAPH alone; Lanes 3-6:
AAPH + [12]-dehydroshogaol, AAPH + [12]-dehydrogingerol, AAPH + [12]-shogaol
and AAPH + [12]-gingerol, respectively.

196
D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
4. Conclusion
In summary, we synthesized eight gingerol related compounds
and investigated influence of the side chain structure on their anti-
oxidant potency by four different assays. Their antioxidant activity
depends significantly on the side chain structures including
the functional groups and chain length, the reaction mediums
and substrates. Specifically, introduction of the C1-C2 double bond
decreases their formal hydrogen-transfer and electron-donating
abilities, but increases their DNA damage- protecting ability;
Despite lowering their formal hydrogen-transfer and electron-
donating abilities, existence of the 5-OH strengthens their DNA
damage- and lipid peroxidation-protecting abilities; Extension of
the chain length from six to twelve carbons significantly reduces
the DNA strand breakage-inhibiting activity, however, enhances
their anti-haemolysis activity. The above results provide useful
information for designing [6]-gingerol and [6]-shogaol-directed
antioxidants, and understanding the behaviour of antioxidant in
different media and models.
Fig. 4. Inhibitory effect of [6]-gingerol, [6]-shogaol and their derivatives (40 lM)
against 50 mM AAPH-induced haemolysis of 5% human RBCs in 0.10 mM PBS (pH
7.4) under an aerobic atmosphere at 37 °C. (a) AAPH alone; (b) AAPH + [6]-shogaol;
(c) AAPH + [6]-gingerol; (d) AAPH + [6]-dehydrogingerol; (e) AAPH + [6]-dehy-
droshogaol; (f) AAPH + [12]-shogaol; (g) AAPH + [12]-dehydroshogaol; (h)
AAPH + [12]-dehydrogingerol, and (i) AAPH + [12]-gingerol.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 31100607) and the Fundamental
Research Funds for the Central Universities (lzujbky-2013-52).
(Niki & Noguchi, 2004; Niki, 2010). Human red blood cell mem-
branes are rich in polyunsaturated fatty acids and are very suscep-
tible to free radical-mediated lipid peroxidation, which induces
membrane disturbance and haemoglobin leakage, ultimately lead-
ing to occurrences of haemolysis (Niki et al., 1988). Consequently,
we finally employed the AAPH-induced RBC haemolysis model to
assess inhibitory activity of [6]-gingerol, [6]-shogaol and their
derivatives against lipid peroxidation in heterogeneous media.
The haemolysis extent can be detected by measuring absor-
bance of the haemolysates at 540 nm (Niki et al., 1988). When
human RBCs were incubated in air at 37 °C as a 5% suspension in
buffered saline solution, they were stable and little haemolysis
was observed during 5 h (data not shown). When 50 mM AAPH
was added to the RBC suspension, it induced fast haemolysis after
an inhibition time of 88 min (tinh) due to the presence of the endog-
enous antioxidants in the RBC membranes (line a of Fig. 4)
(Esterbauer & Ramos, 1996). However, the onset of oxidative
haemolysis was significantly inhibited by adding the compounds
tested, as indicated by prolongation of the tinh derived from the
endogenous antioxidants (Fig. 4). Their antioxidant efficacy can
be assessed by the prolongation portion called ‘‘an effective inhibi-
tion time (teff)’’.
Of interest, according to the teff values (Table 1), the best results
regarding the anti-haemolysis activity are again appeared in dehy-
drogingerols and gingerols bearing the 5-OH, a similar result to
that obtained from the DNA strand breakage-inhibiting experi-
ment. This is probably because the binding of the compounds to
RBC membranes could be mediated by hydrogen bonds between
the 5-OH and the membrane component, hence facilitating their
reaction with the propagating lipid peroxyl radicals (LOOÅ) within
membranes. A previous study has also proven that the hydroxyl
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.2014.
05.077.
References
Baliga, M. S., Haniadka, R., Pereira, M. M., D’souza, J. J., Pallaty, P. L., Bhat, H. P., et al.
(2011). Update on the chemopreventive effects of ginger and its
phytochemicals. Critical Reviews in Food Science and Nutrition, 51, 499–523.
Benzie, I. F. F., & Strain, J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power’’: The FRAP assay. Analytical Biochemistry, 239,
70–76.
Cao, X.-Y., Yang, J., Dai, F., Ding, D.-J., Kang, Y.-F., Wang, F., et al. (2012).
Extraordinary radical-scavengers: 4-Mercaptostilbenes. Chemistry
European Journal, 18, 5898–5905.
-
A
de Gaulejac, N. S. C., Provost, C., & Vivas, N. (1999). Comparative study of polyphenol
scavenging activities assessed by different methods. Journal of Agricultural and
Food Chemistry, 47, 425–431.
de Heer, M. I., Mulder, P., Korth, H. G., Ingold, K. U., & Lusztyk, J. (2000). Hydrogen
atom abstraction kinetics from intramolecularly hydrogen bonded ubiquinol-10
and other (poly)methoxy phenols. Journal of the American Chemical Society, 122,
2355–2360.
Dugasani, S., Pichika, M. R., Nadarajah, V. D., Balijepalli, M. K., Tandra, S.,
&
Korlakunta, J. N. (2010). Comparative antioxidant and anti-inflammatory effects
of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. Journal of
Ethnopharmacology, 127, 515–520.
Esterbauer, H., & Ramos, P. (1996). Chemistry and pathophysiology of oxidation of
LDL. Reviews of Physiology, Biochemistry and Pharmacology, 127, 31–64.
Fleming, S. A., Dyer, C. W., & Eggington, J. (1999). A convenient one-step gingerol
synthesis. Synthetic Communications, 29, 1933–1939.
Gan, F.-F., Nagle, A. A., Ang, X., Ho, O. H., Tan, S.-H., Yang, H., et al. (2011). Shogaols at
proapoptotic concentrations induce G2/M arrest and aberrant mitotic cell death
associated with tubulin aggregation. Apoptosis, 16, 856–867.
Gundala, S. R., Mukkavilli1, R., Yang, C., Yadav, P., Tandon, V., Vangala, S., et al.
(2014). Enterohepatic recirculation of bioactive ginger phytochemicals is
associated with enhanced tumor growth-inhibitory activity of ginger extract.
Carcinogenesis. http://dx.doi.org/10.1093/carcin/bgu011.
Halvorsen, B. L., Carlsen, M. H., Phillips, K. M., Bøhn, S. K., Holte, K., Jacobs, D. R., et al.
(2006). Content of redox-active compounds (ie, antioxidants) in foods
consumed in the United States. The American Journal of Clinical Nutrition, 84,
95–135.
groups at carbons 1, 3 and 4 of D-glucose play important roles for
its binding to isolated human erythrocyte membranes by hydrogen
bond interaction (Kahlenberg & Dolansky, 1972). On the other
hand, the extension from six- to twelve-carbon unbranched alkyl
chains strengthened their anti-haemolysis activity. Notably, this
result is entirely opposite to that of the DNA strand breakage-
inhibiting experiment and the FRAP assay, suggesting that the
microenvironment change significantly influences antioxidant effi-
cacy. This is probably due to the fact that the modest extension in
the chain length facilitates their uptake into the membranes or
assists in their appropriate localisation in the membranes to
increase the LOOÅ-scavenging efficiency.
Hussain, S. P., Hofseth, L. J., & Harris, C. C. (2003). Radical causes of cancer. Nature
Reviews Cancer, 3, 276–285.
Ishiguro, K., Ando, T., Watanabe, O., & Goto, H. (2008). Specific reaction of
a,b-
unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in
tubulin leading to microtubule damage. FEBS Letters, 582, 3531–3536.

D.-L. Lu et al. / Food Chemistry 165 (2014) 191–197
197
Jolad, S. D., Lantz, R. C., Chen, G. J., Bates, R. B., & Timmermann, B. N. (2005).
Commercially processed dry ginger (Zingiber officinale): Composition and effects
on LPS-stimulated PGE2 production. Phytochemistry, 66, 1614–1635.
Reuter, S., Gupta, S. C., Chaturvedi, M. M., & Aggarwal, B. B. (2010). Oxidative stress,
inflammation, and cancer: How are they linked? Free Radical Biology & Medicine,
49, 1603–1616.
Kahlenberg, A., & Dolansky, D. (1972). Structural requirements of
D
-glucose for its
Sang, S., Hong, J., Wu, H., Liu, J., Yang, C. S., Pan, M.-H., et al. (2009). Increased growth
inhibitory effects on human cancer cells and anti-inflammatory potency of
shogaols from Zingiber officinale relative to gingerols. Journal of Agricultural and
Food Chemistry, 57, 10645–10650.
Shang, Y.-J., Jin, X.-L., Shang, X.-L., Tang, J.-J., Liu, G.-Y., Dai, F., et al. (2010).
Antioxidant capacity of curcumin-directed analogues: Structure-activity
relationship and influence of microenvironment. Food Chemistry, 119,
1435–1442.
Shang, Y.-J., Qian, Y.-P., Liu, X.-D., Dai, F., Shang, X.-L., Jia, W.-Q., et al. (2009).
Radical-scavenging activity and mechanism of resveratrol-oriented analogues:
Influence of the solvent, radical, and substitution. The Journal of Organic
Chemistry, 74, 5025–5031.
binding to iaolated human erythrocyte membranes. Canadian Journal of
Biochemistry, 50, 638–643.
Kikuzaki, H., & Nakatani, N. (1993). Antioxidant effects of some ginger constituents.
Journal of Food Science, 6, 1407–1410.
Li, Y., Dai, F., Jin, X.-L., Ma, M.-M., Wang, Y.-H., Ren, X.-R., et al. (2014). An effective
strategy to develop active cinnamic acid-directed antioxidants based on
elongating the conjugated chains. Food Chemistry, 158, 41–47.
Li, F., Nitteranon, V., Tang, X., Liang, J., Zhang, G., Parkin, K. L., et al. (2012). In vitro
antioxidant and anti-inflammatory activities of 1-dehydro-[6]-gingerdione, 6-
shogaol, 6-dehydroshogaol and hexahydrocurcumin. Food Chemistry, 135,
332–337.
Masuda, Y., Kikuzaki, H., Hisamoto, M.,
&
Nakatani, N. (2004). Antioxidant
Shukla, Y., & Singh, M. (2007). Cancer preventive properties of ginger: A brief
review. Food and Chemical Toxicology, 45, 683–690.
Tang, J.-J., Fan, G.-J., Dai, F., Ding, D.-J., Wang, Q., Lu, D.-L., et al. (2011). Finding more
active antioxidants and cancer chemoprevention agents by elongating the
conjugated links of resveratrol. Free Radical Biology & Medicine, 50, 1447–1457.
properties of gingerol related compounds from ginger. BioFactors, 21, 293–296.
Niki, E. (2010). Assessment of antioxidant capacity in vitro and in vivo. Free Radical
Biology & Medicine, 49, 503–515.
Niki, E., Komuro, E., Takahashi, M., Uranoz, S., Itos, E., & Terao, K. (1988). Oxidative
hemolysis of erythrocytes and its inhibition by free radical scavengers. The
Journal of Biological Chemistry, 263, 19809–19814.
Niki, E., & Noguchi, N. (2004). Dynamics of antioxidant action of vitamin E. Accounts
of Chemical Research, 37, 45–51.
Pan, M.-H., Hsieh, M.-C., Hsu, P.-C., Ho, S.-Y., Lai, C.-S., Wu, H., et al. (2008). 6-
Shogaol suppressed lipopolysaccharide-induced up-expression of iNOS and
Weng, C.-J., Chou, C.-P., Ho, C.-T.,
& Yen, G.-C. (2012). Molecular mechanism
inhibiting human hepatocarcinoma cell invasion by 6-shogaol and 6-gingerol.
Molecular Nutrition & Food Research, 56, 1304–1314.
Weng, C.-J., Wu, C.-F., Huang, H.-W., Ho, C.-T., & Yen, G.-C. (2010). Anti-invasion
effects of 6-shogaol and 6-gingerol, two active components in ginger, on human
hepatocarcinoma cells. Molecular Nutrition & Food Research, 54, 1618–1627.
Wu, L. L., Chiou, C. C., Chang, P. Y., & Wu, J. T. (2004). Urinary 8-OHdG: A marker of
COX-2 in murine macrophages. Molecular Nutrition
& Food Research, 52,
1467–1477.
oxidative stress to DNA and a risk factor for cancer, atherosclerosis and
Pan, M.-H., Hsieh, M.-C., Kuo, J.-M., Lai, C.-S., Wu, H., Sang, S., et al. (2008). 6-Shogaol
induces apoptosis in human colorectal carcinoma cells via ROS production,
diabetics. Clinica Chimica Acta, 339, 1–9.
Wu, H., Hsieh, M.-C., Lo, C.-Y., Liu, C. B., Sang, S., Ho, C.-T., et al. (2010). 6-Shogaol is
more effective than 6-gingerol and curcumin in inhibiting 12-O-
tetradecanoylphorbol 13-acetate-induced tumor promotion in mice. Molecular
Nutrition & Food Research, 54, 1296–1306.
Yeh, H.-Y., Chuang, C.-H., Chen, H.-C., Wan, C.-J., Chen, T.-L., & Lin, L.-Y. (2014).
Bioactive components analysis of two various gingers (Zingiber officinale
Roscoe) and antioxidant effect of ginger extracts. LWT - Food Science and
Technology, 55, 329–334.
caspase activation, and GADD 153 expression. Molecular Nutrition
Research, 52, 527–537.
& Food
Peng, F., Tao, Q., Wu, X., Dou, H., Spencer, S., Mang, C., et al. (2012). Cytotoxic,
cytoprotective and antioxidant effects of isolated phenolic compounds from
fresh ginger. Fitoterapia, 83, 568–585.
Qian, Y.-P., Shang, Y.-J., Teng, Q.-F., Chang, J., Fan, G.-J., Wei, X., et al. (2011).
Hydroxychalcones as potent antioxidants: Structure-activity relationship
analysis and mechanism considerations. Food Chemistry, 126, 241–248.
Ramachandra, M. S., & Subbaraju, G. V. (2006). Synthesis and bioactivity of novel
caffeic acid esters from Zuccagnia punctata. Journal of Asian Natural Products
Research, 8, 683–688.
Zhou, B., Miao, Q., Yang, L., & Liu, Z.-L. (2005). Antioxidative effects of flavonols and
their glycosides against the free-radical-induced peroxidation of linoleic acid in
solution and in micelles. Chemistry - A European Journal, 11, 680–691.