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Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish
Journal Pre-proof Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish Jian Li, Shi-Ying Huang, Qianying Deng, Guiling Li, Guocheng Su, Jingwen Liu, HuiMin David Wang PII:
S0278-6915(19)30840-3
DOI:
https://doi.org/10.1016/j.fct.2019.111050
Reference:
FCT 111050
To appear in:
Food and Chemical Toxicology
Received Date: 17 September 2019 Revised Date:
3 December 2019
Accepted Date: 8 December 2019
Please cite this article as: Li, J., Huang, S.-Y., Deng, Q., Li, G., Su, G., Liu, J., David Wang, H.-M., Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2019.111050. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Authors’ Contributions Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang conceived and designed the experiments; Qianying Deng, Guilin Li, Guocheng Su, and Jingwen Liu performed the experiments and analyzed the data; Jian Li and Hui-Min David Wang contributed the reagents, materials, and analysis tools; Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang wrote the paper.
Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish
Jian Lia,b,*, Shi-Ying Huanga, Qianying Denga, Guiling Lia,b, Guocheng Sua, Jingwen Liua, Hui-Min David Wanga,c, d,e,*
a
College of Food and biological engineering, Jimei University, Xiamen 361021, China
b
Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen 361021, China
c
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan
d
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung City 404, Taiwan
e
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan Graphic Abstract
Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish
Jian Lia,b,*, Shi-Ying Huanga, Qianying Denga, Guiling Lia,b, Guocheng Sua, Jingwen Liua, Hui-Min David Wanga,c, d,e,*
a
College of Food and biological engineering, Jimei University, Xiamen 361021, China
b
Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen 361021, China
c
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan
d
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung City 404, Taiwan
e
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
*Corresponding Authors: Jian Li, Ph.D. Associate Professor, College of Biological Engineering, Jimei University Address: No.43, Yindou Rd., Xiamen city, Fujian Province, 361021, China Tel: 0086-592-6181915 Fax: 86-592-6180470 E-mail: [email protected]
Hui-Min David Wang, Ph.D. 1
Professor, Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan Address: No.145, Xingda Rd., South Dist., Taichung City 402, Taiwan Mobil: 886-935753718 TEL: 886-4-2284-0733#651 Fax: 886-4-22852242 E-mail: [email protected] https://sites.google.com/site/davidbiocosme/home
2
ABSTRACT The pickled radish can be kept at room temperature for years without spoilage. 2,6-dihydroxyacetophenone (DHAP), 4-hydroxybenzaldehyde (HBA), and 4-hydroxyphenethyl alcohol (4-HPEA) were first found from the pickled radish. The structures of three phenolic compounds were elucidated by analysis of their nuclear magnetic resonance and high-resolution electro-spray ionization mass spectrometry data. All these phenolic compounds showed good free radical scavenging capacity except HBA. Both DHAP and 4-HPEA also showed high ferric reducing ability. DHAP showed good antimicrobial activity against Escherichia coli, Bacillus subtilis, and Canidia albicans. HBA demonstrated antimicrobial activity against E. coli and C. albicans but not B. subtilis. Based on the results of MTT assay, these compounds did not show cytotoxicity to LO2 cell line. All results indicated the pickled radish had antioxidant and antimicrobial phenolic compounds. To the best of our knowledge, this report is the first to answer partially the question of why pickled foods can be kept at room temperature for years without spoilage based on the evidence of three phenolic compounds.
Keywords Pickled radish; Phenolic compounds; Antioxidant; Antimicrobial; Shelf life; Stability
3
1
1. Introduction
2
Pickled radish was processed by traditional method in China. Fresh white radish is dehydrated
3
with sea salt and pressed, and then pickled in ceramic container without any other ingredients. The
4
pickled radish has a very long shelf life, and it can be kept at room temperature up to 20 years
5
without package. The antioxidant activity of radish is partly due to acylated pelargonidin derivatives
6
(Wang et al., 2010). Complex biochemical reactions occur in the pickling and fermentation of
7
kimchi (Cheigh et al., 1994). Pickled radish has particular nutritional content and health benefits
8
(Kumakura et al., 2017). Many bioactive components with antimicrobial and/or antioxidant
9
generate from the pickling process, such as phenolic compounds (Jing et al., 2014). However, there
10
was no study on the phenolic compounds of pickled radish.
11
Plant phenolic compounds have been extensively studied due to their bioactive activities and
12
also healthy benefits for human beings (Zheng and Wang, 2001; Derakhshan et al., 2018; Qin et al.,
13
2019). The aromatic ring having the hydroxyl groups is a family characteristic of these compounds,
14
and these structures include high-molecular weight polymers or small phenolic molecules
15
(Balasundram et al., 2006; Wang et al., 2019). Phenolic compounds are usually used as natural
16
antioxidants in foods to extend the shelf life (Caponio et al., 2001; Estevinho et al., 2008;
17
Llorent-Martinez et al., 2017). Phenolic compounds in cooked ground walnut showed high
18
antioxidant activities during refrigerated storage (Ahn et al., 2002). Phenolics in clove essential oil
19
can enhance the stability of cake and other lipid foods for storage (Ibrahium et al., 2013). Phenolic
20
compounds can also inhibit the growth of microorganisms, such as E. coli, K. pneumoniae, B.
21
cereus, A. flavus, and A. parasiticus (Aziz et al., 1998). Different concentrations of phenolic
22
compounds exhibit different sensitivities towards different microorganisms (Vaquero et al., 2007).
23
However, antimicrobial activity of phenolic compounds from pickled radish has not been shown in
24
the previous literature.
25
In this study, the compounds with antimicrobial activities from pickled radish were
26
characterized. The antioxidant activity and cytotoxicity of the compounds from the pickled radish
27
were evaluated. All the results indicated that these phenolic compounds play an important role in
28
pickled radish shelf life than other factors. These phenolic compounds extracted from pickled radish
29
might also be considered be used in other food preservation.
30 31
2. Materials and Methods
32
2.1. Materials
33
Pickled radish was obtained from stores in Zhangpu, Fujian Province. Reverse phase (RP-18)
34
Silica gel was purchased from YMC (Kyoto, Japan). Silica gel (Silia Flash P60) for column
35
chromatography was purchased from SiliCycle (Quebec City, Canada). Sephadex LH-20 was
36
purchased from GE Healthcare (Uppsala, Sweden). Precoated silica gel GF254 plates were
37
purchased from Qingdao Marine Chemical Factory (Qingdao, China). Membrane filter was
38
purchased from Millipore Co. (Milford, MA). Human liver cell lines (LO2) were obtained from
39
Chinese Academy of Sciences Cell Collection Center (Shanghai, China).
40
2.2. Chemicals
41
We obtained the chemicals from Sigma-Aldrich (St. Louis, MO): dimethyl sulfoxide (DMSO),
42
vitamin
C
(L-ascorbic
acid),
2,2-Diphenyl-1-picrylhydrazyl
43
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We purchased DMEM-high
44
glucose medium, antibiotics (streptomycin and penicillin), and fetal bovine serum (FBS) through
45
HyClone (Northbrook, IL). Total antioxidant capacity assay kit for FRAP and ABTS assay was
46
purchased from Beyotime Institute of Biotechnology (Haimen, China). All other reagents were 5
(DPPH),
and
47
analytical grade and purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). A
48
Milli-Q reagent water system, which was obtained from Millipore Co. (Milford, MA), produced
49
deionized water.
50
2.3. Preparation of crude extract
51
Pickled radish was air-dried at 40 ℃ for 24 h, and was then extracted with methanol, acetone,
52
and ethyl acetate for 12 h. The extraction solution was passed through membrane filter to remove
53
insoluble. Then the extraction solution was vacuumed evaporated to obtain crude extracts at 40 ℃.
54
The antimicrobial activities were measured by oxford cup method.
55
2.4. Purification and identification of antimicrobial compounds from the crude extract
56
Gel column chromatography: Methanol extract was dissolved in a small amount of methanol,
57
loaded on pretreated Sephadex LH-20 gel column, using methanol as eluent, according to the
58
sample amount to control velocity within the limits of 10-15 s/drop. Fractions were collected every
59
30 min in a collection tube by automatic fraction collector. All fractions were analyzed by TLC and
60
pooled separately according to the results of TLC analysis. Antimicrobial activity of the pooled
61
fractions was measured by oxford cup method as described later. Fractions with antimicrobial
62
activity were further purified; however, fractions without antimicrobial activity were not further
63
purified.
64
Reversed phase silica gel column chromatography: After LH-20 gel column chromatography
65
separation, fractions with antimicrobial activities were filtrated through membrane filter and
66
vacuumed evaporated. Fractions were dissolved in methanol, then subjected to the MPLC (RP-18)
67
for further isolation with water and methanol. All fractions were collected and vacuumed
68
evaporated at 40 ℃, and were then dissolved with appropriate amount of methanol for TLC
69
analysis and antimicrobial activity assay. 6
70
Silica gel column chromatography: Fractions with antimicrobial activities were further purified in
71
a silica gel column with petroleum ether and acetone as the eluent solvent. Elution fractions was
72
pooled according to TLC analysis and then vacuumed evaporated at 40 ℃ for further instrumental
73
analysis.
74
Instrumental Analysis: Nuclear magnetic resonance (NMR) spectra from samples were obtained in
75
CDCl3 by a Bruker AVANCE III HD 400MHz (Bruker Biospin GmbH, Karlsruhe, Germany). We
76
reported chemical shifts (δ) in units of parts per million (ppm) versus tetramethylsilane. We
77
corrected chemical shift to residual solvent signal, 7.26 ppm. Mass spectrometry was acquired by
78
Xevo™ G2 Q Tof (Waters MS Technologies, Manchester, UK), a quadrupole and orthogonal
79
acceleration time-of-flight tandem mass spectrometer. The carrier solvent for flow injection analysis
80
was methanol. The scan range was from 50 to 1000 m/z for both positive and negative electrospray
81
modes. All the acquisition and analysis of data were controlled by Waters Mass Lynx v4.1 software,
82
respectively.
83
2.5. Determination of antioxidant activity
84
We measured the antioxidant activity by DPPH assay (Yang et al., 2006; Luo et al., 2009). A
85
DPPH solution (0.25 mM; 99 µL) in methanol was added to each compound’s solution (1 µL). We
86
used L-ascorbic acid (100 µM) as the positive control. We measured the absorption at OD517 nm
87
after 30 min, and obtained the inhibitory effect using the formula: (1)
88
89
We measured total antioxidant capacity using an assay kit of ferric reducing ability of plasma
90
(FRAP) (Benzie and Strain, 1996; Benzie and Szeto, 1999). Prepare the working solution through
91
fully mixing 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) diluents with TPTZ solution in a ratio of 10:1,
7
92
followed by mixing with detection buffer in equal quantities with TPTZ solution, and finally
93
incubated at 37 ℃ before use. Standard curve was prepared by Trolox in order to calculate Trolox
94
equivalent antioxidant capacity (TEAC) values (mM/g). 5 µL of compounds solution with different
95
concentrations were mixed with 180 µL working solution, which were incubated at 37 ℃ for 5
96
min. We measured absorbance at 593 nm from mixture.
97
Total antioxidant capacity was measured by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
98
acid) (ABTS) assay kit (Luo et al., 2012; Zhao et al., 2015). We produced the working solution
99
through mixing stock ABTS solution with stock oxidant solution in equal quantities, and the stock
100
solutions reacted for 12-16 h in the dark. Then, the stock solutions were diluted 40 times with PBS
101
to get working solution, and subsequently 10 µL of compounds (50 µg/mL) solution were mixed
102
with 200 µL working solution. At 6 min after incubation at room temperature, we measured
103
absorbance with 734 nm, and expressed results as TEAC mM/g.
104
2.6. Microbial cultures and antimicrobial assay
105
We evaluated antimicrobial effects of compounds on Escherichia coli, Bacillus subtilis, and
106
Canidia albicans, which were purchased from China General Microbiological Culture Collection
107
Center (CGMCC). E. coli and B. subtilis were grown in Luria-Bertani (LB) medium at 37 ℃, and
108
C. albicans was cultured in potato dextrose agar medium at 27 ℃. The antimicrobial assay of
109
compounds was performed by the oxford cup method (Soković et al., 2010; Huang et al., 2012).
110
The bacterial suspensions were inoculated to soft agar media to a final concentration of 1.0 × 105
111
CFU/mL. After the soft agar medium is solidified, the oxford cup containing compounds solution,
112
negative control (methanol), and positive control (chloromycetin and amphotericin B) were placed
113
on the agar plates. After 24 h incubation, the diameter of the inhibition zones was measured.
114
2.7. Minimum inhibitory concentration (MIC) of compounds 8
115
Minimum inhibitory concentration (MIC) of compounds were determine by broth
116
micro-dilution method (Golestani et al., 2015; Wu et al., 2015). In 96-well microplates, we prepared
117
the compounds (2-fold serial dilutions) and positive control. Take a few colonies from the agar plate
118
for creating the inoculum by a sterile swab, and dilute the McFarland standard into media. Dispense
119
the inocula with the test compounds, which were incubated for 24 h at 27 or 37 ℃. Determine the
120
MIC value by reading the microdilution plate. The lowest concentration with no visible growth and
121
with ∆OD less than 0.05 was defined as minimum inhibitory concentration (MIC).
122
2.8. Cytotoxicity assay
123
Cells were cultured at 37 ℃ with 5 % CO2. The medium was changed every 2 days. The
124
cytotoxicity
of
compounds
to
LO2
cells
was
determined
by
125
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosmann, 1983). LO2
126
cells were seeded into a 96-well plate at 104 cells per well. After 24 h, the cells were incubated with
127
the compounds at serial dilutions for additional 24 h incubation. Add 10 µL MTT stock solution (5
128
mg/mL) to each well at 37 ℃ for 4 h. A negative control was prepared by mixing 10 µL of the
129
MTT stock solution and 100 µL of medium alone, and was incubated at 37 ℃ for 4 h. Add 50 µL
130
of DMSO to each well and mix thoroughly using the pipette. Incubate the microplate at 37 ℃ for
131
10 min. Mix each sample again using a pipette and read absorbance at 540 nm.
132
2.9. Statistical analysis
133
Statistical analysis of the data (mean ± SD) was performed using t one-way ANOVA and
134
Duncan test (SPSS statistics 17.0 software program). The present study considered P < 0.05 for
135
significantly different between groups.
136 137
3. Results and Discussion 9
138
3.1. Characterization for purified compounds
139
Compound 1 was obtained as a beige powder and was soluble in methanol, acetone, ethyl
140
acetate, and chloroform. In 1H NMR spectra data from compound 1, there were 3 signals: 1 Me
141
[δ(H) 2.75 (s)] and 2 aromatic CH groups [δ(H) 6.41(d), 7.24(d)] (Table 1). In 13C NMR spectrum
142
from compound 1, there were 6 signals: a Me, 2 CH, and 3 quaternary carbons. 1H and
143
data suggested that there was a symmetry trisubstituted aromatic ring. There was [M-H]- with m/z =
144
151.0393 in HR-ESI mass spectrum data, suggesting a molecular formula C8H8O3. Based on all the
145
information of compound 1, we elucidated this structure as 2,6-dihydroxyacetophenone (DHAP)
146
(Fig. 1).
13
C NMR
147
Compound 2 was a colorless oily compound and soluble in methanol, chloroform, DMSO,
148
acetone, ethyl acetate, and water. In 1H NMR spectra from compound 2 (Table 1), there were 2 CH2
149
moieties [δ(H) 2.82 (t), 3.82 (t)] and 2 aromatic CH groups [δ(H) 6.79(d), 7.11(d)]. In
150
(DEPT) spectrum from compound 2, there were 6 signals: 2 CH2, CH, and 2 quaternary carbons,
151
suggesting that there was a symmetry disubstituted aromatic ring. The HR-ESI mass spectrum data
152
displayed [M-H]- with m/z = 137.0603, suggesting a molecular formula C8H10O2. Based on all data,
153
the structure of compound 2 was elucidated as 4-hydroxyphenethyl alcohol (4-HPEA) (Fig. 1).
13
C NMR
154
Compound 3 was a white powder and soluble in methanol, DMSO, ethyl acetate, chloroform,
155
and water. 1H NMR spectra of compound 3 showed 2 aromatic CH groups [δ(H) 6.79(d), 7.11(d)]
156
and aldehyde group proton (Table 1). 13C NMR spectrum from compound 3 displayed two CH and
157
three quaternary carbons, suggesting that there was a symmetry disubstituted aromatic ring. HR-ESI
158
mass spectrum data displayed [M-H]- with m/z = 121.0287, suggesting a molecular formula C7H6O2.
159
We elucidated this structure (compound 3) as 4-hydroxybenzaldehyde, C7H6O2 (HBA) (Fig. 1).
160
3.2. Antioxidant activity of the purified compounds 10
161
More than one type of antioxidant activity determination needs to be performed to investigate
162
the various modes of action of antioxidants (Prior et al., 1999; Huang et al., 2005; Dudonne et al.,
163
2009). We determined free radical scavenging rate using assays of DPPH and ABTS, while ferric
164
reducing ability was measured by FRAP assay. Results are showed in Fig. 2 and 3.
165
In DPPH assay, the antioxidant is single-electron paired with DPPH free radicals to make its
166
strong absorption at 517 nm gradually disappear, and its degree of fading is quantitatively related to
167
the number of electrons accepted. Each of these 3 compounds was tested with same concentration
168
(2 mg/mL) by the DPPH analysis. It was demonstrated that these three phenolic compounds
169
possessed different degree of antioxidant activity. DHAP had the highest antioxidant activity
170
(66.21 %) which was close to the capability of VC (97.13 %). The higher activity of DHAP was due
171
to the amount of hydroxyl groups within phenyl ring which can be donated to stabilize the free
172
radicals (Rice-Evans et al., 1996). 4-HPEA also showed relatively good free radical scavenging
173
ability. HBA did not show strong effects.
174
In FRAP assay, the antioxidant could reduce Fe3+ into Fe2+ which can combine with TPTZ for
175
forming a blue complex with light absorption (593 nm). High absorbance indicates strong reducing
176
ability and antioxidant activity. Because the FRAP method is not aimed at free radical scavenging
177
ability, but the total reducing ability of the sample, which can be used to reflect the total antioxidant
178
activity of the sample (Benzie and Strain, 1996; Pulido et al., 2000; Halvorsen et al., 2002). Similar
179
to the trend of DPPH radical scavenging results, DHAP showed the greatest antioxidant activity
180
among three phenolic compounds, which had the capacity equivalent to 0.188 mM Trolox and its
181
total antioxidant capacity was 0.752 mM/g. Antioxidant activity difference of these three
182
compounds may depend on the degree of hydroxylation and extent of conjugation (Pulido et al.,
183
2000) In addition, the reducing ability is connected to redox potentials of compounds while 11
184
phenolic compounds usually possess lower redox potentials (Simic and Jovanović, 1994; Hagerman
185
et al., 1998).
186
Unlike the DPPH free radical scavenging principle, the principle of ABTS assay is the electron
187
transfer process, which is more reactive than DPPH free radicals (Gil et al., 2000; Prieto et al.,
188
2015). In this assay, DHAP and 4-HPEA still showed higher antioxidant activity than HBA which
189
possess only one phenolic hydroxyl group. The relationship of structure-antioxidant capacity was
190
similar as DPPH radical scavenging activity as discussed previously. The ABTS radical cation
191
scavenging capacities of three compounds were showed in the decreasing order: 4-HPEA, DHAP,
192
and HBA. Furthermore, the capacity of both DHAP and 4-HPEA had significantly higher capacity
193
than VC which was used as positive control (P < 0.001).
194
3.3. Inhibition of microbial growth by three phenolic compounds from pickled radish
195
The results of the antimicrobial activity of the compounds are presented in Fig. 4 and Table 2.
196
DHAP and HBA had high antimicrobial activity against E. coli with inhibition zones of 25.93-23.67
197
mm and 22.77-19.83 mm, respectively. For B. subtilis, DHAP and HBA had inhibition zones of
198
25.39-21.87 mm and 17.36-11.51 mm, respectively. DHAP and HBA also inhibited the growth of C.
199
albicans with inhibition zones of 26.31-18.15 mm and 23.28-15.66 mm, respectively. However,
200
4-HPEA did not have effects on these three bacteria by the Oxford cup method. DHAP showed the
201
lowest MIC at 0.25 mg/mL, 0.06 mg/mL, and 0.03 mg/mL against E. coli, B. subtilis, and C.
202
albicans, respectively. HBA was demonstrated to have higher MIC at 1.25 mg/mL, 2.5 mg/mL, and
203
0.32 mg/mL. In contrast, 4-HPEA had the highest MIC (2.5 mg/mL, 2.5 mg/mL, and 1.25 mg/mL).
204
Microorganisms often have significant impact on food safety and organoleptic features during
205
food storage (Lv et al., 2011). The antimicrobial activity of phenolic compounds may involve
206
various modes of action. For instance, phenolic compounds can degrade cell walls, destroy the 12
207
plasma membrane, cause cellular components to leak, alter fatty acid and phospholipid components,
208
affect DNA and RNA synthesis, and disrupt protein translocations (Shan et al., 2007). Phenolic
209
compounds in plant were reported to have antimicrobial properties (Pereira et al., 2007;
210
Smith-Palmer et al., 2010; Qin et al., 2019). Grape seeds and bagasse extract rich in phenolic
211
compounds can also have antimicrobial effects against Bacillus amyloliquefaciens, Bacillus cereus,
212
and other microorganisms (Baydar et al., 2004). These three phenolic compounds were isolated
213
from pickled radish for the first time. Two of them (DHAP and HBA) possessed antimicrobial
214
activity against the three pathogenic bacteria. These phenolic compounds are the main component
215
of antimicrobial activities in the pickled radish. These phenolic compounds also contribute to
216
extend the shelf life of pickled radish as antimicrobial and antioxidant compounds naturally
217
produced. These three phenolic compounds might also be used as potential natural additives in
218
other food products to extend the shelf life.
219
3.4. Safety evaluation of compounds using LO2 cells
220
To confirm cytotoxicity of DHAP, 4-HPEA, and HBA on LO2 cells, cell viability was assessed
221
by MTT assay. After cells were treated with a series of concentrations of three compounds for 24 h.
222
The results demonstrated that as the increasing concentration of DHAP and HBA, LO2 cell viability
223
gradually decreased while 4-HPEA did not significantly affect cell viability (Fig. 5). 200 µg/mL and
224
400 µg/mL of DHAP, 400 µg/mL and 800 µg/mL of HBA were significantly toxic to LO2 cell with
225
viability decreased to about 85.24 %, 62.70 %, 92.90 %, and 80.16 %, respectively (**P < 0.01 and
226
***P < 0.001). According to the results, the IC50 of three compounds on LO2 were calculated. The
227
IC50 of DHAP, 4-HPEA, and HBA showed as 0.546 mg/mL, 26.073 mg/mL, and 1.368 mg/mL,
228
respectively. These three phenolic compounds do not show significant toxic effects at low
229
concentrations, which means pickled radish is safe as a daily food. 13
230
3.5. Future research directions
231
Although these three compounds in this study have been evaluated in several in vivo models in
232
the previous studies (Kanchanapoo et al., 2006; Jang et al., 2014; Kang et al., 2017), antioxidant or
233
antibacterial effects from these three compounds still needs to be further studied in future research
234
in the in vivo models. Previous studies have indicated that phenolic compounds may have the
235
potential to improve diabetes, which was also supported by several natural product development
236
studies (Mollica et al., 2017a and 2017b; Picot et al., 2017). Moreover, phenolic compounds from
237
plants display protection against hydrogen peroxide-induced oxidative damage (Ju et al., 2012), and
238
also show anti-inflammatory effects in HT29 and PC3 cells (Kogiannou et al., 2013). More studies
239
need to evaluate the other biological functions of these phenolic compounds in this research.
240 241
4. Conclusions
242
All results indicated pickled radish had antioxidant and antimicrobial phenolic compounds,
243
which might contribute to the preservation of this product during long term storage. To the best of
244
our knowledge, this report is the first to answer partially the question of why pickled foods can be
245
kept at room temperature for years without spoilage based on the evidence of three phenolic
246
compounds. The results from this study are also expected to help scientists in the future direction
247
for exploring other potential biological activities in three phenolic compounds.
248 249 250
Abbreviations Used DHAP,
2,6-Dihydroxyacetophenone; DPPH,
4-HPEA,
4-Hydroxyphenethyl
2,2-Diphenyl-1-picrylhydrazyl;
vitamin
C;
HBA,
251
4-Hydroxybenzaldehyde;
252
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; TEAC, Trolox equivalent antioxidant capacity; 14
VC,
alcohol;
ABTS,
253
FRAP, ferric reducing ability of plasma; TPTZ, 2,4,6-tri(2-pyridyl)-s-triazine; LB, Luria-Bertani;
254
PDA, potato dextrose agar; MIC, minimum inhibitory concentration.
255 256
Acknowledgment
257
This work was supported by the Natural Science Foundation of Fujian Province, China (Grant
258
No. 2017J01636); we also thank the projects of Ministry of Science and Technology (MOST
259
108-2221-E-005-044).
260
261
262
Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article.
263
264
AUTHORS’ CONTRIBUTIONS
265
Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min
266
David Wang conceived and designed the experiments; Qianying Deng, Guilin Li, Guocheng Su, and
267
Jingwen Liu performed the experiments and analyzed the data; Jian Li and Hui-Min David Wang
268
contributed the reagents, materials, and analysis tools; Jian Li, Shi-Ying Huang, Qianying Deng,
269
Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang wrote the paper.
270 271
Supporting Information description
272
Supplementary materials can be found at Supporting information.
273 274
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Figure captions Fig. 1. Structures of DHAP, 4-HPEA, and HBA. Fig. 2. Antioxidant capacity of three compounds was measured by DPPH assay, and VC was used as positive control. Values expressed are means ± SD. Fig. 3. Antioxidant activities of three phenolic compounds were determined by the FRAP assay and ABTS assay. Determine statistical significance between groups using one-way ANOVA and the values represent mean ± SD. ***P < 0.01 vs. VC. Fig. 4. The inhibition activity of three phenolic compounds against (A) E. coli, (B) B. subtilis, and (C) C. albicans. Vehicle was methanol and negative control. Fig. 5. Cytotoxicity of three phenolic compounds on LO2 cells was measured by MTT assay. Determine statistical significance between groups using one-way ANOVA and the values represent mean ± SD. **P < 0.01 and ***P < 0.01 vs. vehicle.
23
Figures Fig. 1
24
Fig. 2
25
Fig. 3
26
Fig. 4
27
Fig. 5
28
Table 1. NMR data from 3 compounds at 400, 100 MHz, in CDCl3 (δ in ppm). 1 (DHAP) Position
13
C
1
2 (4-HPEA) 13
H
C
1
H
3 (HBA) 13
C
1
H
1
110.2
-
130.5
-
129.2
-
2
161.3
-
130.2
7.11
132.4
7.83
3
108.4
6.41
115.5
6.79
116.0
6.97
4
136.1
7.24
154.2
-
168.7
-
5
108.4
6.41
115.5
6.79
116.0
6.97
6
161.3
-
130.2
7.11
132.4
7.83
7
205.3
-
29.7
2.82
190.9
9.87
8
33.5
2.75
38.2
3.82
-
-
-
9.62
-
-
-
-
29
Table 2. Antibacterial activity of compounds against E. coli, B. subtilis, and C. albicans. E. coli
B. subtilis
C. albicans
Compounds Inhibition zone
MIC
Inhibition zone
MIC
Inhibition zone
MIC
DHAP
24.80±1.13
0.25
23.63±1.76
0.06
22.23±4.08
0.03
4-HPEA
<8
2.5
<8
2.5
<8
1.25
HBA
21.31±1.47
1.25
14.43±2.93
2.5
19.46±3.81
0.32
Note：Inhibition zone in (mm)，MIC in (mg/mL).
30
Highlights: 3 phenolic compounds are purified from the pickled radish for the first time. DHAP displays good abilities of free radical scavenging and ferric reducing power. 4-HPEA has good abilities of free radical scavenging and ferric reducing power. DHAP shows antimicrobial activity against E. coli, B. subtilis and C. albicans. HBA demonstrates antimicrobial activity against E. coli and C. albicans.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0278-6915(19)30840-3
DOI:
https://doi.org/10.1016/j.fct.2019.111050
Reference:
FCT 111050
To appear in:
Food and Chemical Toxicology
Received Date: 17 September 2019 Revised Date:
3 December 2019
Accepted Date: 8 December 2019
Please cite this article as: Li, J., Huang, S.-Y., Deng, Q., Li, G., Su, G., Liu, J., David Wang, H.-M., Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2019.111050. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Authors’ Contributions Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang conceived and designed the experiments; Qianying Deng, Guilin Li, Guocheng Su, and Jingwen Liu performed the experiments and analyzed the data; Jian Li and Hui-Min David Wang contributed the reagents, materials, and analysis tools; Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang wrote the paper.
Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish
Jian Lia,b,*, Shi-Ying Huanga, Qianying Denga, Guiling Lia,b, Guocheng Sua, Jingwen Liua, Hui-Min David Wanga,c, d,e,*
a
College of Food and biological engineering, Jimei University, Xiamen 361021, China
b
Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen 361021, China
c
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan
d
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung City 404, Taiwan
e
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan Graphic Abstract
Extraction and characterization of phenolic compounds with antioxidant and antimicrobial activities from pickled radish
Jian Lia,b,*, Shi-Ying Huanga, Qianying Denga, Guiling Lia,b, Guocheng Sua, Jingwen Liua, Hui-Min David Wanga,c, d,e,*
a
College of Food and biological engineering, Jimei University, Xiamen 361021, China
b
Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen 361021, China
c
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan
d
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung City 404, Taiwan
e
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
*Corresponding Authors: Jian Li, Ph.D. Associate Professor, College of Biological Engineering, Jimei University Address: No.43, Yindou Rd., Xiamen city, Fujian Province, 361021, China Tel: 0086-592-6181915 Fax: 86-592-6180470 E-mail: [email protected]
Hui-Min David Wang, Ph.D. 1
Professor, Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan Address: No.145, Xingda Rd., South Dist., Taichung City 402, Taiwan Mobil: 886-935753718 TEL: 886-4-2284-0733#651 Fax: 886-4-22852242 E-mail: [email protected] https://sites.google.com/site/davidbiocosme/home
2
ABSTRACT The pickled radish can be kept at room temperature for years without spoilage. 2,6-dihydroxyacetophenone (DHAP), 4-hydroxybenzaldehyde (HBA), and 4-hydroxyphenethyl alcohol (4-HPEA) were first found from the pickled radish. The structures of three phenolic compounds were elucidated by analysis of their nuclear magnetic resonance and high-resolution electro-spray ionization mass spectrometry data. All these phenolic compounds showed good free radical scavenging capacity except HBA. Both DHAP and 4-HPEA also showed high ferric reducing ability. DHAP showed good antimicrobial activity against Escherichia coli, Bacillus subtilis, and Canidia albicans. HBA demonstrated antimicrobial activity against E. coli and C. albicans but not B. subtilis. Based on the results of MTT assay, these compounds did not show cytotoxicity to LO2 cell line. All results indicated the pickled radish had antioxidant and antimicrobial phenolic compounds. To the best of our knowledge, this report is the first to answer partially the question of why pickled foods can be kept at room temperature for years without spoilage based on the evidence of three phenolic compounds.
Keywords Pickled radish; Phenolic compounds; Antioxidant; Antimicrobial; Shelf life; Stability
3
1
1. Introduction
2
Pickled radish was processed by traditional method in China. Fresh white radish is dehydrated
3
with sea salt and pressed, and then pickled in ceramic container without any other ingredients. The
4
pickled radish has a very long shelf life, and it can be kept at room temperature up to 20 years
5
without package. The antioxidant activity of radish is partly due to acylated pelargonidin derivatives
6
(Wang et al., 2010). Complex biochemical reactions occur in the pickling and fermentation of
7
kimchi (Cheigh et al., 1994). Pickled radish has particular nutritional content and health benefits
8
(Kumakura et al., 2017). Many bioactive components with antimicrobial and/or antioxidant
9
generate from the pickling process, such as phenolic compounds (Jing et al., 2014). However, there
10
was no study on the phenolic compounds of pickled radish.
11
Plant phenolic compounds have been extensively studied due to their bioactive activities and
12
also healthy benefits for human beings (Zheng and Wang, 2001; Derakhshan et al., 2018; Qin et al.,
13
2019). The aromatic ring having the hydroxyl groups is a family characteristic of these compounds,
14
and these structures include high-molecular weight polymers or small phenolic molecules
15
(Balasundram et al., 2006; Wang et al., 2019). Phenolic compounds are usually used as natural
16
antioxidants in foods to extend the shelf life (Caponio et al., 2001; Estevinho et al., 2008;
17
Llorent-Martinez et al., 2017). Phenolic compounds in cooked ground walnut showed high
18
antioxidant activities during refrigerated storage (Ahn et al., 2002). Phenolics in clove essential oil
19
can enhance the stability of cake and other lipid foods for storage (Ibrahium et al., 2013). Phenolic
20
compounds can also inhibit the growth of microorganisms, such as E. coli, K. pneumoniae, B.
21
cereus, A. flavus, and A. parasiticus (Aziz et al., 1998). Different concentrations of phenolic
22
compounds exhibit different sensitivities towards different microorganisms (Vaquero et al., 2007).
23
However, antimicrobial activity of phenolic compounds from pickled radish has not been shown in
24
the previous literature.
25
In this study, the compounds with antimicrobial activities from pickled radish were
26
characterized. The antioxidant activity and cytotoxicity of the compounds from the pickled radish
27
were evaluated. All the results indicated that these phenolic compounds play an important role in
28
pickled radish shelf life than other factors. These phenolic compounds extracted from pickled radish
29
might also be considered be used in other food preservation.
30 31
2. Materials and Methods
32
2.1. Materials
33
Pickled radish was obtained from stores in Zhangpu, Fujian Province. Reverse phase (RP-18)
34
Silica gel was purchased from YMC (Kyoto, Japan). Silica gel (Silia Flash P60) for column
35
chromatography was purchased from SiliCycle (Quebec City, Canada). Sephadex LH-20 was
36
purchased from GE Healthcare (Uppsala, Sweden). Precoated silica gel GF254 plates were
37
purchased from Qingdao Marine Chemical Factory (Qingdao, China). Membrane filter was
38
purchased from Millipore Co. (Milford, MA). Human liver cell lines (LO2) were obtained from
39
Chinese Academy of Sciences Cell Collection Center (Shanghai, China).
40
2.2. Chemicals
41
We obtained the chemicals from Sigma-Aldrich (St. Louis, MO): dimethyl sulfoxide (DMSO),
42
vitamin
C
(L-ascorbic
acid),
2,2-Diphenyl-1-picrylhydrazyl
43
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We purchased DMEM-high
44
glucose medium, antibiotics (streptomycin and penicillin), and fetal bovine serum (FBS) through
45
HyClone (Northbrook, IL). Total antioxidant capacity assay kit for FRAP and ABTS assay was
46
purchased from Beyotime Institute of Biotechnology (Haimen, China). All other reagents were 5
(DPPH),
and
47
analytical grade and purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). A
48
Milli-Q reagent water system, which was obtained from Millipore Co. (Milford, MA), produced
49
deionized water.
50
2.3. Preparation of crude extract
51
Pickled radish was air-dried at 40 ℃ for 24 h, and was then extracted with methanol, acetone,
52
and ethyl acetate for 12 h. The extraction solution was passed through membrane filter to remove
53
insoluble. Then the extraction solution was vacuumed evaporated to obtain crude extracts at 40 ℃.
54
The antimicrobial activities were measured by oxford cup method.
55
2.4. Purification and identification of antimicrobial compounds from the crude extract
56
Gel column chromatography: Methanol extract was dissolved in a small amount of methanol,
57
loaded on pretreated Sephadex LH-20 gel column, using methanol as eluent, according to the
58
sample amount to control velocity within the limits of 10-15 s/drop. Fractions were collected every
59
30 min in a collection tube by automatic fraction collector. All fractions were analyzed by TLC and
60
pooled separately according to the results of TLC analysis. Antimicrobial activity of the pooled
61
fractions was measured by oxford cup method as described later. Fractions with antimicrobial
62
activity were further purified; however, fractions without antimicrobial activity were not further
63
purified.
64
Reversed phase silica gel column chromatography: After LH-20 gel column chromatography
65
separation, fractions with antimicrobial activities were filtrated through membrane filter and
66
vacuumed evaporated. Fractions were dissolved in methanol, then subjected to the MPLC (RP-18)
67
for further isolation with water and methanol. All fractions were collected and vacuumed
68
evaporated at 40 ℃, and were then dissolved with appropriate amount of methanol for TLC
69
analysis and antimicrobial activity assay. 6
70
Silica gel column chromatography: Fractions with antimicrobial activities were further purified in
71
a silica gel column with petroleum ether and acetone as the eluent solvent. Elution fractions was
72
pooled according to TLC analysis and then vacuumed evaporated at 40 ℃ for further instrumental
73
analysis.
74
Instrumental Analysis: Nuclear magnetic resonance (NMR) spectra from samples were obtained in
75
CDCl3 by a Bruker AVANCE III HD 400MHz (Bruker Biospin GmbH, Karlsruhe, Germany). We
76
reported chemical shifts (δ) in units of parts per million (ppm) versus tetramethylsilane. We
77
corrected chemical shift to residual solvent signal, 7.26 ppm. Mass spectrometry was acquired by
78
Xevo™ G2 Q Tof (Waters MS Technologies, Manchester, UK), a quadrupole and orthogonal
79
acceleration time-of-flight tandem mass spectrometer. The carrier solvent for flow injection analysis
80
was methanol. The scan range was from 50 to 1000 m/z for both positive and negative electrospray
81
modes. All the acquisition and analysis of data were controlled by Waters Mass Lynx v4.1 software,
82
respectively.
83
2.5. Determination of antioxidant activity
84
We measured the antioxidant activity by DPPH assay (Yang et al., 2006; Luo et al., 2009). A
85
DPPH solution (0.25 mM; 99 µL) in methanol was added to each compound’s solution (1 µL). We
86
used L-ascorbic acid (100 µM) as the positive control. We measured the absorption at OD517 nm
87
after 30 min, and obtained the inhibitory effect using the formula: (1)
88
89
We measured total antioxidant capacity using an assay kit of ferric reducing ability of plasma
90
(FRAP) (Benzie and Strain, 1996; Benzie and Szeto, 1999). Prepare the working solution through
91
fully mixing 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) diluents with TPTZ solution in a ratio of 10:1,
7
92
followed by mixing with detection buffer in equal quantities with TPTZ solution, and finally
93
incubated at 37 ℃ before use. Standard curve was prepared by Trolox in order to calculate Trolox
94
equivalent antioxidant capacity (TEAC) values (mM/g). 5 µL of compounds solution with different
95
concentrations were mixed with 180 µL working solution, which were incubated at 37 ℃ for 5
96
min. We measured absorbance at 593 nm from mixture.
97
Total antioxidant capacity was measured by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
98
acid) (ABTS) assay kit (Luo et al., 2012; Zhao et al., 2015). We produced the working solution
99
through mixing stock ABTS solution with stock oxidant solution in equal quantities, and the stock
100
solutions reacted for 12-16 h in the dark. Then, the stock solutions were diluted 40 times with PBS
101
to get working solution, and subsequently 10 µL of compounds (50 µg/mL) solution were mixed
102
with 200 µL working solution. At 6 min after incubation at room temperature, we measured
103
absorbance with 734 nm, and expressed results as TEAC mM/g.
104
2.6. Microbial cultures and antimicrobial assay
105
We evaluated antimicrobial effects of compounds on Escherichia coli, Bacillus subtilis, and
106
Canidia albicans, which were purchased from China General Microbiological Culture Collection
107
Center (CGMCC). E. coli and B. subtilis were grown in Luria-Bertani (LB) medium at 37 ℃, and
108
C. albicans was cultured in potato dextrose agar medium at 27 ℃. The antimicrobial assay of
109
compounds was performed by the oxford cup method (Soković et al., 2010; Huang et al., 2012).
110
The bacterial suspensions were inoculated to soft agar media to a final concentration of 1.0 × 105
111
CFU/mL. After the soft agar medium is solidified, the oxford cup containing compounds solution,
112
negative control (methanol), and positive control (chloromycetin and amphotericin B) were placed
113
on the agar plates. After 24 h incubation, the diameter of the inhibition zones was measured.
114
2.7. Minimum inhibitory concentration (MIC) of compounds 8
115
Minimum inhibitory concentration (MIC) of compounds were determine by broth
116
micro-dilution method (Golestani et al., 2015; Wu et al., 2015). In 96-well microplates, we prepared
117
the compounds (2-fold serial dilutions) and positive control. Take a few colonies from the agar plate
118
for creating the inoculum by a sterile swab, and dilute the McFarland standard into media. Dispense
119
the inocula with the test compounds, which were incubated for 24 h at 27 or 37 ℃. Determine the
120
MIC value by reading the microdilution plate. The lowest concentration with no visible growth and
121
with ∆OD less than 0.05 was defined as minimum inhibitory concentration (MIC).
122
2.8. Cytotoxicity assay
123
Cells were cultured at 37 ℃ with 5 % CO2. The medium was changed every 2 days. The
124
cytotoxicity
of
compounds
to
LO2
cells
was
determined
by
125
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosmann, 1983). LO2
126
cells were seeded into a 96-well plate at 104 cells per well. After 24 h, the cells were incubated with
127
the compounds at serial dilutions for additional 24 h incubation. Add 10 µL MTT stock solution (5
128
mg/mL) to each well at 37 ℃ for 4 h. A negative control was prepared by mixing 10 µL of the
129
MTT stock solution and 100 µL of medium alone, and was incubated at 37 ℃ for 4 h. Add 50 µL
130
of DMSO to each well and mix thoroughly using the pipette. Incubate the microplate at 37 ℃ for
131
10 min. Mix each sample again using a pipette and read absorbance at 540 nm.
132
2.9. Statistical analysis
133
Statistical analysis of the data (mean ± SD) was performed using t one-way ANOVA and
134
Duncan test (SPSS statistics 17.0 software program). The present study considered P < 0.05 for
135
significantly different between groups.
136 137
3. Results and Discussion 9
138
3.1. Characterization for purified compounds
139
Compound 1 was obtained as a beige powder and was soluble in methanol, acetone, ethyl
140
acetate, and chloroform. In 1H NMR spectra data from compound 1, there were 3 signals: 1 Me
141
[δ(H) 2.75 (s)] and 2 aromatic CH groups [δ(H) 6.41(d), 7.24(d)] (Table 1). In 13C NMR spectrum
142
from compound 1, there were 6 signals: a Me, 2 CH, and 3 quaternary carbons. 1H and
143
data suggested that there was a symmetry trisubstituted aromatic ring. There was [M-H]- with m/z =
144
151.0393 in HR-ESI mass spectrum data, suggesting a molecular formula C8H8O3. Based on all the
145
information of compound 1, we elucidated this structure as 2,6-dihydroxyacetophenone (DHAP)
146
(Fig. 1).
13
C NMR
147
Compound 2 was a colorless oily compound and soluble in methanol, chloroform, DMSO,
148
acetone, ethyl acetate, and water. In 1H NMR spectra from compound 2 (Table 1), there were 2 CH2
149
moieties [δ(H) 2.82 (t), 3.82 (t)] and 2 aromatic CH groups [δ(H) 6.79(d), 7.11(d)]. In
150
(DEPT) spectrum from compound 2, there were 6 signals: 2 CH2, CH, and 2 quaternary carbons,
151
suggesting that there was a symmetry disubstituted aromatic ring. The HR-ESI mass spectrum data
152
displayed [M-H]- with m/z = 137.0603, suggesting a molecular formula C8H10O2. Based on all data,
153
the structure of compound 2 was elucidated as 4-hydroxyphenethyl alcohol (4-HPEA) (Fig. 1).
13
C NMR
154
Compound 3 was a white powder and soluble in methanol, DMSO, ethyl acetate, chloroform,
155
and water. 1H NMR spectra of compound 3 showed 2 aromatic CH groups [δ(H) 6.79(d), 7.11(d)]
156
and aldehyde group proton (Table 1). 13C NMR spectrum from compound 3 displayed two CH and
157
three quaternary carbons, suggesting that there was a symmetry disubstituted aromatic ring. HR-ESI
158
mass spectrum data displayed [M-H]- with m/z = 121.0287, suggesting a molecular formula C7H6O2.
159
We elucidated this structure (compound 3) as 4-hydroxybenzaldehyde, C7H6O2 (HBA) (Fig. 1).
160
3.2. Antioxidant activity of the purified compounds 10
161
More than one type of antioxidant activity determination needs to be performed to investigate
162
the various modes of action of antioxidants (Prior et al., 1999; Huang et al., 2005; Dudonne et al.,
163
2009). We determined free radical scavenging rate using assays of DPPH and ABTS, while ferric
164
reducing ability was measured by FRAP assay. Results are showed in Fig. 2 and 3.
165
In DPPH assay, the antioxidant is single-electron paired with DPPH free radicals to make its
166
strong absorption at 517 nm gradually disappear, and its degree of fading is quantitatively related to
167
the number of electrons accepted. Each of these 3 compounds was tested with same concentration
168
(2 mg/mL) by the DPPH analysis. It was demonstrated that these three phenolic compounds
169
possessed different degree of antioxidant activity. DHAP had the highest antioxidant activity
170
(66.21 %) which was close to the capability of VC (97.13 %). The higher activity of DHAP was due
171
to the amount of hydroxyl groups within phenyl ring which can be donated to stabilize the free
172
radicals (Rice-Evans et al., 1996). 4-HPEA also showed relatively good free radical scavenging
173
ability. HBA did not show strong effects.
174
In FRAP assay, the antioxidant could reduce Fe3+ into Fe2+ which can combine with TPTZ for
175
forming a blue complex with light absorption (593 nm). High absorbance indicates strong reducing
176
ability and antioxidant activity. Because the FRAP method is not aimed at free radical scavenging
177
ability, but the total reducing ability of the sample, which can be used to reflect the total antioxidant
178
activity of the sample (Benzie and Strain, 1996; Pulido et al., 2000; Halvorsen et al., 2002). Similar
179
to the trend of DPPH radical scavenging results, DHAP showed the greatest antioxidant activity
180
among three phenolic compounds, which had the capacity equivalent to 0.188 mM Trolox and its
181
total antioxidant capacity was 0.752 mM/g. Antioxidant activity difference of these three
182
compounds may depend on the degree of hydroxylation and extent of conjugation (Pulido et al.,
183
2000) In addition, the reducing ability is connected to redox potentials of compounds while 11
184
phenolic compounds usually possess lower redox potentials (Simic and Jovanović, 1994; Hagerman
185
et al., 1998).
186
Unlike the DPPH free radical scavenging principle, the principle of ABTS assay is the electron
187
transfer process, which is more reactive than DPPH free radicals (Gil et al., 2000; Prieto et al.,
188
2015). In this assay, DHAP and 4-HPEA still showed higher antioxidant activity than HBA which
189
possess only one phenolic hydroxyl group. The relationship of structure-antioxidant capacity was
190
similar as DPPH radical scavenging activity as discussed previously. The ABTS radical cation
191
scavenging capacities of three compounds were showed in the decreasing order: 4-HPEA, DHAP,
192
and HBA. Furthermore, the capacity of both DHAP and 4-HPEA had significantly higher capacity
193
than VC which was used as positive control (P < 0.001).
194
3.3. Inhibition of microbial growth by three phenolic compounds from pickled radish
195
The results of the antimicrobial activity of the compounds are presented in Fig. 4 and Table 2.
196
DHAP and HBA had high antimicrobial activity against E. coli with inhibition zones of 25.93-23.67
197
mm and 22.77-19.83 mm, respectively. For B. subtilis, DHAP and HBA had inhibition zones of
198
25.39-21.87 mm and 17.36-11.51 mm, respectively. DHAP and HBA also inhibited the growth of C.
199
albicans with inhibition zones of 26.31-18.15 mm and 23.28-15.66 mm, respectively. However,
200
4-HPEA did not have effects on these three bacteria by the Oxford cup method. DHAP showed the
201
lowest MIC at 0.25 mg/mL, 0.06 mg/mL, and 0.03 mg/mL against E. coli, B. subtilis, and C.
202
albicans, respectively. HBA was demonstrated to have higher MIC at 1.25 mg/mL, 2.5 mg/mL, and
203
0.32 mg/mL. In contrast, 4-HPEA had the highest MIC (2.5 mg/mL, 2.5 mg/mL, and 1.25 mg/mL).
204
Microorganisms often have significant impact on food safety and organoleptic features during
205
food storage (Lv et al., 2011). The antimicrobial activity of phenolic compounds may involve
206
various modes of action. For instance, phenolic compounds can degrade cell walls, destroy the 12
207
plasma membrane, cause cellular components to leak, alter fatty acid and phospholipid components,
208
affect DNA and RNA synthesis, and disrupt protein translocations (Shan et al., 2007). Phenolic
209
compounds in plant were reported to have antimicrobial properties (Pereira et al., 2007;
210
Smith-Palmer et al., 2010; Qin et al., 2019). Grape seeds and bagasse extract rich in phenolic
211
compounds can also have antimicrobial effects against Bacillus amyloliquefaciens, Bacillus cereus,
212
and other microorganisms (Baydar et al., 2004). These three phenolic compounds were isolated
213
from pickled radish for the first time. Two of them (DHAP and HBA) possessed antimicrobial
214
activity against the three pathogenic bacteria. These phenolic compounds are the main component
215
of antimicrobial activities in the pickled radish. These phenolic compounds also contribute to
216
extend the shelf life of pickled radish as antimicrobial and antioxidant compounds naturally
217
produced. These three phenolic compounds might also be used as potential natural additives in
218
other food products to extend the shelf life.
219
3.4. Safety evaluation of compounds using LO2 cells
220
To confirm cytotoxicity of DHAP, 4-HPEA, and HBA on LO2 cells, cell viability was assessed
221
by MTT assay. After cells were treated with a series of concentrations of three compounds for 24 h.
222
The results demonstrated that as the increasing concentration of DHAP and HBA, LO2 cell viability
223
gradually decreased while 4-HPEA did not significantly affect cell viability (Fig. 5). 200 µg/mL and
224
400 µg/mL of DHAP, 400 µg/mL and 800 µg/mL of HBA were significantly toxic to LO2 cell with
225
viability decreased to about 85.24 %, 62.70 %, 92.90 %, and 80.16 %, respectively (**P < 0.01 and
226
***P < 0.001). According to the results, the IC50 of three compounds on LO2 were calculated. The
227
IC50 of DHAP, 4-HPEA, and HBA showed as 0.546 mg/mL, 26.073 mg/mL, and 1.368 mg/mL,
228
respectively. These three phenolic compounds do not show significant toxic effects at low
229
concentrations, which means pickled radish is safe as a daily food. 13
230
3.5. Future research directions
231
Although these three compounds in this study have been evaluated in several in vivo models in
232
the previous studies (Kanchanapoo et al., 2006; Jang et al., 2014; Kang et al., 2017), antioxidant or
233
antibacterial effects from these three compounds still needs to be further studied in future research
234
in the in vivo models. Previous studies have indicated that phenolic compounds may have the
235
potential to improve diabetes, which was also supported by several natural product development
236
studies (Mollica et al., 2017a and 2017b; Picot et al., 2017). Moreover, phenolic compounds from
237
plants display protection against hydrogen peroxide-induced oxidative damage (Ju et al., 2012), and
238
also show anti-inflammatory effects in HT29 and PC3 cells (Kogiannou et al., 2013). More studies
239
need to evaluate the other biological functions of these phenolic compounds in this research.
240 241
4. Conclusions
242
All results indicated pickled radish had antioxidant and antimicrobial phenolic compounds,
243
which might contribute to the preservation of this product during long term storage. To the best of
244
our knowledge, this report is the first to answer partially the question of why pickled foods can be
245
kept at room temperature for years without spoilage based on the evidence of three phenolic
246
compounds. The results from this study are also expected to help scientists in the future direction
247
for exploring other potential biological activities in three phenolic compounds.
248 249 250
Abbreviations Used DHAP,
2,6-Dihydroxyacetophenone; DPPH,
4-HPEA,
4-Hydroxyphenethyl
2,2-Diphenyl-1-picrylhydrazyl;
vitamin
C;
HBA,
251
4-Hydroxybenzaldehyde;
252
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; TEAC, Trolox equivalent antioxidant capacity; 14
VC,
alcohol;
ABTS,
253
FRAP, ferric reducing ability of plasma; TPTZ, 2,4,6-tri(2-pyridyl)-s-triazine; LB, Luria-Bertani;
254
PDA, potato dextrose agar; MIC, minimum inhibitory concentration.
255 256
Acknowledgment
257
This work was supported by the Natural Science Foundation of Fujian Province, China (Grant
258
No. 2017J01636); we also thank the projects of Ministry of Science and Technology (MOST
259
108-2221-E-005-044).
260
261
262
Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article.
263
264
AUTHORS’ CONTRIBUTIONS
265
Jian Li, Shi-Ying Huang, Qianying Deng, Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min
266
David Wang conceived and designed the experiments; Qianying Deng, Guilin Li, Guocheng Su, and
267
Jingwen Liu performed the experiments and analyzed the data; Jian Li and Hui-Min David Wang
268
contributed the reagents, materials, and analysis tools; Jian Li, Shi-Ying Huang, Qianying Deng,
269
Guilin Li, Guocheng Su, Jingwen Liu, and Hui-Min David Wang wrote the paper.
270 271
Supporting Information description
272
Supplementary materials can be found at Supporting information.
273 274
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Figure captions Fig. 1. Structures of DHAP, 4-HPEA, and HBA. Fig. 2. Antioxidant capacity of three compounds was measured by DPPH assay, and VC was used as positive control. Values expressed are means ± SD. Fig. 3. Antioxidant activities of three phenolic compounds were determined by the FRAP assay and ABTS assay. Determine statistical significance between groups using one-way ANOVA and the values represent mean ± SD. ***P < 0.01 vs. VC. Fig. 4. The inhibition activity of three phenolic compounds against (A) E. coli, (B) B. subtilis, and (C) C. albicans. Vehicle was methanol and negative control. Fig. 5. Cytotoxicity of three phenolic compounds on LO2 cells was measured by MTT assay. Determine statistical significance between groups using one-way ANOVA and the values represent mean ± SD. **P < 0.01 and ***P < 0.01 vs. vehicle.
23
Figures Fig. 1
24
Fig. 2
25
Fig. 3
26
Fig. 4
27
Fig. 5
28
Table 1. NMR data from 3 compounds at 400, 100 MHz, in CDCl3 (δ in ppm). 1 (DHAP) Position
13
C
1
2 (4-HPEA) 13
H
C
1
H
3 (HBA) 13
C
1
H
1
110.2
-
130.5
-
129.2
-
2
161.3
-
130.2
7.11
132.4
7.83
3
108.4
6.41
115.5
6.79
116.0
6.97
4
136.1
7.24
154.2
-
168.7
-
5
108.4
6.41
115.5
6.79
116.0
6.97
6
161.3
-
130.2
7.11
132.4
7.83
7
205.3
-
29.7
2.82
190.9
9.87
8
33.5
2.75
38.2
3.82
-
-
-
9.62
-
-
-
-
29
Table 2. Antibacterial activity of compounds against E. coli, B. subtilis, and C. albicans. E. coli
B. subtilis
C. albicans
Compounds Inhibition zone
MIC
Inhibition zone
MIC
Inhibition zone
MIC
DHAP
24.80±1.13
0.25
23.63±1.76
0.06
22.23±4.08
0.03
4-HPEA
<8
2.5
<8
2.5
<8
1.25
HBA
21.31±1.47
1.25
14.43±2.93
2.5
19.46±3.81
0.32
Note：Inhibition zone in (mm)，MIC in (mg/mL).
30
Highlights: 3 phenolic compounds are purified from the pickled radish for the first time. DHAP displays good abilities of free radical scavenging and ferric reducing power. 4-HPEA has good abilities of free radical scavenging and ferric reducing power. DHAP shows antimicrobial activity against E. coli, B. subtilis and C. albicans. HBA demonstrates antimicrobial activity against E. coli and C. albicans.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: