Development of a Gemini Interfacial Antioxidant for Oil in Water Emulsion with Gallic Acid and Dodecyl Gemini Chains

Article abstract of DOI:10.1021/acs.jafc.0c00807

In this study, development of the gemini gallate (GG) interfacial antioxidant for oil in water (O/W) emulsion was performed employing Steglich esterification with gallic acid and dodecyl gemini chains through a prepacked column and peristaltic pump-based purification system. The structural identity and purity of the prepared GG and monogallate (MG) were confirmed by NMR, Fourier transform infrared spectroscopy, and high-performance liquid chromatography-mass spectrometry. Further evaluation revealed that the GG possessed excellent radical scavenging activity and superior antioxidant activity in an O/W emulsion relative to the MG, especially under the condition of a reduced amount of the emulsifier. Microscopic investigation by a fluorescent probe and transmission electron microscopy (TEM) unveiled that the extraordinary antioxidant performance in the O/W emulsion was presumably attributed to preferable interfacial location because of the unique gemini molecular architecture. The superior antioxidant activity of the gemini antioxidant holds great promise for antioxidation in O/W emulsions.

Full text of DOI:10.1021/acs.jafc.0c00807

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Bioactive Constituents, Metabolites, and Functions
Development of Gemini Interfacial Antioxidant for O/
W Emulsion with Gallic Acid and Dodecyl Gemini Chain
Peifei Wang, and Songbai Liu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00807 • Publication Date (Web): 20 Aug 2020
Downloaded from pubs.acs.org on August 23, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 36
Journal of Agricultural and Food Chemistry
1
2
Development of Gemini Interfacial Antioxidant for O/W Emulsion with Gallic
Acid and Dodecyl Gemini Chain
3
4
Peifei Wang, Songbai Liu*
5
6
Department of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang
Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food
Technology and Equipment, Zhejiang University, 866 Yuhangtang Road, Hangzhou
310058, China
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 36
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
ABSTRACT: In this study, development of the gemini gallate interfacial antioxidant
for O/W emulsion was performed employing Steglich esterification with gallic acid
and dodecyl gemini chain through a prepacked column and peristaltic pump based
purification system. The structural identity and purity of the prepared gemini and
mono gallate were confirmed by NMR, FT-IR, and HPLC-MS. Further evaluation
revealed that the gemini gallate possessed excellent radical scavenging activity and
superior antioxidant activity in O/W emulsion relative to the mono gallate especially
under the condition of reduced amount of emulsifier. Microscopic investigation by
fluorescent probe and transmission electron microscopy (TEM) unveiled that the
extraordinary antioxidant performance in O/W emulsion was presumably attributed to
preferable interfacial location due to the unique gemini molecular architecture. The
superior antioxidant activity of the gemini antioxidant hold a great promise for
antioxidation in O/W emulsion.
KEYWORDS: gallic acid, gemini interfacial antioxidant, gemini antioxidant,
antioxidant activity, O/W emulsion, Steglich esterification
2
ACS Paragon Plus Environment

Page 3 of 36
Journal of Agricultural and Food Chemistry
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
INTRODUCTION
In recent years, functional lipids such as ω-3 fatty acid1,2, vitamin D3,4 and
carotenoids5,6 have received much attention for their health beneficial effects.
However, the functional lipids are highly sensitive to oxidation during processing and
storage especially when they are prepared as O/W emulsion due to increased exposure
to oxidation.7 As a result, great efforts have been made to develop good antioxidants
for O/W emulsion. It is revealed that the oil-water interface plays an important role in
oxidation where the oxidation reaction takes place and propagates, and the interfacial
antioxidants which prefer to stay at the interface have superior activities in O/W
emulsion.8 A variety of examples of interfacial antioxidants have been developed
including emulsifier-antioxidant bioconjugates, grafted ovalbumin-catechin
conjugates and the conjugates of epigallocatechin and phytosterols which
demonstrated excellent antioxidant performance in O/W emulsion.9-11 The exceptional
antioxidant performance of interfacial antioxidants in O/W emulsion has been
attributed to their amphiphilic nature leading to increased effective interfacial
concentration of antioxidants and so more efficient inhibition of lipid oxidation.12-15
It is well documented that a new class of amphiphilic molecules, gemini
surfactants, containing two hydrophilic groups and two hydrophobic chains, are
substantially more active relative to the conventional monomeric compounds.16,17
Gemini's meaning in astronomy is the gemini constellation, which vividly expresses
the molecular structure characteristics of such surfactants as conjoined twin babies.
The unique molecular architectures of gemini surfactants result in their remarkable
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 36
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
activities that hold great promise in food industry, material and biological applications
as emulsifiers, gene and drug delivery agents.18-20 In previous work, our laboratory
developed a novel type of highly effective nonionic gemini alkyl O-glucoside
surfactants with intriguing self-assembly behavior.21 Gallic acid is abundant natural
antioxidant and its derivatives have been proven excellent antioxidants in lipophilic
settings.22-25 Therefore, we reasoned that incorporation of gallic acid in the
architecture of gemini alkyl chain would facilitate location of gallic acid at interfacial
layer and improve antioxidant performance in O/W emulsion accordingly. The
concept of gemini interfacial antioxidant would be useful for design of antioxidants in
complicated settings.
In this work, development of gemini interfacial antioxidant for O/W emulsion was
exercised with gallic acid and dodecyl gemini chain. The gemini gallate was designed
and successfully prepared. A prepacked column and pump system was built for
purification. Then DPPH radical scavenging activity, oxygen radical absorbance
capacity (ORAC), and the antioxidant performance in O/W emulsion were evaluated.
Superior antioxidant activity of the gemini antioxidant in O/W emulsion was observed
relative to the corresponding mono antioxidant. The microscopic structure of the
emulsion was further investigated by fluorescent probe and transmission electron
microscopy (TEM). The details of this work were disclosed as follows.
MATERIALS AND METHODS
Chemicals. Potassium hydroxide, hydrochloric acid (HCl), sodium dihydrogen
4
ACS Paragon Plus Environment

Page 5 of 36
Journal of Agricultural and Food Chemistry
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
phosphate dihydrate, sodium phosphate dibasic dodecahydrate, anhydrous magnesium
sulfate (MgSO4), ethyl acetate, petroleum ether, toluene, and ethanol were purchased
from Sinopharm Chemical Reagent (Shanghai, China). Ethylene glycol diglycidyl
ether was obtained from Energy Chemical Reagent (Shanghai, China). Pyrene was
obtained from Macklin Reagent (Shanghai, China). Gallic acid (GA), isobutyric
anhydride, triethylamine, N,N-dimethylformamide (DMF), 4-dimethyaminopyridine
(DMAP),
dodecanol,
N,N-dicyclohexylcarbodiimide
(DCC),
(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), hydrazinium
hydrate solution (80%), butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), tert-butyl hydroquinone (TBHQ), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
5-aminofluorescein, dodecanoyl chloride, 2,2’-azobis-(2-methylpropionamidine)
dihydrochloride (AAPH), ethyl butyrate, and Tween-80 were purchased from Aladdin
Reagent (Shanghai, China). All chemicals were of analytical grade.
Preparation of Mono Gallate (MG) and Gemini Gallate (GG). Preparation of
Triisobutyryl Gallic Acid. Triisobutyryl gallic acid was prepared according to the
previously reported method with minor modifications.25 Briefly, to gallic acid (6
mmol, 1020.7 mg) and DMAP (0.6 mmol, 73.3 mg) in DMF (3 mL) was added
sequentially isobutyric anhydride (27 mmol, 4.477 mL), triethylamine (27 mmol,
3.763 mL). The reaction mixture was stirred at ambient temperature for 2 h and
quenched by HCl (1 N), followed by aqueous washing to afford the pure triisobutyryl
gallic acid as a white solid.
100
101
102
103
Preparation of the Mono Gallate. To a solution of triisobutyryl gallic acid (0.838
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 36
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
mmol, 318.7 mg), dodecanol (0.559 mmol, 127.1 μL), DMAP (0.056 mmol, 6.8 mg)
in toluene (3 mL) was added a solution of DCC (0.838 mmol, 172.9 mg in 1 mL
toluene). The reaction mixture was stirred at ambient temperature for 3 h. Then
ethanol (95%, 4 mL) was added, followed by addition of hydrazinium hydrate
solution (80%, 203 μL). The reaction mixture was stirred further at ambient
temperature for 1.5 h before quenching with HCl (1 N). After extracted with ethyl
acetate, the organic phase was dried over MgSO4 and evaporated under reduced
pressure. Purification by a silica gel chromatography with petroleum ether/ethyl
acetate (2/1 to 1/1, v/v) afforded the mono gallate as a white solid (182.5 mg, 64.5%).
Preparation of the Gemini Gallate. Dodecanol and ethylene glycol diglycidyl ether
were applied to prepare the dodecyl gemini chain based on the previous method with
improvement of purification protocol.21 To a solution of ethylene glycol diglycidyl
ether (10 mmol, 1558 μL) and dodecanol (30 mmol, 6816 μL) in DMSO (5 mL) was
o
added KOH (20 mmol, 1120 mg). The reaction mixture was stirred at 60 C for 6 h
and then extracted with ethyl acetate and washed by water. The organic phase was
dried over MgSO4 and evaporated under reduced pressure. The purification was
performed on a prepacked column system equipped with a prepacked silica gel
column (SepaFlash 120 g, Lisure Science Inc., Suzhou, China) and two peristaltic
pumps (RZ1030, Runze Fluid Inc., Nanjing, China) pumping petroleum ether (13.2
mL/min) and ethyl acetate (4.4 mL/min) respectively, and produced the dodecyl
gemini chain as a yellow oil (360 mg).
Then the dodecyl gemini chain and triisobutyryl gallic acid were employed to
6
ACS Paragon Plus Environment

Page 7 of 36
Journal of Agricultural and Food Chemistry
126
prepare the gemini gallate. To a solution of triisobutyryl gallic acid (1.98 mmol, 753.2
mg), dodecyl gemini chain (0.66 mmol, 360.0 mg), DMAP (0.066 mmol, 8.1 mg) in
toluene (3.6 mL) was added a solution of DCC (1.98 mmol, 408.5 mg in 0.8 mL
toluene ). The reaction mixture was stirred at ambient temperature for 4 h. After
extracted with ethyl acetate and washed by water, the organic phase was dried over
MgSO4 and evaporated under reduced pressure. Purification by a prepacked silica gel
column (SepaFlash 25 g, Lisure Science Inc., Suzhou, China) with two peristaltic
pumps (RZ1030, Runze Fluid Inc., Nanjing, China) pumping petroleum ether (16.5
mL/min) and ethyl acetate (3 mL/min) respectively, and afforded the product as a
yellow oil (263 mg). After that, the product was dissolved in ethanol (95%, 3 mL),
followed by addition of hydrazinium hydrate solution (80%, 121 μL). The reaction
mixture was stirred further at ambient temperature for 1.5 h before quenching with
HCl (1 N). After extracted with ethyl acetate, the organic phase was dried over
MgSO4 and evaporated under reduced pressure and produced the gemini gallate as a
colorless oil (130 mg).
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
1
13
Structural Determination. The H and C NMR spectra of the prepared mono
gallate and gemini gallate were carried out on a 400 MHz NMR spectrometer (Bruker
Corporation, Fӓllanden, Zürich, Switzerland) at room temperature applying DMSO-d6
13
(1H, 2.50 ppm; C, 39.52 ppm) as solvent.26 FT-IR spectra were performed on an
AVA TAR370 spectrophotometer (Thermo Nicolet Corporation, Madison, WI, USA)
by attenuated total reflectance method within 400-4000 cm−1. Mass spectra were
collected on a Thermo Finnigan LCQ Deca XP Max system (Thermo Fisher
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 36
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
Scientific, Waltham, MA, USA) with application of negative ion electron spray
ionization (ESI) mode within 300−2000 (m/z).
HPLC-MS Analysis. The HPLC-MS analysis was performed by an Agilent 1200
system (Agilent, Santa Clara, CA, USA) employing the Agilent SB-C18 (2.1×150
mm, 3.5 μm) column. The mobile phase A was formic acid solution (0.1%, v/v) and B
was methanol. The flow rate was 0.5 mL/min. 5 μL of sample was injected and
detected at 277 nm. The gradient was starting from 80% B to 100% within 20 min,
followed by 30 min with 100% B. Mass spectra were collected on a Thermo Finnigan
LCQ Deca XP Max system (Thermo Fisher Scientific, Waltham, MA, USA) with
application of negative ion electron spray ionization (ESI) mode within 300−2000
(m/z).
Antioxidant Activity Evaluated by the DPPH Scavenging Activity Assay. The
method described by Wang et al. was applied to evaluate the DPPH radical
scavenging activity with slight modification.11 Briefly, 0.1 mL of the antioxidant
ethanol solution of specified concentration was added to 3.9 mL of DPPH ethanol
solution (0.025 g/L). The mixture was placed in the dark for 30 min at ambient
temperature. Then the absorbance was measured at 515 nm by a UV−vis
spectrophotometer (SP-756, Shanghai Spectrum Corporation, Shanghai, China). The
assay was carried out in triplicate. The DPPH scavenging activity (DPPHSCA %) was
calculated as follows.
퐴푠 ― 퐴푏
168
169
퐷푃푃퐻푆퐶퐴% = 1 ―
× 100%
(
)
퐴푐
where As represents the absorbance of sample mixture (addition of 0.1 mL of
8
ACS Paragon Plus Environment

Page 9 of 36
Journal of Agricultural and Food Chemistry
170
antioxidant ethanol solution to 3.9 mL of DPPH ethanol solution)，Ab the absorbance
of the blank mixture (addition of 0.1 mL of antioxidant ethanol solution to 3.9 mL
ethanol), and Ac the absorbance of the control mixture (addition of 0.1 mL ethanol to
3.9 mL of DPPH ethanol solution).
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
Antioxidant Activity Evaluated by the Oxygen Radical Absorbance Capacity
(ORAC) Assay. Preparation of Dodecanoyl 5-Aminofluorescein (DAF). According to
Chaiyasit et al.27, 5-aminofluorescein (AF) was applied to synthesize fat-soluble
fluorescent probe DAF. Briefly, to a solution of AF (0.58 mmol, 200 mg), dodecanoyl
chloride (1.16 mmol, 266.6 μL) in DMF (10 mL) was added triethylamine (1.73
mmol, 239.2 μL ) dropwise. The reaction mixture was stirred at ambient temperature
for 2.5 h, and then extracted with ethyl acetate and washed by HCl (1 N). Purification
by a silica gel chromatography with petroleum ether/ethyl acetate (2/1 to 1.5/1, v/v)
produced DAF as a yellow oil (30 mg).
ORAC Assay. 1.25 mL of DAF PBS solution (pH 7.4, 0.1 μmol/L) was added to 2.5
mL of antioxidant solution of specified concentration with 1 μL of Tween-80, then
treated by ultrasonication for 5 min. The microplate containing 150 µL of the
prepared solution was incubated at 37 °C for 15 min. Then 50 µL of AAPH ethanol
solution (640 mmol/L) was added to the prepared solution and analyzed every 5 min
for 150 min with an excitation wavelength of 485 nm and an emission wavelength of
495 nm by a microplate reader (SpectraMax M5/M5e, American molecular
instruments, America). The temperature was maintained at 37°C. The assay was
carried out in triplicate. The fluorescence decay area under the curve (AUC), NET
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 36
192
193
194
AUC, and ORAC value were calculated as follows.
[
(
)
]
AUC = 0.5 × 2 × 푓0 + 푓1 + … + 푓푛 ― 1 + 푓푛 ― 푓0 ― fn × ∆t
NET AUC = 퐴푈퐶푠 ― 퐴푈퐶푏
푁퐸푇 퐴푈퐶푠
퐶푇푟표푙표푥
195
ORAC =
×
푁퐸푇 퐴푈퐶푇푟표푙표푥
퐶푠
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
Where fn represents the relative fluorescence intensity at the nth measure point,
∆t
the time interval, AUCs the sample’s or Trolox’s AUC, AUCb the control’s AUC,
NET AUCs the sample’s NET AUC, NET AUCTrolox the Trolox’s AUC, CTrolox the
Trolox’s concentration, Cs the sample’s concentration.
β-Carotene AAPH Bleaching Assay in O/W Emulsion. The protocol was
improved on the basis of the previous study.11 To a round-bottom flask containing 2
mL of β-carotene ethyl butyrate solution (0.2 mg/mL) were added 3.35 or 2.6 mL of
Tween-80 and 2.25 mL of ethanol. The mixture was stirred forcefully at 50 °C and
then added 100 mL of AAPH aqueous solution (0.3 mmol/L) to form the O/W
emulsion. To 4.8 mL of the emulsion was added 0.2 mL of antioxidant ethanol
o
solution of specified concentration. The mixture was incubated in a 50 C water bath
for 2.5 h. Then the absorbance at 470 nm was measured applying a UV−vis
spectrophotometer (SP-756, Shanghai Spectrum Corporation, Shanghai, China). The
assay was carried out in triplicate. The antioxidant activity (AA %) was calculated as
follows.
퐴0 ― 퐴푡
211
212
AA (%) = 1 ―
× 100%
(
)
퐴푂0 ― 퐴푂푡
where A0 represents the initial absorbance of each sample, At the absorbance after
10
ACS Paragon Plus Environment

Page 11 of 36
Journal of Agricultural and Food Chemistry
213
incubation for 2.5 h, and AO the absorbance of the blank (addition of 0.2 mL ethanol
to 4.8 mL of the emulsion).
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
Critical Micelle Concentration (CMC) Determined by Fluorescent Probe. The
CMCs of the mono gallate and gemini gallate were measured by the steady state
fluorescence method. Pyrene was applied as the fluorescent probe. 10 μL of pyrene
ethanol solution (1 mmol/L) was added into a standard volumetric flask, followed by
evaporation of the solvent. The sample in aqueous solution of specified concentration
was added, and kept the pyrene concentration at 10-3 mmol/L. All the samples were
treated by ultrasonication for 5 min before measured at 298.15 K. The fluorescence
emission spectra of pyrene were recorded by a fluorescence spectrometer (Cary
Eclipse, Varian, America). Excitation was done at 335 nm while the emission spectra
were scanned over the range of 350-600 nm. The intensity ratio of the first peak (I1 at
around 371 nm) to the third peak (I3 at around 382 nm) of the emission spectra was
used as an index of the environment around pyrene molecules.28 The CMC was
determined by the intersection point of the extended two lines.
Microscope and Transmission Electron Microscopy (TEM) Analysis. The
samples in β-carotene AAPH bleaching assay were analyzed. Micrographs were
obtained by a microscope (CKX53, Olympus Corporation, Tokyo, Japan) and a
transmission electron microscope (SU-8010, Hitachi, Japan). For microscope analysis,
the samples were dropped on a glass slide before observation. For TEM analysis, the
samples were dropped on a copper grid and placed on a filter paper, and then dried in
air before observation.
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 36
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
Statistical Analysis. The data of all the experiments were presented as mean ±
standard deviation (SD). The results were subjected to least significant difference
(LSD) in one-way analysis of variance (ANOVA) applying the SPSS 20.0 software to
analyze the difference. Differences with a p value less than 0.05 were considered
significant.
RESULTS AND DISCUSSION
Design and Preparation of Gemini Gallate. To realize a gemini gallate
antioxidant, the key issue was how to construct the gemini molecular architecture.
Ethylene glycol and its polymeric forms have been ubiquitously employed as building
blocks for molecular modification owing to biodegradable and biocompatible
nature.29,30 As a result, polymeric ethylene glycol was chosen to build the gemini
architecture in this study. Owing to the complexity in selective manipulation of
galloyl moieties, it was preferred to build the gemini chain first followed by
introduction of galloyl moieties at final stage. In previous study, a class of gemini
chain was developed based on polymeric ethylene glycol with alkyl alcohols, which
was proven very effective to promote the activities of the prepared emulsifiers.
During the developed gemini chains, the dodecyl gemini chain exhibited significantly
higher efficiency than other alkyl gemini chains. Hence, the dodecyl gemini chain was
applied in this study to construct the gemini gallate antioxidant, which could be
readily prepared from ethylene glycol epoxide and dodecanol. The molecular
structure of designed gemini gallate and the proposed synthetic route were
12
ACS Paragon Plus Environment

Page 13 of 36
Journal of Agricultural and Food Chemistry
257
demonstrated in Figure 1A.
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
The synthesis of the gemini gallate commenced with protection of the three
hydroxyl groups of gallic acid. According to previous study, isobutyryl was utilized
for protection due to exceptional stability and operational simplicity. Following the
established protocol,25 the protection was smoothly performed by the action of
isobutyric anhydride. Then the dodecyl gemini chain was prepared from
regioselective ring-opening of ethylene glycol epoxide by the dodecanol.21 This step
produced a mixture of the gemini chain and other unidentified side products which
were not easy to separate by traditional flash chromatography. Therefore, a prepacked
column and peristaltic pump based purification system was developed in this study as
indicated in Figure 1B to facilitate isolation of the dodecyl gemini chain. The
prepacked silica gel column had better column efficiency and the system readily
provided analytically pure dodecyl gemini chain for further synthesis. Then the
protected gallic acid was coupled with the dodecyl gemini chain through Steglich
esterification applying the optimized condition to minimize side reactions.25 Finally,
deprotection of the protected gemini gallate by aqueous hydrazine successfully
provided the gemini gallate antioxidant. After preparation of the gemini gallate, the
mono gallate was also synthesized for comparison from the protected gallic acid and
dodecanol through Steglich esterification and deprotection with the same conditions.
Structural Analysis of the Mono Gallate and the Gemini Gallate. The molecular
structures of the mono gallate (MG) and the gemini gallate (GG) were confirmed by
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 36
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
NMR, FT-IR and HPLC-MS.
1H NMR of MG (400 MHz，DMSO-d6) δ 9.17 (br, 2 H; phenolic proton), 8.88 (br,
1 H; phenolic protons), 6.93 (s, 2 H; aromatic protons of the galloyl group), 4.14 (t, J
= 6.5 Hz, 2 H; oxygen adjacent protons of dodecanol), 1.82-0.72 (m, 23 H; protons of
dodecanol).
13C NMR of MG (100 MHz，DMSO-d6) δ 165.80, 145.47, 138.26, 119.57, 108.44,
63.87, 31.28, 29.03, 29.00, 28.97, 28.70, 28.28, 25.51, 22.08, 13.91. The 13C chemical
shifts of 165.80 ppm corresponded to the signal of the galloyl ester carbon, 145.47 to
108.44 ppm corresponded to the signals of the aromatic carbons of gallic acid, and
63.87 to 13.91 ppm corresponded to the signals of dodecyl carbons.
1H NMR of GG (400 MHz, DMSO-d6) δ 9.23 (s, 4 H; phenolic protons), 8.93 (m,
2 H; phenolic protons), 6.94 (s, 4 H; aromatic protons of the galloyl group), 5.12 (t, J
= 5.2 Hz, 2 H; galloyl oxygen adjacent methine protons of the gemini chain),
3.66-3.28 (m, 16 H; oxygen adjacent methylene protons of the gemini chain),
1.54-0.80 (m, 46 H; oxygen nonadjacent protons of the gemini chain).
13C NMR of GG (100 MHz, DMSO-d6) δ 165.38, 145.56, 138.54, 119.37, 108.68,
71.41, 70.65, 70.14, 69.33, 68.88, 31.35, 29.09, 29.07, 28.88, 28.77, 25.60, 22.15,
13.98. The 13C chemical shifts of 165.38 ppm corresponded to the signals of the
galloyl ester carbons, 145.56 to 108.68 ppm corresponded to the signals of the
aromatic carbons of gallic acid, 71.41 to 13.98 ppm corresponded to the signals of the
gemini chain carbons.
IR of MG (cm-1) 3450 (s, νO−H), 3330 (s, νO−H), 2918 (s, νC−H), 2849 (s, νC−H), 1670
14
ACS Paragon Plus Environment

Page 15 of 36
Journal of Agricultural and Food Chemistry
301
(s, νC=O), 1626 (s, νC=C), 1608 (s, νC=C), 1468 (s, δO−H), 1404 (s, δO−H), 1300 (s, νC−O),
1258 (s, νC−O), 1027 (s, δC−H), 769 (m, δC−H). The absorption bands at 3450 cm−1 and
3330 cm-1 corresponded to the stretching vibrations of the phenolic hydroxyl groups
of gallic acid. The absorption band at 1670 cm−1 was attributed to C＝O stretching
vibration, which confirmed esterification of gallic acid and dodecanol.
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
IR of GG (cm-1) 3420 (m, νO−H), 2924 (s, νC−H), 2853 (s, νC−H), 1632 (w, νC=O),
1463 (m, δO−H), 1378 (m, δO−H), 1083 (w, δC−H), 721 (w, δC−H). The absorption band at
3420 cm−1 corresponded to the stretching vibration of the phenolic hydroxyl groups of
gallic acid. The absorption band at 1632 cm−1 was attributed to C ＝ O stretching
vibration, which confirmed esterification of gallic acid and the gemini chain.
The identity and purity of GSt and GSi were further investigated by HPLC-MS
(Figure 2). ESI-MS spectra of MG and GG (negative ion mode) clearly exhibited
their corresponding structures: MG, [M-H]- 337.33 and [2M-H]- 674.56; GG, [M-H]-
849.33. Then HPLC-MS analysis revealed that the purities of MG and GG were
99.2% and 93.6%, respectively. The confirmation of structure and purity assured
further antioxidant activity studies.
Radical Scavenging Activities of the Mono Gallate and Gemini Gallate
Evaluated by DPPH and ORAC Assays. Radical scavenging activities of the mono
and gemini gallates (MG, GG) were evaluated by the generally applied DPPH assay,
which was developed by Blois who first identified DPPH as a source of stable free
radicals.31 In the study, the unmodified gallic acid and the typical lipophilic
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 36
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
antioxidants including BHA, BHT and TBHQ were also investigated for comparison.
As shown in Figure 3A, MG and GG exhibited outstanding antioxidant activities
compared with other antioxidants, suggesting successful inheritance of the antioxidant
activity from GA. Then the EC50 value expressed as Trolox equivalent (TE) was
employed to evaluate their DPPH radical scavenging activity.32 According to Table 1,
the EC50 values of the antioxidants were described as follows: BHA (0.794 TE), BHT
(0.397 TE), TBHQ (1.059 TE), GA (2.060 TE), MG (1.806 TE) and GG (3.835 TE).
The EC50 values demonstrated that the antioxidant activities of MG and GG were
substantially higher than those of the frequently employed antioxidants, BHA, BHT
and even TBHQ. The EC50 value of the gemini gallate was around twice of that of the
mono gallate, which suggested that the antioxidant behavior of mono and gemini
antioxidants was similar in the homogeneous system because the antioxidant activity
was just proportional to the molar ratio of the galloyl group (MG, 1 galloyl group;
GG, 2 galloyl groups).
Furthermore, the antioxidant activities of MG and GG were evaluated by the
frequently used ORAC assay. Initially, this method was proposed and named by Cao
et al.33 who took β-phycoerythrin as an indicator protein, AAPH as a peroxyl radical
generator and Trolox as a standard. Later fluorescein rather than β-phycoerythrin was
applied as the indicator owing to excellent photostability and notable reduction of
cost.34 To study the lipophilic MG and GG antioxidants, a previously developed
fat-soluble fluorescent probe, dodecanoyl 5-aminofluorescein (DAF), was employed
as the indicator,27 and Tween-80 was added to promote the aqueous solubility in this
16
ACS Paragon Plus Environment

Page 17 of 36
Journal of Agricultural and Food Chemistry
345
investigation. The concentration of the MG was set to twice of that of the GG to
facilitate comparison of their antioxidant capacities. As shown in Figure 3B, the
antioxidant activity (indicated as Net AUC) of GG was basically twice of that of MG
during the examined concentration (3.12 to 50 μmol/L). The ORAC values were
further expressed as Trolox equivalent (TE) as follows: MG (3.05 TE), GG (6.22 TE).
As a result, the antioxidant activity of ORAC assay was proportional to the molar
ratio of the galloyl group, demonstrating that there was no antioxidant behavior
difference between MG and GG in a sufficiently solubilized system which was
consistent with the DPPH assay. Presumably, the difference of self-assembly behavior
of MG and GG was masked in a sufficiently solubilized or homogeneous system.
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
Inhibition of β-Carotene Oxidation in O/W Emulsion. After demonstration of
the extraordinary radical scavenging activities of MG and GG, their antioxidant
activities in heterogeneous O/W emulsion system were further explored. The
β-carotene O/W emulsion was applied as the model to investigate the antioxidant
activity in emulsion.11 Traditionally, linoleic acid was often employed to generate free
radicals and bleach the unsaturated β-carotene in emulsion. However, the
autooxidation of linoleic acid was not easy to control and so inconvenient for further
structural analysis. In previous study, an AAPH-based β-carotene O/W emulsion
assay was developed employing AAPH as the aqueous radical initiator.11 Thermal
initiation of AAPH was readily controllable. As a result, AAPH-based β-carotene
bleaching assay was applied in this study to facilitate further investigation.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 36
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
As disclosed in Figure 4A, GG exhibited outstanding antioxidant activity in O/W
emulsion though slightly lower than TBHQ but higher than other antioxidants. It was
not a surprise that the antioxidant activity of more lipophilic MG was lower than those
of BHA and BHT at low concentration since the radical was produced in aqueous
phase by hydrophilic AAPH. As demonstrated in previous study, the performance of
an antioxidant was greatly influenced by either location of the radical source or
distribution of the antioxidant.11 The less lipophilic BHA and BHT were more
efficient to scavenge radicals in aqueous phase at low concentration. These results
were consistent with the previous study. As expected, MG would be more efficient
when the radicals were generated in oil phase as indicated in previous study.11
Although GG demonstrated eminent antioxidant activity in O/W emulsion, the
activity relative to MG was still proportional to the molar ratio of the galloyl group
probably owing to that excessive addition of Tween-80 emulsifier diluted the
effective interfacial concentration of the antioxidant and also masked the difference of
self-assembly behavior.12-15
To unmask the difference of MG and GG, their antioxidant activities in β-carotene
O/W emulsion were further observed with reduced amount of Tween-80. To our
delight, exceptional antioxidant activity of GG (5 to 2 times relative to MG) was
unveiled at low concentration (<0.5 mmol/L) in the test (Figure 4B). The antioxidant
activity ratio (GG to MG: 5, 0.25 mmol/L) was significantly higher than the molar
ratio of the galloyl group (GG to MG: 2), demonstrating notable contribution from the
difference of self-assembly behavior. The significant promotion of the antioxidant
18
ACS Paragon Plus Environment

Page 19 of 36
Journal of Agricultural and Food Chemistry
389
activity in O/W emulsion under the condition of reduced amount of the emulsifier
suggested that the gemini antioxidant more readily assembled at interfacial layers and
so increased the interfacial concentration and also helped to maintain the integrity of
oil droplets when the emulsifier was limited.12-15,21 As a result, these results exhibited
the superiority of the gemini antioxidant relative to the traditional mono antioxidant
especially under the condition of low concentration of emulsifier.
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
Microscopic Investigation of Antioxidant Activity in O/W Emulsion. To further
understand details of the antioxidant behavior in O/W emulsion, critical micelle
concentrations (CMC) of the antioxidants were determined by a steady-state
fluorescence method employing pyrene as the fluorescent probe. The ratio of
fluorescence intensity (I1/I3) reflected the hydrophobicity of the microenvironment of
pyrene. A surface-active compound would cause an abrupt change of the curve of I1/I3
at the point of micelle formation. As shown in Figure 5, there were no inflection
points observed for lipophilic BHA, BHT, TBHQ and hydrophilic GA, which
confirmed that they were not surface-active and so had no micellization activity.
Notable surface activity was observed for MG, and the CMC of MG was determined
as 0.13 mmol/L through calculation of the intersection of the trend lines. The CMC of
GG determined as 0.04 mmol/L was significantly smaller than that of MG, which
confirmed superior micellization activity of the gemini antioxidant. The superior
micellization capacity presumably increased effective interfacial concentration of GG
and promoted antioxidant activity accordingly.12-15
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 36
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
Then the microstructure of β-carotene emulsions of MG and GG with normal and
reduced amount of Tween-80 was explored by optical microscopy and transmission
electron microscopy (TEM) to further understand the antioxidant behavior. As
revealed in microscopic bright field images, both MG emulsion (Figure 6a1, 6b1)
and GG emulsion (Figure 6a2, 6b2) had a consistent dispersion of the oil droplets in
aqueous phase. The oil droplets in GG emulsion were generally larger than those in
MG emulsion. Moreover, the oil droplets in MG or GG emulsion with normal amount
of Tween-80 (Figure 6a1 or 6a2) were larger than those in the corresponding
emulsion with reduced amount (Figure 6b1 or 6b2). The details of the microstructure
of MG and GG emulsions were further scrutinized by TEM. The darker inner area of
oil droplets in GG emulsion (Figure 6a4) clearly demonstrated better encapsulation
of β-carotene relative to MG emulsion (Figure 6a3). Under the condition of reduced
amount of Tween-80, the oil droplets in GG emulsion (Figure 6b4) exhibited
significantly better encapsulation of β-carotene compared with MG emulsion (Figure
6b3). Hence, the microscopic images of the O/W emulsions confirmed great
micellization ability of GG which resulted in the outstanding antioxidant
performance.
Therefore, these results proved the concept that gemini architecture would improve
antioxidant performance in O/W emulsion presumably through increase of interfacial
concentration, thus referred to as gemini interfacial antioxidant. The great
micellization capacity of the gemini interfacial antioxidant could be attributed to the
tighter connection enforced by the ethylene glycol chain through elimination of the
20
ACS Paragon Plus Environment

Page 21 of 36
Journal of Agricultural and Food Chemistry
433
steric interaction between bulky galloyl moieties.21 The outstanding micellization
capacity of the gemini interfacial antioxidant eventually brought about preferable
location at interfacial layer and enhancement of antioxidant performance in
heterogeneous system. Reduction application of food additives in a food system was
generally welcome because food additives often produced undesired off-flavor. The
gemini interfacial antioxidant was of the activity of emulsifier and so highly desirable.
Hence, the gemini interfacial antioxidant was very useful to lower the amount of
emulsifier and particularly as the low concentration of emulsifier was required. The
developed concept of gemini interfacial antioxidant would be useful for design of
antioxidants in complicated settings.
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
In conclusion, the gemini gallate interfacial antioxidant was designed and
successfully prepared by Steglich esterification with an improved purification
protocol through a prepacked column and peristaltic pump based purification system.
The gemini gallate had excellent radical scavenging activity and superior antioxidant
activity in O/W emulsion relative to the mono gallate especially under the condition
of reduced amount of emulsifier. Further studies revealed that the extraordinary
antioxidant performance of the gemini interfacial antioxidant in O/W emulsion was
presumably attributed to increased interfacial concentration of the antioxidant
enforced by the gemini molecular architecture. The superior antioxidant activity of the
gemini antioxidant hold a great promise for antioxidation in O/W emulsion.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 36
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
AUTHOR INFORMATION
Corresponding Author
*Songbai Liu. Phone: +86-15168319078. E-mail: songbailiu@zju.edu.cn.
Funding
This work was supported by the National Key Research and Development Program
(2016YFD0400805, 2017YFF0207800), Zhejiang Public Welfare Technology
Research Program (LGN18C200009), Qinghai Science and Technology Program
(2017-ZJ-Y06, 2016-NK-C22, 2015-NK-502), Foundation of Fuli Institute of Food
Science at Zhejiang University, Zhejiang Science and Technology Program
(2017C26004).
Notes
The authors declare no conflict of interest.
REFERENCE
(1) Azizi, M.; Li, Y. T.; Kaul, N.; Abbaspourrad, A. Study of the Physicochemical
Properties of Fish Oil Solid Lipid Nanoparticle in the Presence of Palmitic Acid and
Quercetin. J. Agric. Food Chem. 2019, 67, 671-679.
(2) Carlson, S. J.; Fallon, E. M.; Kalish, B. T.; Gura, K. M.; Puder, M. The Role of the
ω-3 Fatty Acid DHA in the Human Life Cycle. J. Parenter. Enteral. Nutr. 2013, 37,
15-22.
(3) Gil, A.; Plaza-Diaz, J.; Mesa, M. D. Vitamin D: Classic and Novel Actions. Ann.
22
ACS Paragon Plus Environment

Page 23 of 36
Journal of Agricultural and Food Chemistry
477
Nutr. Metab. 2018, 72, 87-95.
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
(4) Tippetts, M.; Martini, S.; Brothersen, C.; McMahon, D. J. Fortification of cheese
with vitamin D-3 using dairy protein emulsions as delivery systems. J. Dairy Sci.
2012, 95, 4768-4774.
(5) Igielska-Kalwat, J. Studies on stability of emulsions containing carotenoids. Acta
Biochim. Pol. 2018, 65, 455-463.
(6) Kopec, R. E.; Failla, M. L. Recent advances in the bioaccessibility and
bioavailability of carotenoids and effects of other dietary lipophiles. J. Food Compos.
Anal. 2018, 68, 16-30.
(7) Laguerre, M.; Bayrasy, C.; Panya, A.; Weiss, J.; McClements, D. J.; Lecomte, J.;
Decker, E. A.; Villeneuve, P. What Makes Good Antioxidants in Lipid-Based
Systems? The Next Theories Beyond the Polar Paradox, Crit. Rev. Food Sci. Nutr.
2015, 55, 183-201.
(8) McClements, D. J.; Decker, E. Interfacial Antioxidants: A Review of Natural and
Synthetic Emulsifiers and Coemulsifiers That Can Inhibit Lipid Oxidation. J. Agric.
Food Chem. 2018, 66, 20-35.
(9) Hunneche, C. S.; Lund, M. N.; Skibsted, L. H.; Nielsen, J. Antioxidant activity of
a combinatorial library of emulsifier - Antioxidant bioconjugates. J. Agric. Food
Chem. 2008, 56, 9258-9268.
(10) Feng, J.; Cai, H.; Wang, H.; Li, C. Y.; Liu, S. B. Improved oxidative stability of
fish oil emulsion by grafted ovalbumin-catechin conjugates. Food Chem. 2018, 241,
60-69.
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 36
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
(11) Wang, S. S.; Fan, J. W.; Xu, L. J.; Ye, K.; Shu, T.; Liu, S. B. Enhancement of
Antioxidant Activity in O/W Emulsion and Cholesterol-Reducing Capacity of
Epigallocatechin by Derivatization with Representative Phytosterols. J. Agric. Food
Chem. 2019, 67, 12461-12471.
(12) Mitrus, O.; Zuraw, M.; Losada-Barreiro, S.; Bravo-Diaz, C.; Paiva-Martins, F.
Targeting Antioxidants to Interfaces: Control of the Oxidative Stability of
Lipid-Based Emulsions. J. Agric. Food Chem. 2019, 67, 3266-3274.
(13) Costa, M.; Losada-Barreiro, S.; Paiva-Martins, F.; Bravo-Diaz, C. Physical
evidence that the variations in the efficiency of homologous series of antioxidants in
emulsions are a result of differences in their distribution. J. Sci. Food Agric. 2017, 97,
564-571.
(14) Freiria-Gandara, J.; Losada-Barreiro, S.; Paiva-Martins, F.; Bravo-Diaz, C.
Enhancement of the antioxidant efficiency of gallic acid derivatives in intact fish
oil-in-water emulsions through optimization of their interfacial concentrations. Food
Funct. 2018, 9, 4429-4442.
(15) Ferreira, I.; Costa, M.; Losada-Barreiro, S.; Paiva-Martins, F.; Bravo-Diaz, C.
Modulating the interfacial concentration of gallates to improve the oxidative stability
of fish oil-in-water emulsions. Food Res. Int. 2018, 112, 192-198.
(16) Sakai, K.; Sakai, H.; Abe, M. Recent Advances in Gemini Surfactants: Oleic
Acid-Based Gemini Surfactants and Polymerizable Gemini Surfactants. J. Oleo Sci.
2011, 60, 159-163.
(17) Menger, F. M.; Keiper, J. S. Gemini surfactants. Angew. Chem., Int. Ed. 2000,
24
ACS Paragon Plus Environment

Page 25 of 36
Journal of Agricultural and Food Chemistry
521
39, 1907-1920.
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
(18) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.;
derman, O. S.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; García Rodríguez, C. L.;
Guédat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P.
Gemini Surfactants: New Synthetic Vectors for Gene Transfection. Angew. Chem. Int.
Ed. 2003, 42, 1448-1457.
(19) Sharma, R.; Kamal, A.; Abdinejad, M.; Mahajan, R. K.; Kraatz, H. B. Advances
in the synthesis, molecular architectures and potential applications of gemini
surfactants. Adv. Colloid Interface 2017, 248, 35-68.
(20) Zhao, W. W.; Wang, Y. L. Coacervation with surfactants: From single-chain
surfactants to gemini surfactants. Adv. Colloid Interface 2017, 239, 199-212.
(21) Liu, S. B.; Sang, R. C.; Hong, S.; Cai, Y. J.; Wang, H. A Novel Type of Highly
Effective Nonionic Gemini Alkyl O-Glucoside Surfactants: A Versatile Strategy of
Design. Langmuir 2013, 29, 8511-8516.
(22) Choubey, S.; Varughese, L. R.; Kumar, V.; Beniwal, V. Medicinal importance of
gallic acid and its ester derivatives: a patent review. Pharm. Pat. Anal. 2015, 4,
305-315.
(23) Locatelli, C.; Filippin-Monteiro, F. B.; Creczynski-Pasa, T. B. Alkyl esters of
gallic acid as anticancer agents: A review. Eur. J. Med. Chem. 2013, 60, 233-239.
(24) Fu, Y.; Zhang, Y.; Hu, H.; Chen, Y.; Wang, R.; Li, D.; Liu, S. Design and
straightforward synthesis of novel galloyl phytosterols with excellent antioxidant
activity. Food Chem. 2014, 163, 171-177.
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 36
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
(25) Wang, S. S.; Ye, K.; Shu, T.; Tang, X. W.; Wang, X. J.; Liu, S. B. Enhancement
of Galloylation Efficacy of Stigmasterol and beta-Sitosterol Followed by Evaluation
of Cholesterol-Reducing Activity. J. Agric. Food Chem. 2019, 67, 3179-3187.
(26) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common
laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.
(27) Chaiyasit, W.; McClements, D. J.; Weiss, J.; Decker, E. A. Impact of
surface-active compounds on physicochemical and oxidative properties of edible oil.
J. Agric. Food Chem. 2008, 56, 550-556.
(28) Wilhelm, M.; Zhao, C. L.; Wang, Y. C.; Xu, R. L.; Winnik, M. A.; Mura, J. L.;
Riess, G.; Croucher, M. D. Polymer Micelle Formation .3. Poly(Styrene-Ethylene
Oxide) Block Copolymer Micelle Formation in Water - a Fluorescence Probe Study.
Macromolecules 1991, 24, 1033-1040.
(29) Kutikov, A. B.; Song, J. Biodegradable PEG-Based Amphiphilic Block
Copolymers for Tissue Engineering Applications. ACS Biomater. Sci. Eng. 2015, 1,
463-480.
(30) Ulbrich, K.; Holá, K.; Šubr, V.; Bakandritsos, A.; Tuček, J.; Zbořil, R.; Targeted
Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent
Approaches, Release Control, and Clinical Studies. Chem. Rev. 2016, 116,
5338-5431.
(31) Blois, M. S. Antioxidant Determinations by the Use of a Stable Free Radical.
Nature 1958, 181, 1199-1200.
(32) Chen, Z.; Bertin, R.; Froldi, G. EC50 estimation of antioxidant activity in DPPH
26
ACS Paragon Plus Environment

Page 27 of 36
Journal of Agricultural and Food Chemistry
565
center dot assay using several statistical programs. Food Chem. 2013, 138, 414-420.
(33) Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay
for antioxidants. Free Radical Biol. Med. 1993, 14, 303-11.
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
(34) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and Validation of an
Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the
Fluorescent Probe. J. Agric. Food Chem. 2001, 49, 4619-4626.
Figure 1. (A) Synthetic route of the gemini gallate and (B) the prepacked column and
peristaltic pump based purification system.
Figure 2. HPLC-MS of (A) the mono gallate (MG) and (B) the gemini gallate (GG).
Figure 3. (A) DPPH radical scavenging activities and (B) Net AUC of ORAC assay.
Table 1. EC50 Values of BHA, BHT, TBHQ, GA, MG, and GG in DPPH Assay.
Figure 4. (A) Inhibition of β-carotene oxidation in O/W emulsion and (B) ratio of the
antioxidant activity of GG to MG with reduced amount of Tween-80.
Figure 5. Critical micelle concentration (CMC) of the antioxidants determined by
pyrene fluorescent probe.
Figure 6. Images of O/W emulsions in β-carotene bleaching assay: (a1-a4) the
emulsions with normal amount of Tween-80; (b1-b4) the emulsions with reduced
amount of Tween-80; (a1, b1) micrographs of MG emulsions under bright field; (a2,
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 36
587
588
589
b2) micrographs of GG emulsions under bright field; (a3, b3) TEM images of MG
emulsions; (a4, b4) TEM images of GG emulsions.
28
ACS Paragon Plus Environment

Page 29 of 36
Journal of Agricultural and Food Chemistry
Figure 1
ACS Paragon Plus Environment