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IDENTIFICATION OF ANTIOXIDANTS IN DARK SOY SAUCE
WANG HUANSONG
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCES
DEPARTMENT OF BIOCHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2010
Acknowledgements
This journey is not possible without the help of so many people. First and foremost, I
would especially like to express my most sincere and profound appreciation to my
supervisor, Professor Barry Halliwell, for his constant guidance, invaluable
suggestion and critical comments throughout this work.
I am also grateful to all my colleagues and friends inside/outside Professor
Halliwell’s group, who help me in this way or that way, especially to Dr Tang Soon
Yew for his useful discussions and many technical support in cell culture, Dr Andrew
Jenner and Dr Lee Chung-Yung for their useful suggestions, Dr Shui Guanghou,
Professor Markus Wenk’s group, for his help in using LC-TOF-MS, Dr Koh Hwee
Ling, Department of Pharmacy, for her help in using FT-IR, Dr Mark Richards,
Agilent Technologies Singapore, for his help in LC-APCI-Ion trap -MS/MS
measurement, and Professor Yong Eu Leong, Department of Obstetrics &
Gynecology, for his help in using triple quadrupole LC-MS/MS.
My heartfelt thanks also go to the Department of Biochemistry, the Centre of Life
Sciences, the National University of Singapore for holding so many exciting talks and
creating a wonderful academic atmosphere.
Last but not least, I thank my wife, Shen Ping, for her patience and consistent support.
I would like to dedicate this thesis to my son, Bo Qian.
i
Table of Contents
Acknowledgements
i
Table of Contents
ii
Summary
iv
List of Tables
v
List of Figures and Chart
vi
List of Symbols
ix
Chapter 1 Introduction
1
1.1 A brief history of soy sauce
1
1.2 The methodology of preparation of soy sauce
3
1.3 The functional components of soy sauce
5
1.4 The balance of free radicals/reactive species and antioxidants
6
Chapter 2 Materials and Methods
8
2.1 Chemicals
8
2.2 ABTS assay
8
2.3 Isolation of low molecular mass components from ethyl acetate extract
9
2.4 Fraction of colored components
9
2.5 HPLC determination of maltol in dark soy sauce
11
2.6 Mass spectrometry
12
2.7 Fourier transfer infrared spectrometry (FTIR)
13
2.8 Nuclear Magnetic resonance spectrometry (NMR)
13
2.9 Detection and determination of maltol metabolites in human urine samples
13
2.9.1 Standard preparation
13
2.9.2 Sample preparation
14
ii
2.9.3 HPLC-DAD detection of maltol metabolites and determination
of total maltol content in urine
14
2.9.4 HPLC-MS/MS detection of maltol metabolites
15
2.10 GC-MS analysis of DNA base modification
16
2.10.1 Sample preparation
16
2.10.2 GC-MS analysis
16
2.11 Cell culture
17
2.12 Assessment of cell viability
17
2.13 Western blot analysis
18
2.14 Statistical analysis
18
Chapter 4 Results and Discussion
19
4.1 Separation and characterization of low molecular mass components
19
4.2 Content of maltol and its contribution to the total antioxidant activity
of dark soy sauce
26
4.3 Maltol excretion in urine
32
4.4 Fractionation and characterization of the colored components
42
4.5 Protection against HOCl-induced DNA damage
49
4.6 Cytotoxicity on HT-29 cells
51
4.7 Inhibition of COX-2 protein expression in LPS-induced HT-29 cells
54
4.8 Discussion
56
Chapter 5 Conclusion
61
Bibliography
62
Appendices
70
iii
Summary
Soy sauce is a traditional fermented seasoning in Asian countries, that has high
antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to
identify the major antioxidants present, using the 2,2’-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl
acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations
in DSS. However, most of the antioxidant activity was present in colored fractions,
two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their
structural characteristics based on nuclear magnetic resonance (NMR) and
electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis
suggest that carbohydrate-containing pigments such as melanoidins are the major
contributors to the high antioxidant capacity of DSS. In vitro, maltol, CP1 and CP2
can protect against hypochlorous acid (HOCl)-mediated DNA damage dosedependently. Furthermore, dark soy sauce potentially inhibits the growth of colon
cancer HT 29 cells at high concentrations, while it decreases the up-regulation of
cyclooxygenase-2 (COX-2) expression in LPS (lipopolysaccharide) -induced HT 29
cells at low concentrations.
iv
List of Tables
Table I. 1H- and 13C-NMR data of Compound 1.
25
Table II. Within- and between-assay precision and recoveries of the assay
used to measure maltol.
27
Table III. The observed ions in TOF-MS spectrum of CP1. Three series were
observed. Within each series, m/zs of the doubly-charged or the singly-charged
ions consistently increase by 81 or 162, respectively.
48
Table IV. Inhibition of HOCl-induced DNA damage by maltol, the colored
product 1 (CP1) and the colored product 2 (CP2).
50
v
List of Figures and Chart
Figure 1. Trolox equivalent antioxidant capacity (TEAC) values
per g/ml of dark soy sauce (DSS) and three fractions: methanol
extract residue (MeOH-R), ethyl acetate extract (EtOAc-extract)
and ethyl acetate extract residue (EtoAc-R). Values are mean ±
SD, n=3.
21
Figure 2. (A) Typical HPLC chromatogram of ethyl acetate extract.
The absorbance was monitored at 270 nm. (B) Trolox equivalent
antioxidant capacity (TEAC) values of 10 fractions of ethyl acetate
extract per μg/mL. Results are mean ± SD, n=3.
22
Figure 3. EI-MS spectrum of compound 1 and the proposed mechanism
for the formation of fragment ions.
23
Figure 4. The HPLC chromatograms of (a) ethyl acetate extract of dark
soy sauce, (b) compound1 and (c) authentic maltol. (d) The overlaid spectra
of maltol and compound 1; the match factor is 999.967 (It is generally
considered to be matched well, if the match factor is no less than 990.).
24
Figure 5. Structure of 3-hydroxy-2-methyl-4H-pyran-4-one (maltol).
25
Figure 6. Standard curve of maltol at five concentrations, 0.25, 0.5 1.0,
1.5 and 2.0 mM .
28
Figure 7. (a) limit of detection (LOD): a typical chromatogram of maltol
of 8 μM;.(b) limit of quantification (LOQ): a typical chromatogram of
maltol of 25 μM.
29
Figure 8. Typical chromatograms of (a) dark soy sauce extract and
(b) maltol standard (1.0mM) under the following HPLC conditions,
Mobile phase: A: 0.1% formic acid; B: methanol. 10% of B for 15min,
10%-90% of B in 6min, 90% of B for another 5 min; Flow rate:
1.0 ml/min; Column: Agilent ZORBAX SB-C18 (4.6 mm i.d. × 250mm);
Injection volume: 10 μl; Detection wavelength: 270nm.
30
Figure 9. The scavenging effects of dark soy sauce (a), maltol and trolox (b),
on ABTS•+. Results are mean±SD, n≥3.
31
Figure 10. Typical HPLC chromatograms of (a) urine sample collected at
1 hour after the subject orally taken 70 mg maltol, and (b) the same urine
sample as in (a) digested with β-glucuronidase. And the UV spectrum of
(c) peak 1, whose retention time (RT) at 5.12 min, agrees well with that of
(d) synthesized maltol sulphate.
34
Figure 11. (A) The total ion chromatogram of MS/MS scanning of maltol
glucuronide from urine sample. (B) The mass spectrum of the peak with
vi
retention time at 3.12 min in total ion chromatogram A: The ion with
m/z 303 is the protonated ion of maltol glucuronide; whereas the product ion
with m/z 127 is protonated ion of maltol.
35
Figure 12. (A).Total ion chromatogram of MS/MS scanning of maltol sulfate
in urine sample. (B). MS spectrum of the peak with retention time at 5.2 min
in total ion chromatogram A. (C). MS spectrum of the peak with retention time
at 1.7 min in total ion chromatogram A: The ion with m/z 207 is the protonated
ion of maltol sulfate; whereas the product ion with m/z 127 is protonated ion
of maltol.
36
Figure 13. (A). Total ion chromatogram of multiple reaction monitoring (MRM)
scan of maltol glucuronide (303/127) and maltol sulfate (207/127) in
urine sample.(B). Extract ion chromatogram of MRM scan of maltol
glucuronide (303/127). (C). Extract ion chromatogram of MRM scan of
maltol sulfate (207/127).
37
Figure 14. The typical HPLC chromatograms of (a) urine sample with subject
taking 30 ml dark soy sauce and (b) that urine sample digested with enzyme:
inset is the UV spectrum of peak with RT at 14.8 min which agrees with that
of maltol.
38
Figure 15. Time course of digestion of urine samples with 5000U
β-glucuronidase at 37°C.
39
Figure 16. (A). The maltol amount excreted in urine after one subject took
6 mg maltol or 30 ml dark soy sauce mixed with plain boiled rice. The
accumulated maltol amounts excreted in urine for such two cases are also
shown in (B).
40
Figure 17. The average total maltol (standardized with creatinine) measured
in the different time point urine samples of 24 young healthy subjects who
orally took 30 ml of dark soy sauce mixed with 200 gram of plain boiled rice.
Data are mean±SD, n=24 (**P < 0.01 vs 0 h and 3 h).
41
Figure 18. The absorbance and ABTS•+ scavenging activity of MeOH-R
fraction under acidic and basic conditions. The MeOH-R samples were
incubated in 6 M HCl or 4.2 M NaOH in vacuo at 110°C for 18 hours.
The colored components became insoluble in acidic condition, while in
alkaline condition they were stable. Removal of insoluble colored
components dramatically decreased the antioxidant activity of the acidic
hydrolysate, indicating that the colored components could greatly contribute
to the total antioxidant activity (TAA) of dark soy sauce. Values are
mean ± SD, n=3. ***Comparision between the ABTS•+ scavenging activity
of sample + 6 M HCl and that of sample + H2O (*** p < 0.001).
43
Figure 19. (A) Overlain Sephadex G-75 gel filtration chromatograms of
EtOAc-R and MeOH-R of dark soy sauce. Fraction 26 to 34 of EtOAc-R
were combined as Colored Product 1 (CP1), and fraction 3 to 9 of MeOH-R
vii
were combined as Colored Product 2 (CP2). The fragmentation range of
Sephadex G75 gel filtration chromatography is 1000 ~ 50 000 Dalton.
(B) The correlation of ABTS•+ scavenging activity and absorbance of
CP1 and CP2 at 470 nm. Values are mean ± SD, n=3.
44
Figure 20. (a) 1H-NMR spectrum of CP1, (b) 1H-NMR spectrum of
CP2, and (c) 13C-NMR spectrum of CP1.
45
Figure 21. TOF-MS spectrum of CP1. The inset shows a typical doubly
charged ion. The peaks observed at around 1001.3 show the isotopic
pattern at 0.5 Dalton distance.
47
Figure 22. Decrease of cell viability of HT 29 cells 24, 48 and 72 hours
after incubation with various concentrations of dark soy sauce (DSS) (A),
and DSS Nondialysable fraction (B). Results are mean±SD, n=3.
52
Figure 23. Cell viability tested on dark soy sauce only or plus catalase
(1000 units/ml).
53
Figure 24. Time course of COX-2 expression and its inhibition by dark
soy sauce in lipopolysaccharide (LPS)-induced HT-29 cells. Cells were
pretreated with dark soy sauce (5 μl/ml) for half of an hour and then
induced with LPS (100ng/ml) for various time indicated. Protein levels
were estimated by Western Blot analysis as described in “Materials and
Methods”. Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells treated
with LPS (100 ng/ml) only); and lane 3, HT-29 cells simultaneously
treated with dark soy sauce (5 μl/ml) and LPS (100 ng/ml).
54
Figure 25. Inhibitory effect of dark soy on COX-2 expression in LPSinduced HT-29 cells . Lane 1, untreated HT-29 cells; Lane 2, HT-29
cells treated with 1 μl/ml dark soy sauce(DSS); Lane 3, with 5 μl/ml
DSS; Lane 4, with LPS (100 ng/ml); Lane 5, with DSS (1 μl/ml) and
LPS (100 ng/ml); Lane 6, with DSS (5 μl/ml) and LPS (100 ng/ml).
The western blot is from a single experiment, but is representative of 3
independent experiments.
55
Figure 26. The effect of maltol on COX-2 expression in LPS-induced
HT-29 cells.Lane 1 is untrated HT-29 cells; lane 2, HT-29 cells treated
with maltol (100 μM); lane 3, maltol (500 μM); lane 4; treated with LPS
(100 ng/ml); lane 5, maltol (100 μM) and LPS (100 ng/ml); lane 6, maltol
(500 μM) and LPS (100 ng/ml).
55
Chart 1. The flow chart of fractionation and isolation of maltol, CP1 and
CP2 from dark soy sauce.
10
viii
List of Symbols
ABTS
2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
APCI-ITMS
atmospheric pressure chemical ionization-ion trap mass spectrometry
COX-2
cyclooxygenase-2
CP1/2
the colored product 1/2
DAD
diode array detection
DSS
dark soy sauce
EI-MS
electron impact – mass spectrometry
ESI-MS
electrospray-ionization mass spectrometry
EtOAc-R
ethyl acetate extract residue
FTIR
Fourier transform infrared
GIT
gastrointestinal tract
HEMF
4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone
HPLC
high performance liquid chromatography
LC-MS/MS
liquid chromatography-mass spectrometry/mass spectrometry
LOD
limit of detection
LOQ
limit of quantification
LPS
lipopolysaccharide
MeOH-R
methanol extract – residue
MRM
multiple reaction monitoring
NMR
nuclear magnetic resonance
RP
reverse phase
RT
retention time
ix
SPE
solid phase extraction
TAA
total antioxidant activity
TEAC
trolox equivalent antioxidant capacity
TOF
time-of-flight
UV
ultraviolet
x
Chapter 1 Introduction
Pour it into soup, and watch the artful
dark tongue mix its own remedy. It is the same
as looking at hard men cry as they watch
cream weave through coffee.
(From Tina Chang’s poem, Ode to Soy Sauce [1])
Soy sauce is a traditional fermented seasoning of East Asian countries and is currently
used in cooking worldwide. Not only do the flavor components of soy sauce improve
the taste of many types of foods, but its coloring ingredients can enhance the
appearance of the dipped food or the mixed soup. Moreover, recent studies indicate
that some ingredients in soy sauce are potentially beneficial to human health, showing
effects such as anticarcinogenesis, antihypertension and antihyperlipidemia [2-4].
1.1 A brief history of soy sauce
The history of soy sauce goes back over three thousand years. The origin of soy sauce
is generally considered to be in China, where soy sauce is called jiang-you, the extract
of jiang. Jiang was first recorded in the books of Zhou dynasty (1121-256 B.C.),
while Jiang-you was first mentioned in a book of ‘Qimenyaoshu’, Jia Sixie, Bei-Wei
dynasty (220-265) [5].
It is speculated that, to preserve foodstuffs against periods of scarcity, the ancient
Chinese accidently discovered Jiang when they mixed salt with meat, fish and
vegetables, and innocently incorporated some harmless aeroborne fungi, which
fermented the raw material after a long period of culture. With the introduction of
Buddism to China, meat was excluded from the diet of the Buddist monks. Plant1
origin materials, such as soy bean and wheat, then began to be widely used to make
sauce [6].
Buddist monks are thought to have played an important role in spreading soy sauce
from China to Japan. Soy sauce was first introduced into Japan by a Buddist monk,
Jian Zhen, in Tang Dynasty (618-907) [7]. But some consider that Japanese soy
sauce originated from that brought back by a Japanese Buddist monk, Kakushin, from
China in Song Dynasty (960-1279) [8-10]. It is in Japan that the making of soy sauce
was modernized and exported to Europe and North America. Between 17th and 18th
century, a large quantity of soy sauce was exported from Japan by a Dutch company
to Europe [8]. As early as in 1867 Japanese soy sauce was taken along by immigrants
to Hawaii [11]. In 1972, the Kikkoman Company opened a modern soy sauce plant at
Walworth, Wisconsin [8].
Soy sauce was brought to East Asian countries by the Chinese immigrants. In
Singapore, it is said that the small-scale manufacturing of soy sauce started by a small
number of Xin-hui Cantonese, a sub-dialect group from Guangdong Province of
South China, just a couple of years after the first arrival of Chinese immigrants in the
early nineteenth century [12]. After being successful, many of them shifted their
business to other more profitable fields. Only a few survived. For example, Chuen
Cheong Food Industries, which produces Tiger brand sauce, was established in 1930,
earlier than other local famous soy sauce manufacturers, such as Yeo Hiap Seng
(1938) and Tai Hua Food Industries (1949). The business and technologies of soy
sauce fermentation have been passed from father to son. Now the fourth generation is
in the charge of the business [13, 14].
2
1.2 The methodology of preparation of soy sauce
The ratios of starting raw materials, soy bean and wheat, are different for Chinese
style and Japanese style: around 50:50 for Japanese style, while approximately 60:40
for Chinese style [15]. For both styles of traditionally fermented soy sauce, the basic
procedure of production is very similar. The method of soy sauce production
practiced locally is based on the traditional Chinese fermentation process, the main
steps of which are as follows [16]:
Raw material preparation:
Soy beans or defatted soy flakes are soaked and then cooked, while wheat is roasted
The Koji fermentation process
The raw materials are mixed and inoculated with mold (seed Koji), and then cultured
for 2-3 days with controlled temperature and moisture. The mold grows enough to
provide the enzymes necessary to hydrolyze the raw materials, thus, then, also called
sauce Koji.
The mash fermentation
The sauce Koji is poured into fermentation tanks and mixed with saline water, aging
for 3 months to 2 years, upon the culture temperature. During the fermentation period,
the enzymes from Koji mold hydrolyze most of the protein to amino acids and lowmolecular-weight peptides, and some polysaccharides into simple sugars.
Refinement
The aged mash is filtered. The filtrate is called raw soy sauce. The raw soy sauce is
further pasteurized and finally bottled. To make dark soy sauce, the aging process is
3
further extended for another 2-4 months, and caramel is added to make the final
product thicker and the color deeper.
Traditionally, the making of soy sauce is time-consuming and labor-consuming. The
mash is usually fermented in earthenware, and sunlight provides the thermal energy
for aging. So in the North of China or Japan, the process of manufacturing can last as
long as 2 years. To save time, in some modern soy sauce factories, thermal-controlled
fermentation tanks are used. The making process of soy sauce can be shortened to
three months [17].
After World War II, when the supplies of soy beans were scarce, many manufacturers
applied chemical soy sauce, acid hydrolyzed vegetable proteins. But the flavor of such
products is poor. To keep some flavor as of fermented soy sauce, semi-chemical and
blended soy sauce were produced. Semi-chemical soy sauce is produced by further
fermentation of acid hydrolyzed products; while blended soy sauce is produced by
mixing fermented soy sauce with chemical soy sauce and/or enzymatically
hydrolyzed vegetable protein. As Asian economies have grown, fermented soy sauce
becomes the main product in Asian markets. The United States is left to be the largest
chemical soy sauce producer in the world. Japanese producers once proposed the
world-trade regulations that the manufacturing methods should be included in the
label. But the US suggested that individual countries should make their own decisions
on labeling [18]. In Singapore, soy sauce can be made from soy beans with or
without other foodstuffs, by “either enzymic reaction or acid hydrolysis or by both
methods” [19], although local products are traditionally fermented. But there is no
labeling requirement on the manufacturing process.
4
1.3 The Functional components of soy sauce
In China, soy sauce has been traditionally used for treatment of anorexia, ulcers post
thermal burn etc, recorded in Traditional Chinese medicinal books (Su Jing,
Xinxiubencao, Tang Dynasty, 659; Sun Simiao, Qianjinfang, Tang Dynasty, 625)
[7]. Modern scientific research has provided some supporting evidence for such usage:
e.g. soy sauce can promote gastric juice secretion in humans; soy sauce has
antimicrobial activity, due to the synergistic effects of NaCl, ethanol, pH, and
preservatives [20].
Soy sauce has a variety of biologically active effects, such as hypotensive,
anticarcinogenic, anticataract, and antiplatelet . Nicotianamine was found to be the
major bioactive components inhibiting angiotensin I-converting enzyme. 4-hydroxy3(2H)-furanone derivatives, namely, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)furanone, 4-hydroxy-5-methyl-3(2H)-furanone and 4-hydroxy-2,5-dimethyl-3(2H)furanone, are antioxidants, also having anticarcinogenic and anticataract activities.
Shoyu-flavones, derivatives of daidzein, genistein, and 8-hydroxygenistein, are
antioxidants and histidine decarboxylase inhibitors. 1-methyl-1,2,3,4,-tetrahydro-βcarboline and 1-methyl-β-carboline are found to be the active antiplatelet components
[20].
During the fermentation process, the soybean and wheat proteins are degraded into
peptides and amino acids. Thus the IgE-mediated hypersensitive response to wheat is
markedly reduced in soy sauce. Furthermore, the soy sauce polysaccharides that
cannot be hydrolyzed during the fermentation process can inhibit hyaluronidase
activity and histamine release. In vitro, these polysaccharides can increase the
production of IgA from Peyer’s patch cells. In vivo, soy sauce polysaccharides can
5
increase the concentration of IgA in the intestines of mice, suppress passive cutaneous
anaphylaxis reaction in the ears of allergy model mice, and regulate the balance of
Th1/Th2 cell response in mice, potentially enhancing host defenses [21-23]. Clinically,
oral administration of soy sauce polysaccharides can improve allergic symptoms of
patients with perennial allergic rhinitis or seasonal allergic rhinitis [23]. Soy sauce
polysaccharides can also inhibit pancreatic lipase activity and reduce the absorption of
lipid in mice and humans [4].
1.4 The balance of free radicals/reactive species and antioxidants
A free radical is any species capable of independent existence containing one or more
unpaired electrons [24]. Free radicals can be formed by homolytic or heterolytic
fission of a covalent bond. For example, UV-induced homolytic fission of the O-O
bond in H2O2 can produce hydroxyl radical, OH•. Reactive oxygen species (ROS)
includes not only the oxygen radicals (O2• − and OH•) but also non-radical derivatives
of O2 (H2O2, HOCl and O3). Univalent reduction of molecular oxygen can form a
variety of ROS. Mitochondrial electron transport chain is the main cellular source of
ROS. Various oxidases in cell membranes, cytosol, and other organelles can transfer
single electrons onto dioxygen,e.g. Cytochrome P450, a monooxygenase in the
endoplasmic reticulum, is able to reduce dioxygen to superoxide radicals. Transition
metals, e.g. iron, having the capability of donating and/or receiving electrons, play an
important role in oxygen radical formation. Inflammation can also result in excessive
production of free radicals. Free radicals and ROS are not only formed endogenously,
but also introduced outside [25].
Our body has evolved antioxidant defense systems. Small molecules and antioxidant
enzymes are two major categories. Antioxidant enzymes, such as superoxide
6
dismutase, catalase, and glutathione reductase, are important for antioxidant defense
[24].
Imbalance between formation of free radicals/reactive oxygen species and levels of
antioxidants in vivo has been suggested to play a role in the development of various
diseases, such as atherosclerosis, diabetes, rheumatoid arthritis, cancer and
neurodegenerative diseases [24, 26]. Some (but not all) studies show that nutritional
antioxidants can decrease oxidative damage in the human body and may have
beneficial effects on disease prevention [26 – 29]. This has led to a growing interest in
antioxidants from natural products [26 – 31]. Several papers have alluded to the
presence of antioxidants in soy sauce [32 – 36].
Tiger brand soy sauce is a local brand sauce with an 80-years history [13, 14]. Our
group’s studies showed that dark soy sauce, especially Tiger brand products, had
extremely high total antioxidant activity (TAA) in vitro [36] as judged by the ability
to scavenge the nitrogen-centred ABTS•+ radical, an assay that is frequently used to
assess the antioxidant activity of beverages, food extracts and body fluids [37]. In vivo
Dark soy sauce of this brand also decreased lipid peroxidation in human volunteers
[38]. In this study, using Tiger brand products, we attempted to identify the major
components that contribute to the high antioxidant activity of dark soy sauce.
7
Chapter 2 Materials and Methods
2.1 Chemicals
All chemicals were obtained from Sigma-Aldrich, Singapore unless otherwise stated.
Ethyl acetate, formic acid 37% [Guaranted Reagent (GR) for analysis], sodium
hydroxide (GR for analysis), were from Merck, Germany; methanol (HPLC grade)
was from Labscan Analytical Science, Thailand.
Dark soy sauce (Tiger brand, Chuen Cheong Food Industries, Singapore) was
purchased from a local supermarket.
2.2 ABTS assay
This was carried out as described in Ref. [36, 37]. 2,2ʹ-Azino-bis[3ethylbenzothiazoline-6-sulfonate] (ABTS) in water (7 mM final concentration) was
oxidized using potassium persulfate (2.45 mM final concentration) for at least 12 h in
the dark. The ABTS•+ solution was diluted to an absorbance of 0.70 ± 0.02 at 734 nm
(Beckman UV – VIS spectrophotometer, Model DU640B, UK) with phosphate
buffered saline (PBS 10 mM, pH 7.4). Extracts of dark soy sauce (10 μl) or trolox
standard (10 μl) were added to 1 ml of ABTS•+ solution. Absorbance was measured
1 min after initial mixing. Antioxidant properties of fractions of dark soy sauce
extracts were expressed as Trolox equivalent antioxidant capacity (TEAC), calculated
from at least three different concentrations of extract tested in the assay and giving a
linear response.
8
2.3 Isolation of low molecular mass components from ethyl acetate extract
Dark soy sauce (6.4 L) was extracted three times with a four-fold volume of methanol.
The methanol extracts were combined, filtered by filter paper and evaporated to
dryness under vacuum at 40ºC (MeOH-extract, yield 2.6 kg). No significant retention
of antioxidant components from soy sauce by the filter paper was detected.
The residue after methanol extraction (MeOH-R) yielded 1.4 kg. The MeOH-extract
was suspended in water and partitioned with ethyl acetate three times.
The ethyl acetate fractions were combined and evaporated to dryness under vacuum at
30ºC (EtOAc-extract, yield 11.6 g). The remaining aqueous fraction (EtOAc-R)
yielded 2.6 kg (Chart 1.).
As the EtOAc-extract exhibited strong ABTS radical scavenging activity, it was
subjected to flash chromatographic separation with a silica gel RP18 (particle size 40
– 63 µm, Merck KGaA, Darmsadt, Germany) packed column (6 × 42 cm) eluting with
methanol and water. Fractions eluted with 10% methanol had the highest ABTS•+
radical scavenging activity and were further purified by a prep-HPLC (Agilent 1100
Series, equipped with a fraction collector) using a ZORBAX SB-C18 PreP HT
column (21.2 × 250 mm, 7 µm) (Agilent, USA) at 20 ml/min with methanol—0.1%
formic acid in MilliQ water (10:90, v/v) as mobile phase. Ten fractions showing
antioxidant activity in the ABTS assay, Fr.1 to 10, were obtained. Fraction 9 was
found to have the most ABTS scavenging activity and yielded Compound 1 (41mg).
2.4 Fractionation of colored components
Approximately, 1 gm of ethyl acetate extract residue (EtOAc-R) was resuspended in
25 ml distilled water and dialyzed against distilled water for seven days, using a
9
Dark soy sauce
(1) extracted with 4 fold methanol (MeOH)
3 times
MeOH-R
MeOH-extract
(1) dialyzed against distilled water
(2) fractionated with gel filtration
chromatography
(1) filtered by filter paper, evaporated to
dryness under vacuum at 40 ºC
(2) and suspended in water;
(3) partitioned with ethyl acetate (EtOAc)
three times
Colored Product 2
EtOAc-extract
(1) Fractionated with RP18
column chromatography
(2) Further fractionated with
prep HPLC. Eluted with
methanol – 0.1% formic acid
in milli Q water (10:90)
Fr.1 to Fr.10
(Fr. 9 Æ maltol)
EtOAc-R
(1) dialyzed against distilled water
(2) fractionated with gel filtration
chromatography
Colored Product 1
(CP1)
Chart 1. The flow chart of fractionation and isolation of maltol, CP1 and CP2 from dark soy sauce.
10
cellulose dialysis tubing (Pierce, Rockford, USA; molecular weight cutoff 3500).
Initially, we investigated the influence of dialysis time and temperature (room
temperature, ~25ºC, and cold-room temperature, ~4ºC), on the antioxidant capacity of
the colored products as measured by the ABTS assay and found no significant effect.
For convenience, the dialysis experiments were carried out at room temperature. The
non-dialyzable fraction was freeze-dried.
Approximately, 85 mg of the freeze-dried product was dissolved in 5 ml of water and
loaded onto a fine Sephadex G-75 gel filtration chromatography column (2 × 100 cm).
The colored fractions (absorbance at 470 nm), 4 ml per tube, were collected. The
fractions from No. 26 to 34 possessed significantly higher TEAC values and
consequently were combined as Colored Product 1 (CP1) (yield 61 mg).
The MeOH-R fraction (approximately 2 g) was dialyzed against distilled water. The
non-dialyzable fraction (85mg) was fractionated with gel chromatography in the same
way as EtOAc-R. The fractions from No. 3 to 9 were combined as Colored Product 2
(CP2) (yield 47mg) (Chart 1.).
2.5 HPLC determination of maltol in dark soy sauce
Dark soy sauce (10 ml) was extracted with 40 ml methanol on an orbital shaker
(SLOS-20, Seoulin Bioscience, Seoul, Korea) at a speed of 150 rpm for 24 h, and then
centrifuged at 3000g for 30 min. This procedure was repeated three times. The
supernatants were pooled and dried using a rotary evaporator under vacuum at 40ºC.
The residue was dissolved in 20 ml water and extracted three times with 20 ml ethyl
acetate. The ethyl acetate extracts were combined and evaporated to dryness at 30ºC.
Prior to HPLC analysis, the dried samples were dissolved in 10 ml methanol-0.1%
11
formic acid (1:9) and then filtered through 0.45 µm disposable nylon filters (Agilent
Technology, USA).
Analysis was performed using an Agilent 1100 HPLC with an Agilent ZORBAX SBC18 column (4.6 × 250 mm, 5 µm), maintained at 35ºC. The mobile phase was formic
acid in MilliQ water (0.1%, v/v) (A) and methanol (B) with a gradient program as
follows: 10% of B for 15 min, 10 – 90% of B in 6 min, 90% of B for another 5 min,
with flow rate at 1 ml/min. The injection volume for all samples was 10 μl and
absorbance was monitored at 270 nm. Spectra were recorded from 190 to 400 nm.
2.6 Mass spectrometry
An Agilent XCT Plus ion trap mass spectrometer (ITMS) (Agilent Technology, US)
was used to analyze the fractions, Fr. 1 – 10, obtained from ethyl acetate extracts.
Atmospheric pressure chemical ionization (APCI) MS was performed in the positive
mode. The dry gas and vaporizer temperatures were 350 and 400ºC, respectively.
Compound 1 was also analyzed by an electron impact (EI) MS spectrometer (Agilent
Technologies), sample dissolved in methanol and introduced by a gas
chromatography (GC) interface (Agilent Technologies).
For the analysis of CP1, Electrospray-Ionization Mass Spectrometry (ESI-MS) was
performed using a Waters Micromass Q-Tof micro mass spectrometer (Waters, USA).
For acquiring mass spectra, sample was directly infused at a speed of 10 µl/min. The
capillary and sample cone voltages were maintained at 3.0 kV and 50 V, respectively.
The source and desolvation temperatures were 80 and 250ºC, respectively. The mass
spectra were acquired from m/z 100 to 5000 in the positive ion mode.
12
2.7 Fourier transfer infrared spectrometry (FTIR)
IR spectra (KBr disc) were recorded on a JASCO FT/IR-430 spectrometer (Japan).
2.8 Nuclear magnetic resonance spectrometry (NMR)
The NMR spectra of CP1 and CP2 and compound 1 were recorded on a Bruker
Advance AMX500 NMR spectrometer (Rheinstetten, Germany) at 500.13 MHz (1H)
and 125.75 MHz (13C), respectively. Compound 1 was dissolved in methanol-d4; CP1
and CP2 were dissolved in D2O.
2.9 Detection and determination of maltol metabolites in human urine samples
2.9.1 Standard preparation
Maltol glucuronide was isolated and puried from the urine samples of a healthy
volunteer using solid phase extraction (SPE), reverse-phase (RP) C-18 flash column
chromatography and preparative HPLC. The purity of the isolated compound was
checked on an analytical HPLC system. Its identity was confirmed based on NMR
data.
Maltol glucuronide,
1
H-NMR, 2.39 (s), 3.56 (m), 3.86 (d), 4.95 (d,dd), 6.48 (d), 7.98 (d)
13
C-NMR, 15.12, 71.24, 73.11, 75.23, 102.48, 115.98, 141.39, 156.70, 164.73, 172.53,
176.62
Maltol sulfate was synthesized based on a published method [39]. To the solution of
200 mg maltol in pyridine, 300 mg pyridine-sulfur trioxide complex was added and
the mixture was stirred at 4 °C for 60 hours. The precipitating solid in the reaction
mixture was filtered off and washed with CHCl3, then dried in vacuo to give 390 mg
13
solid. The solid was then dissolved in water and further purified using a preparative
HPLC (Agilent Technologies).
A series of different concentrations (0.05 mM - 1 mM) of maltol standard solutions
were prepared in 10% methanol/0.1% formic acid- MilliQ water.
2.9.2 Sample preparation
To test how fast maltol is metabolized, the urine samples at different time points (0.5,
1, 2, 3, 4, 5 and 6h) were collected from a human volunteer who had consumed 6 mg
maltol or 30 ml dark soy sauce mixed with 200 gram of plain boiled rice. The
washtime between these two experiments were more than one week. The urine
samples were also collected from 24 young health volunteers in an observer-blinded,
randomized, placebo controlled, crossover clinical study [38].
The urine samples were deproteinized with three volumes of methanol, then
centrifuged at 1500 × g for 10 min. For detection of maltol metabolites, the
supernatants were directly injected onto HPLC column. For determination of total
maltol content in urine, these supernatants were dried under N2 flow, digested with
200 μl 11.1 mg/ml β-D-galactonidase in 0.5 mM acetate buffer at 37°C overnight, and
then centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were collected and
analyzed immediately using HPLC.
2.9.3 HPLC-DAD detection of maltol metabolites and determination of total maltol
content in urine
HPLC analysis was performed on an agilent 1100 HPLC system, equipped with a
diode array detector. The samples were introduced by an autosampler. A
Phenomenex column (4.6 × 250 mm, 5.0 μm) was used, with temperature kept at
14
35 °C in a thermostat column compartment. UV spectra (190-400 nm) were recorded.
The mobile phase was composed of 10% methanol in 0.1% formic acid, with a
running rate of 1ml/min.
2.9.4 HPLC-MS/MS detection of maltol metabolites
LC-MS/MS analysis of maltol metabolites in urine was performed on an Agilent 1200
LC system (Palo Alto, CA, USA) coupled to an API3200 Triple-Quadrupole mass
spectrometer (Applied Biosystems/MDS SCIEX, Foster City, USA). A Turbo V
source with ESI prober was used for the analysis. Data acquisition was performed
with Analyst 1.4.2 software (AB MDS Sciex).
The chromatographic separation was performed on a 150 × 2.0 (i.d.) mm Phenomenex
Synergi 4µ Polar-RP 80 Å column (Phenomenex, CA, USA) with a column oven
temperature of 35°C. The mobile phase was premixed 10% methanol in 0.1% formic
acid. Flow rate was 0.4 ml/min. Standards or samples were introduced into the LC
using an Agilent 1200 G1367B autosampler and injection volume was 10.0 μl.
The ion source was operated in the positive mode. For survey scan (Q1 MS scan
mode), ion-spray voltage and temperature were set at 5000 V and 600oC. Ion source
gas 1(GS1) and ion source gas 2 (GS2) were set at 40 and 45 psi. Interface heater (ihe)
was set as on. The curtain gas (CUR), declustering potential (CE) and entrance energy
(EP) settings were at 10 psi, 50 V and 10 V, respectively.
In product ion scan (MS2) mode, for maltol glucuronide, parent ion 303 was used,
and scan range was set at 50 – 400; whereas for maltol sulphate, parent ion 207, scan
range was 50 -300. For both chemicals, collision energy (CE) was set at 5 V. In
multiple reaction monitoring (MRM) mode, the ion pairs monitored for maltol
glucuronide and maltol sulphate were 303/127 and 207/127 respectively.
15
2.10 GC-MS analysis of DNA base modification
2.10.1 Sample preparation
Salmon testis DNA was dissolved in 100mM phosphate buffer (final concentration 1
mg/ml). 1 ml of DNA sample was pretreated with or without various concentrations
of soy sauce extracts before hypochlorous acid (HOCl) exposure. HOCl concentration
was quantified spectrophotometrically (290 nm, pH 12, ∈ = 350 M-1cm-1) [40]. HOCl
dilutions were prepared in Krebs buffer (mmol/l: NaCl 130, KCl 5.7, NaHCO3 25,
NaH2PO4 10, D-glucose 5, CaCl2 1, and MgCl2 0.5, pH 7.4) immediately before use.
Here we use the hypochlorous acid (pKa 7.46) to refer to an ≈50% ionized mixture of
HOCl and OCl‾ (hypochlorite). After incubation with HOCl (500 μM) at 37 °C for 1
hour, the DNA samples were dialyzed against distilled water for 24 hours in a cold
room. The concentration of DNA was measured spectrophotometrically at 260 nm
(E260 = 50 μg/ml). Aliquots of 100 μg DNA were hydrolyzed by adding 0.5 ml of 60%
(v/v) formic acid and heating at 140 °C for 45 min in an evacuated, sealed hydrolysis
tube. The internal standards were added to the cooled hydrolyzed samples and freezedried. Derivatization was carried out for 2 hour at 25 °C using BSTFA (+1%
TMCS)/acetonitrile/ethanethiol (16:3:1(v/v)) mixture.
2.10.2 GC-MS analysis
The GC-MS analysis was performed as previously reported [41, 42]. Derivatized
samples were analyzed by GC-MS (Agilent gas chromatography 6890 interfaced with
Agilent 5973 mass selective detector). The injection port and the GC-MS interface
were kept at 250 and 290 °C, respectively. Separations were carried out on a fused
silica gel capillary column (12 m × 0.2 mm i.d.) coated with cross-linked 5%
phenylmethylsiloxane (film thickness 0.33 μm) (Agilent). Helium was the carrier gas
16
with a flow of 1.0 ml/min. Derivatized samples (1.0 μl) were injected into the GC port
using a split ratio of 8:1. Column temperature was increased from 125 to 175 °C at
8 °C/min after 2 min at 125 °C, then increased from 175 to 220 °C at 30 °C/min, held
at 220 °C for 1 min, and finally increased from 220 to 290 °C at 40 °C/min and held
at 290 °C for 2 min. Selected-ion monitoring was performed using the electronionization mode with the ion source maintained at 189 °C.
2.11 Cell culture
Human colorectal adenocarcinoma cells (HT29), obtained from the American Type
Culture Collection, were cultured in McCoy’s 5A media with 1% (v/v) penicillin, 10%
(v/v) fetal bovine serum (FBS), 5% CO2 at 37 °C. The cells were maintained in the
logarithmic growth phase by routine passage every 2-3 days.
2.12 Assessment of cell viability
Viability was assessed by the MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium
bromide) method as previously reported [43, 44]. Cells in McCoy’s 5A full media
were seeded overnight at a density around of 1.5 × 104 cells per well in 96-well plates.
After treatment with various concentration of dark soy sauce in full medium, 200 μl
of MTT (0.5mg/ml final concentration) dissolved in McCoy’s 5A only (with FBS).
Cells were incubated at 37 °C in darkness for 1 hour and then MTT was removed. 200
μl of dimethyl sulfoxide (DMSO) was added to solubilize the formazan formed. After
shaking in the dark for 15 min, absorbance at 550 nm was measured using a
microplate reader (SpectraMax190, Molecular Device).
17
2.13 Western blot analysis
Experiments were performed as describe in [43, 45]. Control and treated cells were
washed twice with cold PBS buffer, incubated in lysis buffer on ice for 30 min, and
then scraped into Eppendorf tubes. Cells were centrifuged at 13800 rpm at 4 °C for 15
min using a desktop centrifuge (Eppendorf) to remove unbroken cells, nuclei and
other organelles. The protein concentration of the resulting supernatant was measured
with the DC protein assay Kit (Bio-Rad), and 20 μg of protein was boiled for 10 min,
and then loaded in a 12.5% (v/v) SDS – PAGE gel. The separated proteins were then
transferred to nitrocellulose membranes and probed with antibodies against COX-2,
β-actin, followed by the appropriate horseradish peroxidase-conjugated secondary
antibodies.
2.14 Statistical analysis
The mean values were calculated from data taken from at least three separate
experiments. Where significance testing was performed, a Student’s t-test was used;
P-values of, 0.05 were considered to be statistically significant.
18
Chapter 4
Results and Discussion
4.1 Separation and characterization of low molecular mass components
Dark soy sauce was extracted with methanol, and the methanol extract was further
partitioned with ethyl acetate as described in Experimental section (Chart 1). The
three fractions of dark soy sauce, methanol extract residue (MeOH-R), ethyl acetate
extract (EtOAc-extract), and ethyl acetate extract residue (EtOAc-R), were subjected
to the ABTS assay. The MeOH-R fraction contributed about 60% of the total
antioxidant activity (TAA) of dark soy sauce. However, the EtOAc-extract showed
the strongest antioxidant activity, approximately 12-fold higher than that of the dark
soy sauce (Figure 1).
After pretreatment with a reverse phase (RP)-C18 column (mainly to remove benzoic
acid, which has no antioxidant activity in the ABTS assay), the EtOAc-extract was
separated with preparative high performance liquid chromatography (HPLC) resulting
in 10 fractions (Figure 2A). Among them, the ABTS assay (Figure 2B) showed that
Fr.9 has by far the strongest activity. Fr.9 was further purified to yield compound 1.
The positive atmospheric pressure chemical ionization - ion trap mass spectrometry
(APCI-ITMS) spectrum of Compound 1 showed a protonated molecular ion peak at
m/z 127 [M + H]+. The electron impact – mass spectrometry (EI-MS) spectrum also
shows its molecular ion at m/z 126 (Figure 3). The major fragment ions (m/z 97, 71,
55) were observed. The molecular ion m/z 126 can be decomposed to a furan ion (m/z
97) by losing one H and CO. The furan fragment ion (m/z 97) can be further
decomposed to ion m/z 55 by losing CH3CO. Via another pathway, the molecular ion
19
(m/z 126) can give the fragment ion m/z 71 by losing CH2CH2 and CO. The
mechanism for these fragment ion formation is proposed in Figure 3. The ion
fragment pattern agrees with that previously reported [46]. The proton and carbon 13
nuclear magnetic resonance (1H- and 13C-NMR) spectral data of compound 1 are
listed in Table I. The 1H-NMR spectrum of compound 1 (500MHz, methanol-d4)
showed two cis-olefinic proton signals at δH 7.94 (1H, d, J = 5.5 Hz) and 6.39 (1H, d,
J = 5.5 Hz). In addition, the signal of one methyl group directly attached to an olefinic
carbon at δH 2.35 (3H, s) was observed. The 13C-NMR spectrum showed one
carbonyl carbon signal at δC175.3, and four olefinic carbon signals at δc 156.3, 152.2,
144.6 and 114.4, indicating the presence of a pyranone ring. The signal at δc 14.2
corresponded to the carbon of the methyl group. The Fourier transform infrared (FTIR)
spectrum of compound 1 shows the major absorbance bands (cm-1): 3258, 3069, 1655,
1617, 1560, 1459, 1257, 1199. Compound 1 was also analyzed by high performance
liquid chromatography – diode array detection (HPLC-DAD). Its retention time and
ultraviolet (UV) spectra (absorption maxima at 210 nm and 275 nm) agreed very
closely with those obtained from authentic maltol (Figure 4). So the MS spectrum,
NMR data, FTIR spectrum, the retention time and UV spectrum support the identity
of compound 1 as 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) (Figure 5).
Fractions 1-8 and 10 were also analyzed by APCI-ITMS, suggesting these fractions
contain molecules with molecular weight ranging from 124-214 (Appendix: Table A1).
20
1800
1580
1600
TEAC (mM)
1400
1200
1000
800
600
400
200
309
122
51
0
D SS
MeO H -R
EtoA cextract
EtoA c-R
Figure 1. Trolox equivalent antioxidant capacity (TEAC) values per g/ml of dark soy
sauce (DSS) and three fractions: methanol extract residue (MeOH-R), ethyl acetate
extract (EtOAc-extract) and ethyl acetate extract residue (EtoAc-R). Values are mean
± SD, n=3.
21
mA U
250
9
5
(A)
200
150
1
3
100
10
7
50
2
8
6
4
0
0
5
10
15
20
25
30
35
40
min
Retention Time
25.00
21.21
(B)
T EA C (mM)
20.00
15.00
10.00
5.00
0.00
1.85
1.81
1.27
0.39
0.34
0.58
2
3
4
0.22
0.43
5
6
1
0.88
7
Fraction Number
8
9
10
Figure 2. (A) Typical HPLC chromatogram of ethyl acetate extract. The absorbance
was monitored at 270 nm. (B) Trolox equivalent antioxidant capacity (TEAC) values
of 10 fractions of ethyl acetate extract per μg/mL. Results are mean ± SD, n=3.
22
+.
O
OH
+
O
_
H.
_
O
CH 3
CH 3
O
+
+
_ CH CO
2
CO
O
O
OH
O
CH 3
m/e = 55
m/e = 97
m/e = 126
O
O
O
O
+
_C H
2 2
C
O
CH 3
O
+
+
O
CH 3
O
O
O
_ CO
CH 3
O
CH 3
+
m/e = 71
A bundance
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
126
71
55
50
97
60
70
80
90
100
m/z
110
120
130
140
Figure 3. EI-MS spectrum of compound 1 and the proposed mechanism for the
formation of fragment ions.
23
N orm
160
*Fr.2a9
*maltol
(d)
Match factor:
999.967
120
80
40
0
200
mA
U
700
250
300
350
nm
(a)
600
500
400
(b)
300
200
100
(c)
0
2.5
5
7.5
10
12.5
15
17.5
min
Figure 4. The HPLC chromatograms of (a) ethyl acetate extract of dark soy sauce, (b)
compound1 and (c) authentic maltol. (d) The overlaid spectra of maltol and
compound 1; the match factor is 999.967 (It is generally considered to be matched
well, if the match factor is no less than 990.).
24
Figure 5. Structure of 3-hydroxy-2-methyl-4H-pyran-4-one (maltol)
Table I. 1H- and 13C-NMR data of Compound 1.
C/H number
δC (ppm)
δH (ppm)
2
152.2
3
144.6
4
175.3
5
114.4
6.39 (1H, d, J = 5.5 Hz)
6
156.3
7.94 (1H, d, J = 5.5 Hz)
7
14.2
2.35 (3H, s)
25
4.2 Content of maltol and its contribution to the TAA of dark soy sauce
A considerable part of the TAA of the dark soy sauce was contained in MeOH-R and
EtOAc-R fractions and in fractions 1-8 and 10 (Figure 1, 2). Although maltol was
identified as the active fraction of Fr. 9, we needed to determine its overall
contribution to TAA. Thus an HPLC method was developed for determination of
maltol in dark soy sauce.
Maltol standard was dissolved in methanol-0.1% formic acid (10:90, v/v) yielding
concentrations of 0.25, 0.5, 1.0, 1.5, 2.0 mM. Three different sets of standard
solutions were prepared and analyzed each day and in three continuous days.
Calibration curves for the quantification of maltol were obtained by plotting amount
(mM) against peak area. The calibration curve (Figure 6) was linear within the
investigated concentration range (0.25 – 2.0 mM) with the following regression
equation:
A = 4869.4C – 43.532 (R2 = 0.9999, n=9)
where A is the peak area (mAU*S), and C is the concentration of maltol solution
(mM).
The within- and between-assay precision were established by injecting three sets of
samples at three spike concentration levels (0.25, 1.0 and 2.0 mM) within one and
three days, respectively. The within-assay and between-assay was in the range 0.3-1.8%
and 1.4-3.4%, respectively (Table II). The limit of detection (LOD) was determined as
the amount that resulted in a peak with a height two or three times that of the baseline
noise. The limit of quantification (LOQ) was determined as the amount that resulted
in a peak with a height ten times that of the baseline noise. The LOD and LOQ were 8
26
and 25 μM respectively. Figure 7 shows a typical chromatogram at such concentration.
The accuracy was evaluated through recovery studies by adding known amounts of
maltol at different levels (0.25, 0.5, 1.0, 1.5, 2.0 mM) to the sample. The unspiked
samples and each of the spiked samples were analyzed in triplicate. The recoveries at
the different concentration levels were in the range 89.8 to 94.2% (Table II). Using
this validated method, the average concentration of maltol in dark soy sauce was
determined to 1.15 ± 0.04 mM (n=5). One typical HPLC chromatogram of ethyl
acetate extract of dark soy sauce is shown in Figure 8.
Based on the ABTS assay, the total antioxidant activity of dark soy sauce and matol
was evaluated. The antioxidant activity of maltol (TEAC value) was 2.67 ± 0.05 mM
(n=5) (Figure 9). This concentration of maltol was calculated to contribute around
2.42% of the TAA of dark soy sauce.
Table II. Within- and between-assay precision and recoveries of the assay used to
measure maltol.
Concentration
level/mM
0.25
Within-assay
precision (%
RSD, n=3)
1.8
Between-assay
precision (% RSD,
n=3)
3.4
0.5
1.0
89.8
89.9
0.4
2.4
1.5
2.0
Recovery (%)
92.9
94.2
0.3
1.4
93.9
27
12000
y = 4869.4x - 43.532
R 2 = 0.9999
(n=9)
UV absorbance (mAU)
10000
8000
6000
4000
2000
0
0
0.5
1
1.5
2
2.5
Concentration (mM)
Figure 6. Standard curve of maltol at five concentrations, 0.25, 0.5 1.0, 1.5 and 2.0
mM, with UV detection at 270nm.
28
mA U
0.16
0.14
(a) Maltol 8μM; injection 10μl;
Signal/N oise ≈ 3
0.12
0.1
0.08
0.06
5
7.5
10
12.5
15
17.5 min
mA U
0.35
0.3
(b) Maltol 25μM; injection 10μl;
Signal/N oise ≈ 10
0.25
0.2
0.15
0.1
5
7.5
10
12.5
15
17.5 min
Figure 7. (a) limit of detection (LOD): a typical chromatogram of maltol of 8 μM;.(b)
limit of quantification (LOQ): a typical chromatogram of maltol of 25 μM.
29
22.856
13.932
8.007
mA U
(a)
300
50
15.618
10.068
100
6.213
6.846
2.591
150
8.597
200
11.955
250
0
5
0
10
15
20
min
13.974
mA U
(b)
250
200
150
100
50
0
0
5
10
15
20
min
Figure 8. Typical chromatograms of (a) dark soy sauce extract and (b) maltol standard
(1.0mM) under the following HPLC conditions, Mobile phase: A: 0.1% formic acid;
B: methanol. 10% of B for 15min, 10%-90% of B in 6min, 90% of B for another 5
min; Flow rate: 1.0 ml/min; Column: Agilent ZORBAX SB-C18 (4.6 mm i.d. ×
250mm); Injection volume: 10 μl; Detection wavelength: 270nm.
30
60
y = 423.12x
2
R = 0.9947
(n=9)
(a)
50
%inhibition
40
30
20
10
0
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
dark soy sauce con (mg)
(b)
70
y = 10.944x
2
R = 0.9991
60
y = 4.1053x
2
R = 0.9992
%inhibition
50
40
maltol std
trolox
30
20
10
0
0
5
10
15
20
con μM
Figure 9. The scavenging effects of dark soy sauce (a), maltol and trolox (b), on
ABTS•+. Results are mean±SD, n≥3.
31
4.3 Maltol excretion in urine
To test how fast maltol is metabolized, we measured the content of maltol in urine
samples after a human volunteer had consumed dark soy sauce. The total volume of
urine at different time points (0.5, 1, 2, 3, 4, 5, 6 h) were collected. No free form of
maltol was found in urine samples, but after the urine sample was digested with β-Dgalactonidase, the HPLC chromatogram shows two main peaks (RT at 5.12, 8.13 min
respectively) disappeared, while the peak of maltol (RT at 14.75 min) was observable
(Figure 10 a, b). Peak 1 was tentatively identified as maltol sulfate by comparing this
peak with that of synthesized maltol sulfate. Its RT and UV spectrum agreed well
with those of the synthesized maltol sulfate (Figure 10 c, d). Peak 2 was isolated and
purified using solid phase extraction, RP C-18 flash chromatography and identified as
maltol glucuronide based on NMR data (Appendix Figure A-2).
The presence of these two major metabolites of maltol was further confirmed with
LC-MS/MS analysis. The total ion chromatogram of MS/MS scanning of maltol
glucuronide of the urine sample shows a peak with RT at 3.12 min, whose mass
spectrum presents two typical ions with m/z 303 and 270, the protonated parent ion
maltol glucuronide and fragment ion after losing glucuronide chain, respectively
(Figure 11). Although the total ion chromatogram of MS/MS scanning of maltol
sulfate shows two peaks with RTs at 1.7 and 5.2 min, only the peak with RT at 1.7
min presents the desirable mass fragment pattern with protonated parent ion (m/z 207)
and fragment ion after losing sulfate (m/z 127) (Figure 12). The multiple reaction
monitoring (MRM) scan of maltol glucuronide (303/127) and maltol sulfate (207/127)
also confirmed the presence of these two components in the urine samples (Figure 13).
32
The total amount of maltol excreted in urine was estimated by enzyme digestion of
the urine samples. The HPLC method for determination of maltol in dark soy sauce
was modified here for this purpose. Figure 14 shows the typical HPLC
chromatograms of the urine sample with or without enzyme digestion. The urine
samples were extracted with methanol and then dried up and digested with βglucuronidase. The time course of digestion was investigated. After 12 hours of
digestion, the concentration of free form of maltol in the samples reached a plateau
(Figure 15). After a subject took 6 mg of maltol only or 30 ml of dark soy sauce
mixed with 200 gram of plain boiled rice, the amount of maltol excreted in urine at
different time points was estimated. The maltol was excreted faster after taking maltol
only than taking dark soy sauce mixed with plain boiled rice. When 6 mg maltol only
was taken, around 0.9 mg maltol as its derivatives (sulfate and glucuronide) was
found in the urine during 0.5 to 1.0 hour interval; whereas, when 30 ml of dark soy
sauce mixed with 200 gram of plain boiled rice was taken, similar amount of maltol
was excreted in the urine during 1 to 2 hour interval (Figure 16 A). The excretion of
maltol is fast. Within 3 hour the accumulated maltol excreted in urine almost reached
a plateau (Figure 16 B). In an observer-blinded, randomized, placebo controlled,
crossover clinical study [38], the average amount of total maltol excreted in 24 young
health volunteers was measured for different time points. Standardized with creatinine,
the concentrations of total maltol in urine samples collected at 1 hour and 2 hour were
significantly higher than those collected at 0 hour and 3 hour (Figure 17).
33
1200
Æpeak 1
800
600
400
200
Æpeak 2
(a)
7.656
2.749
3.164
3.517
4.001
4.507
4.815
1000
8.131
m AU
1400
5.122
D AD 1 D , S ig=258,4 R ef=600,100 (WAN G H S \MAU C 7050.D )
0
0
5
10
15
D AD 1 C , S ig=274,4 R ef=600,100 (WAN G H S \MAU C 7064.D )
14.754
4.514
m AU
1400
20
1200
1000
25
m in
25
m in
(b)
400
200
7.680
2.756
3.525
4.008
600
5.188
5.580
800
0
0
5
10
15
20
*D AD 1, 5.124 (1634 m AU ,A
m AU
*D AD 1, 5.224 (245 m AU ,Ap
m AU
1400
1200
1000
800
600
400
200
0
(c)
200
(d)
150
100
50
0
200
250
300
350 nm
200
250
300
350 nm
Figure 10. Typical HPLC chromatograms of (a) urine sample collected at 1 hour after
the subject orally taken 70 mg maltol, and (b) the same urine sample as in (a) digested
with β-glucuronidase. And the UV spectrum of (c) peak 1, whose retention time (RT)
at 5.12 min, agrees well with that of (d) synthesized maltol sulphate.
34
(A)
(
(B)
Figure 11. (A)
( The totaal ion chrom
matogram of
o MS/MS scanning of m
maltol gluccuronide
from urine sample. (B)) The mass spectrum of the peak with
w retentioon time at 3.12
3
min
in total ion chromatogrram A: Thee ion with m/z
m 303 is thhe protonateed ion of maaltol
glucuronidee; whereas the
t product ion with m/z
m 127 is prrotonated ioon of maltol.
35
(A)
(B)
(C)
Figure 12. (A).Total ion chromatogram of MS/MS scanning of maltol sulfate in urine
sample. (B). MS spectrum of the peak with retention time at 5.2 min in total ion
chromatogram A. (C). MS spectrum of the peak with retention time at 1.7 min in total
ion chromatogram A: The ion with m/z 207 is the protonated ion of maltol sulfate;
whereas the product ion with m/z 127 is protonated ion of maltol.
36
(A)
(B)
(C)
Figure 13. (A). Total ion chromatogram of multiple reaction monitoring (MRM) scan
of maltol glucuronide (303/127) and maltol sulfate (207/127) in urine sample.(B).
Extract ion chromatogram of MRM scan of maltol glucuronide (303/127). (C).
Extract ion chromatogram of MRM scan of maltol sulfate (207/127).
37
35
6.633
2.732
3.522
3.973
4.494
5.202
m AU
9.457
D AD 1 D , S ig=258,4 R ef=600,100 (WAN G H S \MAU C 7018.D )
30
21.747
11.720
7.661
9.052
10
10.054
10.687
15
5.992
6.347
7.190
7.855
8.115
2.932
3.017
3.316
3.732
3.880
4.323
20
4.813
3.183
25
5
0
0
5
10
15
D AD 1 C , S ig=274,4 R ef=600,100 (WAN G H S \MAU C 7030.D )
2.730
3.521
3.967
4.496
m AU
20
25
m in
*D AD 1, 14.787 (10.2 m AU ,
35
m AU
8
5.255
30
6
25
14.784
0
11.727
6.616
9.443
9.717
10.061
10
2
7.852 7.661
15
6.008
2.926
3.046
3.120
3.316
3.730
3.876
20
4.815
5.360
4
200
250
300
350 nm
5
0
0
5
10
15
20
25
m in
Figure 14. The typical HPLC chromatograms of (a) urine sample with subject taking
30 ml of dark soy sauce and (b) that urine sample digested with enzyme: inset is the
UV spectrum of peak with RT at 14.8 min which agrees with that of maltol.
38
0.6
0.5
Conc (mM)
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
30
35
Time (hr)
Figure 15. Time course of digestion of urine samples with 5000U β-glucuronidase at
37°C.
39
(A)
1.20
maltol amount (mg)
1.00
0.80
Maltol (6mg)
0.60
D SS (30mL)
0.40
0.20
0.00
0~0.5
0.5~1
1~2
2~3
3~4
4~6
Time (h)
(B)
Maltol amount secreted (mg)
2.50
2.00
1.50
maltol
(6mg)
1.00
0.50
0.00
0
1
2
3
4
Time (h)
5
6
7
Figure 16. (A). The maltol amount excreted in urine after one subject took 6 mg of
maltol or 30 ml of dark soy sauce mixed with plain boiled rice. The accumulated
maltol amounts excreted in urine for such two cases are also shown in (B).
40
maltol concentration (mmol/mol creatinine)
30.0
25.0
**
**
20.0
15.0
10.0
5.0
0.0
0
1
2
3
4
24
Time point (hour)
Figure 17. The average total maltol (standardized with creatinine) measured in the
different time point urine samples of 24 young healthy subjects who orally took 30 ml
of dark soy sauce mixed with 200 gram of plain boiled rice. Data are mean±SD, n=24
(**p < 0.01 vs 0 h and 3 h).
41
4.4 Fractionation and characterization of the colored components
When the MeOH-R fractions of dark soy sauce were incubated in 6 M HCl in vacuo
at 110°C for 18 h, the colored components became insoluble (the solvent became
transparent, and colored precipitate was present at the tube bottom), while in 4.2 M
NaOH they were stable. Removal of colored components dramatically decreased the
antioxidant activity of the hydrolysate (Figure 18), suggesting that the colored
components could greatly contribute to the TAA of dark soy sauce.
To further investigate the colored components, we used gel filtration chromatography
to fractionate the dark soy sauce. The MeOH-R and EtOAc-R fractions were
separately dialyzed against distilled water and further fractionated with gel filtration
chromatography as described in Experimental section. Figure 19A shows the gel
filtration chromatograms of these two fractions. We obtained a high-molecular-mass
fraction (CP2) from the MeOH-R, and a lower-molecular-mass fraction (CP1) from
the EtOAc-R. Indeed, the absorbance at 470 nm and ABTS•+ scavenging activity of
CP1 and CP2 were highly correlated, r = 0.9945 for CP1 and 0.9999 for CP2 (Figure
19B). However, the ratios of scavenging activity to absorbance for the two fractions
were not equal, 1629.5 for CP1 and 1125.9 for CP2, suggesting that different
compositions are present.
The 1H-NMR and 13C-NMR spectra of CP1 (Figure 20a and 20c) are very complex.
Despite this, it was possible to observe the resonance signals related to carbonyl
groups (δ 166.8). The spectra also indicated the presence of carbohydrates: the proton
signals at δ 5.36-5.33 and carbon signals at δ 99.9-98.5 were due to the anomeric
hydrogens and carbons, and a complex of multiplet between δ 3.93 and δ 3.37 and the
42
carbon signals at the range of δ 77.6 to δ 60.5 corresponded to other sugar backbone
protons and carbons, respectively.
Inhibition
2.5
50
45
40
35
30
25
20
15
10
5
0
Abs at 470 nm
2
1.5
1
***
0.5
0
Sa
mp
le+
Sa
6M
HC
l
mp
le +
Sa
4 .2
M
mp
Na
le +
%Inhibition
A bs
H2
O
OH
Figure 18. The absorbance and ABTS•+ scavenging activity of MeOH-R fraction
under acidic and basic conditions. The MeOH-R samples were incubated in 6 M HCl
or 4.2 M NaOH in vacuo at 110°C for 18 hours. The colored components became
insoluble in acidic condition, while in alkaline condition they were stable. Removal of
insoluble colored components dramatically decreased the antioxidant activity of the
acidic hydrolysate, indicating that the colored components could greatly contribute to
the total antioxidant activity (TAA) of dark soy sauce. Values are mean ± SD, n=3.
***Comparision between the ABTS•+ scavenging activity of sample + 6 M HCl and
that of sample + H2O (*** p < 0.001).
43
(A)
50 000 1000
Fractionation Range (M
Mr) (Dextrans)
(B
B)
%Inhibition
40
35
C P1
30
C P2
y = 1629.5x
r = 0.9945
p < 0.001
25
20
25.9x
y = 112
r = 0.9
9999
p < 0.001
15
10
5
0
0
0
0.005
0.01
0.015
0.02
0.025
Abs orban ce at 470nm
Figure 19. (A)
( Overlaiin Sephadexx G-75 gel filtration
f
chromatogram
ms of EtOAc-R and
MeOH-R of
o dark soy sauce.
s
Fracttions 26 to 34
3 of EtOA
Ac-R were coombined as
Colored Prooduct 1 (CP
P1), and fracctions 3 to 9 of MeOH--R were com
mbined as Colored
C
Product 2 (CP2). The fragmentati
f
on range off Sephadex G75 gel filttration
chromatogrraphy is 10000 ~ 50 0000 Dalton. (B
B) The correelation of AB
BTS•+ scaveenging
activity andd absorbancce of CP1 annd CP2 at 470 nm. Valuues are meaan ± SD, n=
=3.
44
Figure 20. (a) 1H-NM
MR spectrum
m of CP1, (b
b) 1H-NMR spectrum of CP2, and (c) 13CNMR specttrum of CP11.
45
1
H-NMR spectrum of CP2 (Figure 20b) showed similar signals to those of CP1
(Figure 20a).
However, the ratios of integral of the signal peaks at δ 8.25 to that at δ 5.36-5.33 are
different for these two fractions; around 1:2 for CP1 and 1:1 for CP2. This difference
suggested that their compositions are somewhat different.
The ESI-TOF mass spectrum of CP1 (Figure 21) showed that most of the observed
ions were doubly-charged (as shown in the inset of Figure 21, the signal was observed
at 1001.3 with the peaks of the isotopic pattern at 0.5 Dalton distance), and the m/z
range was from 200 to 2200, centered at 1000, indicating that the average molecular
weight was about 2000. As shown in Table 3, the observed ions can be assigned to
several series. Interestingly, within each series, m/zs of the doubly-charged or the
singly-charged ions consistently increase by 81 or 162, respectively. This moiety with
a mass of 162 could be a hexose losing one molecule of water (180-18). These results
were consistent with our NMR data, suggesting that CP1 is a sugar-containing
fraction.
Using TOF-MS, we also investigated the effect of adding the ABTS•+ free radical on
the structure of the colored components. We compared the mass spectra of the CP1
solution spiked with ABTS•+ stock solution with those without spiking. Interestingly,
we found the intensities of doubly-charged ions, especially those in Series 2 (Table
III), were markedly decreased, while the singly-charged ions have no or little change.
These findings suggest that the sugars contribute little to the antioxidant activity;
while possibly chromophore units are present, contributing to both the color and the
antioxidant activity.
46
1001.3344
100
1001.3344
839.2879
1001.8270
920.2736
100
1001.8270
1002.3366
1082.3546
1175.4012
758.2728
%
1002.7953
1337.4618
0
596.1904
1176.3951
344.1183 425.1577
1662.7813
400
600
m
1003
1661.7953
1338.5027 1500.6201
263.0956
0
200
1002
1499.5386
677.2410
%
1001
1003.3053
800
1000
1200
1400
1600
1824.1770 1986.4733
1800
2000
2200
m/z
Figure 21. TOF-MS spectrum of CP1. The inset shows a typical doubly charged ion.
The peaks observed at around 1001.3 show the isotopic pattern at 0.5 Dalton distance.
47
Table III. The observed ions in TOF-MS spectrum of CP1. Three series were
observed. Within each series, m/zs of the doubly-charged or the singly-charged ions
consistently increase by 81 or 162, respectively.
Series 2†
Series 1
ions
Charge
M.W.
states
(Da)
263.0956
2
528.2
344.1483
2
425.1577
ions
Series 3
Charge
M.W.
states
(Da)
353.1367
2
708.3
690.3
434.1462
2
2
852.3
515.1589
506.1745
2
1014.3
587.2086
2
668.2075
ions
Charge
M.W
states
(Da)
1191.4094
1
1192.4
870.3
1353.4515
1
1354.5
2
1032.3
1515.6155
1
1516.6
596.1904
2
1194.4
1677.6451
1
1678.6
1176.4 *
677.2410
2
1356.5
1839.7515
1
1840.8
2
1338.4*
758.2728
2
1518.6
749.2477
2
1500.5*
839.2879
2
1680.6
1175.4012
1
1176.4*
920.2736
2
1842.5
1337.4618
1
1338.5*
1001.3334
2
2004.7
1499.5386
1
1500.5*
1082.3546
2
2166.7
1661.6138
1
1662.6
1163.4160
2
2328.8
1823.7094
1
1824.7
1244.4283
2
2490.9
1985.7924
1
1986.8
1325.4880
2
2653.0
1406.5306
2
2815.1
1487.5840
2
2977.2
*
Both singly-charged and doubly-charged ions were observed.
Intensities of peaks which were markedly decreased after reaction of CP1 with
ABTS•+.
†
48
4.5 Protection against HOCl-induced DNA damage
Hypochlorous acid (HOCl), a reactive oxidative agent, can be generated by
myeloperoidase that is released by activated phagocytes at sites of inflammation [40].
HOCl can oxidize various biological molecules, such as lipids, amino acids, proteins,
nucleotides and DNA repair enzymes. Chronic inflammatory diseases in GI tracts, e.g.
ulcerative colitis and Crohn’s disease, are thought to cause tissue damage and be
associated with the increased risk of tumorigenesis. Thus we are to tests whether the
components of dark soy sauce can protect against HOCl induced DNA damage.
Treatment of DNA with HOCl led to various detectable base modification products.
Significant increases were observed for 5-Cl uracil, 5-OH uracil, and 5-OH cytosine,
but not for 5-(OH, Me) hydantoin, FAPy adenine, FAPy guanine and 8-OH guanine
(Table IV). The formation of HOCl-induced base modification products was inhibited
dose-dependently by pretreatment of DNA with maltol, CP1 or CP2 (Table IV).
49
Table IV. Inhibition of HOCl-induced DNA damage by maltol, the colored product 1 (CP1) or the colored product 2 (CP2) #.
Oxidized base
Control
DNA+HOCl
DNA+HOCl+maltol
DNA+HOCl+CP1
DNA+HOCl+CP2
50 μM
250 μM
500 μM
25 μg/ml
125 μg/ml
250 μg/ml
25 μg/ml
125 μg/ml
250 μg/ml
5-Cl uracil
0.002±0.002
0.986±0.308
0.111±0.015
0.068±0.005
0.048±0.008
1.317±0.239
0.770±0.062
0.528±0.013
1.321±0.191
0.632±0.040
0.521±0.038
5-(OH,Me) hydantoin
0.079±0.002
0.096±0.013
0.084±0.000
0.079±0.002
0.081±0.011
0.097±0.002
0.092±0.004
0.088±0.005
0.098±0.004
0.093±0.007
0.094±0.006
5-OH uracil
0.004±0.001
0.912±0.325
0.144±0.005
0.074±0.007
0.048±0.006
1.190±0.072
0.673±0.070
0.456±0.036
1.021±0.083
0.547±0.032
0.448±0.014
5-(OH, Me) uracil
0.027±0.015
0.095±0.063
0.085±0.039
0.065±0.029
0.052±0.029
0.109±0.034
0.097±0.043
0.095±0.042
0.120±0.032
0.116±.022
0.107±0.028
5-OH cytosine
0.019±0.009
1.517±0.552
0.201±0.021
0.110±0.020
0.093±0.025
1.224±0.127
0.822±0.055
0.558±0.078
1.217±0.094
0.673±0.030
0.533±0.021
FAPy adenine
0.037±0.007
0.046±0.011
0.060±0.006
0.054±0.006
0.043±0.007
0.054±0.008
0.052±0.006
0.056±0.005
0.059±0.004
0.052±0.002
0.049±0.007
8-OH adenine
0.075±0.012
0.466±0.180
0.123±0.042
0.104±0.019
0.166±0.138
0.475±0.056
0.337±0.051
0.269±0.060
0.457±0.040
0.280±0.018
0.250±0.029
2-OH adenine
0.057±0.012
0.224±0.054
0.146±0.091
0.104±0.046
0.123±0.074
0.286±0.126
0.243±0.135
0.215±0.127
0.304±0.132
0.185±0.054
0.159±0.034
FAPy guanine
0.107±0.025
0.101±0.032
0.175±0.023
0.267±0.217
0.115±0.014
0.176±0.073
0.302±0.221
0.190±0.078
0.135±0.004
0.125±0.009
0.122±0.016
8-OH guanine
0.415±0.067
0.239±0.068
0.394±0.126
0.349±0.054
0.307±0.188
0.389±0.134
0.381±0.128
0.386±0.119
0.290±0.034
0.276±0.053
0.336±0.046
#
Data are mean±SD, n=3.
50
4.6 Cytotoxicity on HT-29 cells
To explore the potent effects of dark soy sauce and its components on GI tract, using
MTT assay, the cytotoxicity of dark soy sauce was tested on HT-29 cells, a human
colon adenocarcinoma cell line. Figure 22A shows that dark soy sauce did not result
in significant cytoxicity at low concentrations, i.e. 1, 2.5, 5 μl/ml, of dark soy sauce,
but at higher concentrations, such as 10 μl/ml of dark soy sauce for 72 hr, only around
30% cells survived. When the concentration of dark soy sauce increased to 20 μl/ml,
the viability was reduced to around 15% after 48 hr exposure. Dark soy sauce has
high concentration of salts, e.g. sodium chloride. In case the salts influenced the
cytotoxicity results, the cell viability was also tested using non-dialysable part of dark
soy sauce. Their cytotoxic effects were dose- and time-dependent: the higher the dose
and the longer exposure used, the lower the cell viability observed (Figure 22B).
In cell culture, it is commonly observed that the cytotoxic effects of adding
compounds are possibly caused by the artificially formed H2O2 in the medium [47].
When 5 μl/ml of dark soy sauce was spiked into the culture medium, around 50 μM
H2O2 was detected after 60 min, while 100 μM H2O2 was produced when the
concentration of spiked dark soy sauce increased to 20 μl/ml. (Figure 23A). But
when exposed to 100 μM H2O2 for 72 hr, around more than 60% of HT-29 cells still
survived (Figure 23B). Furthermore, using catalase to neutralize the effects of H2O2,
the cell viability did not change when exposed to 20 μl/ml of dark soy sauce with or
without catalase (Figure 23C), suggesting that the cytotoxic effects observed was not
caused by H2O2 .
51
.
(A)
140
% Viable Cells
120
100
24hr
80
48hr
60
72hr
40
20
0
0
1
2.5
5
10
20
DSS Concentration (μl/ml)
(B)
120
Cell viability (%)
100
80
24hr
60
48hr
40
72hr
20
0
0
1
2.5
5
10
20
DSS Nondialysable (mg/ml)
Figure 22. Decrease of cell viability of HT 29 cells 24, 48 and 72 hours after
incubation with various concentrations of dark soy sauce (DSS) (A), and DSS
Nondialysable fraction (B). Results are mean±SD, n=3.
52
D SS
D SS+Catalase
120
% Viable Cells
100
80
60
40
20
0
0
5
20
DSS concentration ( ul/ml)
Figure 23. Cell viability tested on dark soy sauce only or plus catalase (1000 units/ml).
53
4.7 Inhibition of COX-2 protein expression in LPS-induced HT-29 cells
HT-29 cells expressed a low basal level of cyclooxygenase-2 (COX-2) protein [48],
which can be obviously up-regulated by lipopolysacchride (LPS) induction at 100
ng/ml. The time course in Figure 24 shows that after 2 hours, the levels of COX-2
protein expression had not much difference among the control, treatment with LPS
only, or treatment with both dark soy sauce and LPS. After 24 hours induction, the
up-regulation of COX-2 expression in HT-29 cells was observable, but such effect
was inhibited by treatment with 5 μl/ml dark soy sauce. The inhibitory effects of dark
soy sauce on the expression of COX-2 protein in LPS-induced HT-29 cells were
further observed at the concentrations of 1 μl/ml and 5 μl/ml (Figure 25), at which
concentrations, the dark soy sauce has no significant cytotoxic effects (Figure 22). But
at the concentrations of 100 μM and 500 μM, maltol did not exhibit remarkable
inhibitory effects (Figure 26).
Figure 24. Time course of COX-2 expression and its inhibition by dark soy sauce in
lipopolysaccharide (LPS)-induced HT-29 cells. Cells were pretreated with dark soy
sauce (5 μl/ml) for 30 min and then induced with LPS (100ng/ml) for various time
indicated. Protein levels were estimated by Western Blot analysis as described in
“Materials and Methods”. Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells
treated with LPS (100 ng/ml) only); and lane 3, HT-29 cells simultaneously treated
with dark soy sauce (5 μl/ml) and LPS (100 ng/ml).
54
Figure 25. Inhibitory effect of dark soy on COX-2 expression in LPS-induced HT-29
cells . Lane 1, untreated HT-29 cells; Lane 2, HT-29 cells treated with 1 μl/ml dark
soy sauce(DSS); Lane 3, with 5 μl/ml DSS; Lane 4, with LPS (100 ng/ml); Lane 5,
with DSS (1 μl/ml) and LPS (100 ng/ml); Lane 6, with DSS (5 μl/ml) and LPS (100
ng/ml). The western blot is from a single experiment, but is representative of 3
independent experiments.
Figure 26. The effect of maltol on COX-2 expression in LPS-induced HT-29
cells.Lane 1 is untreated HT-29 cells; lane 2, HT-29 cells treated with maltol (100
μM); lane 3, maltol (500 μM); lane 4; treated with LPS (100 ng/ml); lane 5, maltol
(100 μM) and LPS (100 ng/ml); lane 6, maltol (500 μM) and LPS (100 ng/ml).
55
4.8 Discussion
Our data show that the ethyl acetate extract of dark soy sauce has the strongest
antioxidant activity based on the ABTS assay which detects a wide range of
antioxidants [35, 36]. Fractionation of that extract produced several components with
antioxidant activity, but the most active was indentified as maltol. We also developed
an HPLC method to determine the maltol level in dark soy sauce. Although maltol has
been identified in many soy sauce products [49-51], to the authors’ knowledge, this is
the first study to determine its concentration in soy sauce and its contribution to the
TAA of dark soy sauce. Maltol can be formed from reducing sugars during food
processing and has been identified in a wide variety of other heated materials such as
bread crusts, coffee, cocoa beans, cereals, dried whey and chicory [51]. Therefore, the
amount of the daily maltol intake can be considerable. Maltol has even been
suggested to be capable of exerting neuroprotective activity by inhibiting oxidative
damage [52-54], and its iron chelate has been used to treat anaemia in humans [55].
At present the pharmacokinetics of maltol after oral dosing have only been
investigated in dogs and indicate rapid and extensive absorbtion, followed by
conjugation and rapid excretion [56]. However, the oral bioavailability of maltol in
humans has not yet been examined. In our study on healthy human subjects, maltol in
conjugated form was at high concentrations in urines 2 hours after oral administration,
consistent with rapid aborption, metabolism and excretion of the products in urine.
The flavonoids, e.g. daidzein and genistein, have been identified in some soy sauce
products and postulated to have antioxidant activities [57], but in our study, we found
the levels in the dark soy sauce to be undetectable. This is not perhaps surprising,
since soy sauce is generally regarded as a poor source of isoflavones [58].
56
However, the major antioxidant activity of dark soy sauce appears to reside in high
molecular weight colored components, some soluble in methanol and others not,
containing carbohydrate residues. These are most probably melanoidins, brown
polymers formed by Maillard reactions between reducing sugars and compounds
possessing a free amino group, such as free amino acids and the amino groups of
peptides [59].
Although soy sauce is a traditional cooking ingredient throughout Asia, the actual
process and the composition of the starting ingredients for making the soy sauce is
known to differ between countries. It is highly likely that differences in the raw
materials, fermentation time and heating processes used during the manufacture of
soy sauce may affect the composition and antioxidant activity of the final products.
Indeed, variation in the antioxidant capacity of different soy sauce products has
previously been demonstrated [36]. Conventional soy sauce making involves a solidstate aerobic fermentation process by Aspergillus species on a soybean and wheat
mixture. Aspergillus produces extracellular enzymes, in particular proteases and
amylases, which hydrolyze the proteins and polysaccharides of soybean and wheat.
Subsequently, the sugars and amino acids produced are further digested during a brine
fermentation process and, depending on the extent of fermentation process that takes
place, the amount of Maillard reaction products can vary. Full details of commercial
soy sauce manufacture tend to be carefully guarded by Asian food companies and the
exact details of manufacture for the brand examined in this study are not known.
The antioxidant characteristics of Maillard related products from fermented soy sauce
have been studied [60] and melanoidins have previously been isolated from soy sauce
[61]. Melanoidins have been characterized in many foods, such as beer [62], bread [63]
and roasted coffee [64,65]. Many studies have attempted to elucidate the structure
57
characteristics of melanoidins [59, 66], using a variety of starting materials. Ando [32]
investigated different soy sauce products, and found that the color of soy sauce was
not proportional to its antioxidant capacity. In our study, even for one soy sauce
product, the ratios of free radical scavenging ability to color density of two different
fractions, CP1 and CP2, were different. This could be due to the variety of chemical
structures of the colored components. In this study, NMR and MS were used to
eludicate the structural characteristics of melanoidins. NMR and MS data of CP1
indicate the presence of carbohydrate residues, which agrees with the finding that
sugar moieties could be part of the melanoidin backbone [66]. The carbonyl
resonance and nonsaturated proton signals of CP1 suggest that amino acids may be
involved in the formation of the chromophore unit. Based on our ESI-TOF-MS data,
the molecular weights of CP1 are in the range of 400 – 4400, which is comparable to
soy sauce pigments fractionated by Motai et al. [67].
Maillard reaction products add the flavor and brown color to soy sauce [68]. One of
these antioxidant flavor components, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)furanone (HEMF) was reported to inhibit benzo[α]pyrene-induced forestomach
neoplasia in mice and reduce the hydrogen peroxide concentration in human
polymorphonuclear leucocytes [34]. The colored components of soy sauce have also
been linked to the antioxidant capacity of soy sauce [32]. For example, soy sauce
melanoidin strongly inhibited NO-induced DNA damage in a dose dependent manner,
as determined by the comet assay [69]. In vivo, the formation of hypochlorous acid
(HOCl) by activated netrophils in defense against microorganisms may cause DNA
damage [70]. Our studies also suggest that, in vitro, maltol and the colored products
of dark soy sauce can reduce HOCl-induced DNA base modification. In an in vitro
study, melanoidin has been demonstrated to affect the growth of human colonic
58
bacteria [71]. Melanoidins extracted from soy sauce have been suggested to inhibit
colon cancer cell growth [72]. Our data also indicate that dark soy sauce exibits some
inhibitory effect on growth of the HT29 colon cancer cell line. As has been suggested
for other nutritional antioxidants [73, 74], melanoidins may play a role in the
antioxidant defence of the gastrointestinal tract (GIT), since they are unlikely to be
absorbed as such. Maltol may well be absorbed, however, and could conceivably
exert systemic antioxidant effects.
Postprandial oxidative stress is thougth to be associated with increased risk for
various metabolic syndromes, such as atherosclerosis, due to an increased
susceptibility of the organism toward oxidative damage after consumption of a meal
rich in lipids and/or carbohydrates [75]. Epidemiological studies suggest that food and
diets rich in antioxidants could be benefitful to decrease the postprandial oxidative
stress. However, clinical studies on the efficacy of antioxidants in the prevention of
atherotic diseases have reported conflicting results. It has reported that the timing of
administration of antioxidants has various effects on postprandial oxidative serum
markers [76]. The failure of observation of antioxidant effects in some clinical studies
could be due to the missed timing. Our data show that maltol can be found in urine at
high concentration two hours after orally taken, indicating it may be rapidly
absorbed. . In our in vivo study, dark soy sauce has a rapid (3-4 h) antioxidant effect
[38]. Thus when dark soy sauce is used as seasoning for high fat diets, maltol and
other small molecule components with similar structures could timely reduce the
postprandial oxidative stress.
Recent findings suggest that tumor microenvironment can be crucial for
carcinogenesis [77]. Many molecular targets in the microenvironment might be
59
selected for chemoprevention, such as cyclooxygenase-2 (COX-2). COX-2
overexpression has been documented in more than 80% of human colon carcinomas
[78]. Population-based studies suggested that long-term intake of non-steroidal antiinfammatory drugs, compounds that inhibit the enzymatic activity of cyclooxygenase
(COX), reduces the relative risk for developing colorectal cancer [79]. COX-2
expression may protect cancer cells from apoptosis induced by a variety of stimuli
and could enhance cell tumorigenic potential. So COX-2 inhibitors might be useful
for colorectal cancer prevention and treatment. Our data suggest that dark soy sauce
can decrease COX-2 protein levels in LPS-induced HT29 cells after 24 hour
incubation.
60
Chapter 5 Conclusion
Our data show that 3-hydroxy-2-methyl-4H-pyran-4-one (maltol) is one major active
compounds in an ethyl acetate extract of dark soy sauce and present at millimolar
concentrations in dark soy sauce. In human urine, maltol is excreted quickly and
mainly in conjugated form.
Our data of nuclear magnetic resonance (NMR) and electrospray-ionization time-offlight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydratecontaining pigments such as melanoidins are the major contributors to the high
antioxidant capacity of DSS.
In vitro, maltol and the colored products can protect against hypochlorous acid
(HOCl)-mediated DNA damage dose-dependently. Furthermore, dark soy sauce
potentially inhibits the growth of colon cancer HT 29 cells at high concentrations,
while decreases the up-regulation of cyclooxygenase-2 (COX-2) expression in LPS
(lipopolysaccharide) -induced HT 29 cells at low concentrations.
Although maltol can scavenge ABTS free radicals and protect HOCl-induced DNA
damage in vitro, it is still unknown whether it exerts such antioxidant effects in vivo.
Since maltol might be readily well absorbed, it is worthwhile to test whether it can
reduce the postprandial oxidative stress in model animals or human subjects.
The potent chemopreventive efficacy of the colored products of dark soy sauce on
colon cancer can be further tested using an inducible inflammation-driven colorectal
carcinogenesis rat/mouse model.
Part of this work has been published [80].
61
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69
Appendices
(A)
(B)
Figure A-1. (A) 1H-NMR and (B) 13C-NMR spectra of compound 1.
70
(A)
(B)
Figure A‐2. (A) 1H‐NMR and (B) 13C‐NMR spectra of maltol glucuronide.
71
(A)
%T
100
90
4000
3
3500
3000
2000
2
1750
1500
Wavenumber (1/cm)
509.23
708.87
691.51
846.79
918.16
1256.68
1250
1198.81
1459.21
1397.49
2500
1617.38
30
40
1655.00
50
3257.91
60
1560.48
70
3068.88
1073.43
1021.35
80
1000
0
750
500
(B)
(C)
ompound 1,, (B) CP1 annd (C) CP2.
Figure A-3. Fourier infrared specttra of (A) co
72
Table A-1. Summary of LC-ITMS/MS parent ion and product ions and their possible
identities of the 10 fractions from dark soy sauce.
Fr No.
Parent ion
and product ions
Possible identities
1
126, 108
182, 164, 136
3-Methoxy-2[1h]pyridone;
2-Amino-3-(4-hydroxyphenyl)-propanoic
acid, tyrosine
2
215, 197, 179, 137
185, 167, 149
2-methyl-Dodecanoic acid;
4-Hydroxy-3,5-dimethoxybenzyl alcohol
3
155, 137 108
4
125, 107 88
2-acetyl-5-methylfuran
5
141, 127 109
199, 181 163 121
3-Hydroxy-4-methoxymandelic acid
6
127, 97 79
7
125‚ 107
2-acetyl-5-methylfuran
8
167, 149
125, 107
2-acetyl-5-methylfuran
9
127
maltol
10
155, 123
141, 123 95 81
2-pentylthiophene
3-methoxypyrocatechol
73
Intens.
x10 8
2
3
2
1 3
4
5
1
0
0
1
2
Intens. 2.
x10 8
3
4
5
6
7
8
T ime [min]
+MS , 2.0-2.0min #136-#138
126.3
2
1
108.4
0
x10 6 2.
6
182.3
+MS 2(126.4), 2.0-2.1min #137-#139
108.4
4
2
0
50
100
150
Intens. 3.
x10 7
200
250
300
350
400
450
m/z
+MS , 2.1-2.2min #144-#146
126.3
182.3
3
2
108.4
1
96.4
140.3
170.3
212.3
0
x10 6 3.
+MS 2(108.4), 2.1-2.2min #145-#147
126.3
1.0
0.5
0.0
50
100
Intens. 4.
x10 7
150
200
250
300
350
400
450
m/z
+MS , 2.1-2.3min #140-#154
126.3
6
182.3
4
108.4
2
96.4
0
x10 4 4.
3
140.3
+MS 2(182.4), 2.1-2.3min #141-#155
136.3
2
164.3
1
183.2
391.4
0
50
100
150
200
250
300
350
400
450
m/z
Figure A-4. Total ion chromatogram (TIC), MS and MS2 [126], MS and MS2 [108] ,
MS and MS2 [182] of fraction 1.
74
Intens.
x10 9
1.25
4
1.00
5
0.75
0.50
0.25
1
0.00
0
1
2
Intens. 4.
x10 8
3
2 3
6
7
8
9
101112
4
5
6
T ime [min]
+MS , 3.7-3.9min #261-#271
185.3 215.3
3
2
1
0
x10 7 4.
3
+MS 2(215.3), 3.8-3.9min #262-#272
197.3
2
179.3
137.3
1
0
100
150
Intens. 8.
x10 8
200
250
300
350
400
450
m/z
+MS , 3.8-4.3min #265-#299
185.3 215.3
1
123.3
0
x10 7 8.
+MS 2(185.4), 3.8-4.3min #266-#300
167.3
1.0
0.5
0.0
149.3
107.4
100
150
Intens. 5.
x10 8
200
250
300
350
400
450
m/z
+MS , 3.9-3.9min #273-#275
185.3
4
215.3
2
123.3
0
x10 5 5.
1.0
+MS 2(123.4), 3.9-4.0min #274-#276
391.3
95.5
0.5
139.3
0.0
100
150
200
250
300
350
400
450
m/z
Figure A-5. Total ion chromatogram (TIC), MS and MS2 [215], MS and MS2 [185] ,
MS and MS2 [123] of fraction 2.
75
Intens.
x10 8
6
3
4
4
2
1
0
0
2
Intens. 4.
x10 8
3
4
56 8
7 9
2
6
8
10 T ime [min]
+MS , 5.9-6.3min #356-#384
155.3
2
1
137.3
0
x10 7 4.
+MS 2(155.4), 5.9-6.3min #357-#385
137.3
2
1
0
100
Intens. 3.
x10 8
150
200
250
300
350
400
450
m/z
+MS , 5.8-6.2min #352-#380
155.3
3
2
137.3
1
0
x10 4 3.
6
+MS 2(137.2), 5.8-6.2min #353-#381
391.3
4
2
108.4
0
100
150
200
250
300
350
400
450
m/z
Figure A-6. Total ion chromatogram (TIC), MS and MS2 [155], MS and MS2 [137]
of fraction 3.
76
Intens.
x10 8
3
1
2
2
1
3
4
56 7
0
0
2
Intens. 2.
x10 8
4
6
8
10
T ime [min]
+MS , 7.1-7.6min #371-#405
125.3
1.0
0.5
107.4
0.0
x10 6 2.
+MS 2(125.5), 7.1-7.6min #372-#406
107.4
1.0
0.5
391.3
0.0
100
Intens. 1.
x10 8
150
200
250
300
350
400
450
m/z
+MS , 7.2-7.5min #375-#395
125.3
2
1
107.4
0
x10 4 1.
+MS 2(107.4), 7.2-7.5min #376-#396
391.2
2
1
0
88.5
100
150
200
250
300
350
400
450
m/z
Figure A-7. Total ion chromatogram (TIC), MS and MS2 [125], MS and MS2 [107]
of fraction 4.
77
I n te n s.
x1 0 8
4
5
3
1 .5
6
1 .0
7
0 .5
1
2
0 .0
0
2
I n te n s. 3 .
x1 0 7
4
6
8
10
T i m e [m i n ]
+ M S , 7 .1 -7 .1 m i n # 3 8 9 -# 3 9 1
1 2 7 .3
1 0 9 .4
4
2
0
x1 0 5
3.
+ M S 2 (1 0 9 .4 ), 7 .1 -7 .1 m i n # 3 9 0 -# 3 9 2
3 8 7 .3
1 .0
8 1 .5
0 .5
2 9 5 .4
0 .0
100
I n te n s. 4 .
x1 0 7
6
150
200
300
4 7 0 .3
350
400
450
m /z
+ M S , 7 .1 -7 .2 m i n # 3 9 3 -# 3 9 5
1 2 7 .3
1 0 9 .4
1 4 1 .3
4
2
0
x1 0 6
250
1 9 9 .3
4.
+ M S 2 (1 4 1 .4 ), 7 .1 -7 .2 m i n # 3 9 4 -# 3 9 6
1 0 9 .3
2
1
3 9 1 .4
0
100
150
200
I n te n s. 5 .
x1 0 7
300
350
400
450
m /z
+ M S , 7 .0 -7 .3 m i n # 3 8 5 -# 4 0 7
1 9 9 .3
1 2 7 .3
1 0 9 .3
2
250
1
0
x1 0 6
1 .5
5.
+ M S 2 (1 2 7 .4 ), 7 .0 -7 .3 m i n # 3 8 6 -# 4 0 8
1 0 9 .3
1 .0
0 .5
3 9 1 .3
0 .0
100
150
200
In te n s. 7 .
x1 0 7
2
1
250
300
350
400
450
m /z
+ M S , 7 .2 -7 .6 m i n # 3 9 7 -# 4 2 3
1 9 9 .3
1 2 7 .3
1 0 9 .3
0
x1 0 6 7 .
+ M S 2 (1 9 9 .4 ), 7 .2 -7 .6 m i n # 3 9 8 -# 4 2 4
1 8 1 .3
1 .0
1 6 3 .3
0 .5
1 2 1 .4
0 .0
100
150
200
250
300
350
400
450
m /z
Figure A-8. Total ion chromatogram (TIC), MS and MS2 [109], MS and MS2 [141],
MS and MS [127], MS and MS2 [199] of fraction 5.
78
Intens.
x10 7
2
5
3
4
1
3
2
1
0
6.0
6.5
Intens. 2.
x10 7
7.0
7.5
8.0
8.5
9.0
9.5
10.0
T ime [min]
+MS , 7.9-8.0min #432-#434
127.3
1.0
0.5
126.3
0.0
x10 5 2.
1.00
0.75
157.3
177.3 205.3
271.3
+MS 2(126.6), 8.0-8.0min #433-#435
391.2
97.4
0.50
0.25
0.00
127.2
79.5
100
Intens. 3.
x10 7
187.4
150
200
250
300
350
400
450
m/z
+MS , 8.0-8.1min #436-#442
125.3
1.0
157.3
0.5
0.0
x10 4 3.
6
126.3
205.3
271.3
+MS 2(126.0), 8.0-8.1min #437-#443
97.4
4
403.1
2
79.5
0
100
150
200
250
300
350
400
450
m/z
Figure A-9. Total ion chromatogram (TIC), MS and MS2 [127], MS and MS2 [126]
of fraction 6.
79
Intens.
x10 8
2
2.5
1
2.0
1.5
1.0
34
0.5
0.0
0
2
Intens. 2.
x10 8
4
6
8
10
12
14
T ime [min]
+MS , 9.1-9.6min #420-#454
125.3
1.5
1.0
0.5
0.0
x10 5 2.
4
+MS 2(125.2), 9.2-9.6min #421-#455
125.3
391.3
2
107.4
187.3
0
100
150
200
250
300
350
400
450
m/z
Figure A-10. Total ion chromatogram (TIC), MS and MS2 [125] of fraction 7.
80
Intens.
x10 7
2 3
5
4
3
1
2
1
0
7
Intens. 2.
x10 7
8
9
10
11
12
T ime [min]
+MS , 9.2-9.4min #437-#447
125.3
1.5
167.3
1.0
149.3
0.5
197.3
0.0
x10 6 2.
+MS 2(167.7), 9.2-9.4min #438-#448
149.3
2
1
0
100
Intens. 3.
x10 7
150
200
250
300
350
400
450
m/z
+MS , 9.3-9.7min #441-#467
125.3
2
1
167.3
0
x10 4 3.
6
+MS 2(125.2), 9.3-9.7min #442-#468
125.4
391.3
4
2
107.4
187.3
0
100
150
200
250
300
350
400
450
m/z
Figure A-11. Total ion chromatogram (TIC), MS and MS2 [167], MS and MS2 [125]
of fraction 8.
81
Intens.
x10 8
1
3
2
1
2
0
0.0
2.5
5.0
7.5
10.0
12.5
3 4 56
15.0
17.5
Intens. 1.
x10 8
20.0
22.5 T ime [min]
+MS , 12.1-12.6min #417-#453
3
127.3
2
1
0
x10 6 1.
+MS 2(127.3), 12.1-12.6min #418-#454
391.3
1.0
0.5
0.0
50
100
150
200
250
300
350
400
450
m/z
Figure A-12. Total ion chromatogram (TIC), MS and MS2 [127] of fraction 9.
82
Intens.
x10 7
5
1
2
3
4
3
2
1
0
0
2
4
Intens. 1.
x10 7
6
8
10
12
14
16
18
T ime [min]
+MS , 14.2min #449
141.3
123.3
2
155.3
1
0
x10 6 1.
1.5
+MS 2(155.4), 14.2min #450
123.3
1.0
0.5
0.0
50
100
Intens. 2.
x10 7
2
150
200
250
300
350
400
450
m/z
+MS , 14.1-14.3min #443-#457
141.3
123.3
1
0
x10 4 2.
+MS 2(123.4), 14.1-14.3min #444-#458
95.4
3
81.4
2
401.3
1
0
50
100
Intens. 3.
x10 7
150
200
250
300
350
400
450
m/z
+MS , 14.0-14.4min #439-#465
141.3
1.5
123.3
1.0
0.5
0.0
x10 6 3.
+MS 2(141.4), 14.0-14.4min #440-#466
123.3
1.5
1.0
0.5
391.2
0.0
50
100
150
200
250
300
350
400
450
m/z
Figure A-13. Total ion chromatogram (TIC), MS and MS2 [155], MS and MS2 [123],
MS and MS2 [141] of fraction 10.
83
(A)
(B)
Figure A-14. UV spectra of (A) CP1 and (B) CP2.
84
[...]... countries by the Chinese immigrants In Singapore, it is said that the small-scale manufacturing of soy sauce started by a small number of Xin-hui Cantonese, a sub-dialect group from Guangdong Province of South China, just a couple of years after the first arrival of Chinese immigrants in the early nineteenth century [12] After being successful, many of them shifted their business to other more profitable fields... components of soy sauce improve the taste of many types of foods, but its coloring ingredients can enhance the appearance of the dipped food or the mixed soup Moreover, recent studies indicate that some ingredients in soy sauce are potentially beneficial to human health, showing effects such as anticarcinogenesis, antihypertension and antihyperlipidemia [2-4] 1.1 A brief history of soy sauce The history of soy. .. sauce [6] Buddist monks are thought to have played an important role in spreading soy sauce from China to Japan Soy sauce was first introduced into Japan by a Buddist monk, Jian Zhen, in Tang Dynasty (618-907) [7] But some consider that Japanese soy sauce originated from that brought back by a Japanese Buddist monk, Kakushin, from China in Song Dynasty (960-1279) [8-10] It is in Japan that the making... hydrolyzed vegetable proteins But the flavor of such products is poor To keep some flavor as of fermented soy sauce, semi-chemical and blended soy sauce were produced Semi-chemical soy sauce is produced by further fermentation of acid hydrolyzed products; while blended soy sauce is produced by mixing fermented soy sauce with chemical soy sauce and/or enzymatically hydrolyzed vegetable protein As Asian economies... polysaccharides can 5 increase the concentration of IgA in the intestines of mice, suppress passive cutaneous anaphylaxis reaction in the ears of allergy model mice, and regulate the balance of Th1/Th2 cell response in mice, potentially enhancing host defenses [21-23] Clinically, oral administration of soy sauce polysaccharides can improve allergic symptoms of patients with perennial allergic rhinitis or seasonal... were injected into the GC port using a split ratio of 8:1 Column temperature was increased from 125 to 175 °C at 8 °C/min after 2 min at 125 °C, then increased from 175 to 220 °C at 30 °C/min, held at 220 °C for 1 min, and finally increased from 220 to 290 °C at 40 °C/min and held at 290 °C for 2 min Selected-ion monitoring was performed using the electronionization mode with the ion source maintained... fermented soy sauce becomes the main product in Asian markets The United States is left to be the largest chemical soy sauce producer in the world Japanese producers once proposed the world-trade regulations that the manufacturing methods should be included in the label But the US suggested that individual countries should make their own decisions on labeling [18] In Singapore, soy sauce can be made from soy. .. labeling requirement on the manufacturing process 4 1.3 The Functional components of soy sauce In China, soy sauce has been traditionally used for treatment of anorexia, ulcers post thermal burn etc, recorded in Traditional Chinese medicinal books (Su Jing, Xinxiubencao, Tang Dynasty, 659; Sun Simiao, Qianjinfang, Tang Dynasty, 625) [7] Modern scientific research has provided some supporting evidence... thermal energy for aging So in the North of China or Japan, the process of manufacturing can last as long as 2 years To save time, in some modern soy sauce factories, thermal-controlled fermentation tanks are used The making process of soy sauce can be shortened to three months [17] After World War II, when the supplies of soy beans were scarce, many manufacturers applied chemical soy sauce, acid hydrolyzed... The filtrate is called raw soy sauce The raw soy sauce is further pasteurized and finally bottled To make dark soy sauce, the aging process is 3 further extended for another 2-4 months, and caramel is added to make the final product thicker and the color deeper Traditionally, the making of soy sauce is time-consuming and labor-consuming The mash is usually fermented in earthenware, and sunlight ... List of Figures and Chart vi List of Symbols ix Chapter Introduction 1.1 A brief history of soy sauce 1.2 The methodology of preparation of soy sauce 1.3 The functional components of soy sauce. .. 4.2 Content of maltol and its contribution to the TAA of dark soy sauce A considerable part of the TAA of the dark soy sauce was contained in MeOH-R and EtOAc-R fractions and in fractions 1-8... modern soy sauce plant at Walworth, Wisconsin [8] Soy sauce was brought to East Asian countries by the Chinese immigrants In Singapore, it is said that the small-scale manufacturing of soy sauce