Flazin as a Promising Nrf2 Pathway Activator

Article abstract of DOI:10.1021/acs.jafc.9b04600

Flazin is a β-carboline-derived alkaloid found in Japanese fermented foods. Here, the potential of flazin as an antioxidant food was studied with particular reference to its effect on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system in human hepatocytes (C3A). Flazin and flazin analogues including the decarboxylated derivative perlolyrine were chemically synthesized and compared with each other and with chlorogenic acid and curcumin. Among these compounds, flazin showed the lowest cytotoxicity (IC₅₀ < 500 μM) and the highest capacity to activate the Keap1-Nrf2 system. It provided the largest (>3-fold of the control) cytoprotection ability against a pro-oxidant, although its radical absorbance capacity was relatively low. Flazin increased the expressions of Nrf2-dependent phase II enzyme genes and their products (NQO1, GSTP, and GSH proteins). The strong cytoprotection ability of flazin associated with low log ?P (0-3) is shared by sulforaphane and 3,5-dihydroxy-4-methoxybenzyl alcohol, suggesting the potential value of flazin and flazin-rich foods for the prevention of oxidation-related health disorders.

Full text of DOI:10.1021/acs.jafc.9b04600

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Food and Beverage Chemistry/Biochemistry
Flazin as a promising Nrf2 pathway activator
Hirotoshi Fuda, Satoshi Miyanaga, Takayuki Furukawa, Satomi Umetsu, Sae Joko,
Yuning Roan, Hirotaka Suzuki, Shu-Ping Hui, Mitsugu Watanabe, and Hitoshi Chiba
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04600 • Publication Date (Web): 31 Oct 2019
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Title: Flazin as a promising Nrf2 pathway activator
a,
Authors: Hirotoshi Fuda 1, Satoshi Miyanaga a, 1, Takayuki Furukawa a, 1, Satomi
a
a
a
a
Umetsu , Sae Joko , Yuning Roan , Hirotaka Suzuki , Shu-Ping Hui a *, Mitsugu
Watanabe b, and Hitoshi Chiba c
a
Affiliations: Faculty of Health Sciences, Hokkaido University, Sapporo 060-0812,
b
Japan; Watanabe Oyster Laboratory Co. Ltd., 490-3, Shimoongata-cho, Hachioji,
c
Tokyo 190-0154, Japan;
Department of Nutrition, Sapporo University of Health
Sciences, Nakanuma Nishi-4-2-1-15, Higashi-ku, Sapporo 007-0894, Japan
1 These authors contributed equally to this work.
* Corresponding author: Shu-Ping Hui
Address: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku,
Sapporo 060-0812, Japan,
+81.11.706.3693, Email: keino@hs.hokudai.ac.jp
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ABSTRACT
Flazin is a -carboline-derived alkaloid found in Japanese fermented foods. Here, the
potential of flazin as an antioxidant food was studied with particular reference to its
effect on the Keap1-Nrf2 system in human hepatocytes (C3A). Flazin, flazin analogues
including the decarboxylated derivative perlolyrine were chemically synthesized and
compared with each other and with chlorogenic acid and curcumin. Among these
compounds, flazin showed the lowest cytotoxicity (IC50<500 M) and the highest
capacity to activate Keap1-Nrf2 system. It provided the largest (>3-fold of control)
cytoprotection ability against a pro-oxidant, although its radical absorbance capacity
was relatively low. Flazin increased the expressions of Nrf2-dependent phase II enzyme
genes, and their products (NQO1, GSTP and GSH proteins). The strong cytoprotection
ability of flazin associated with low logP (0 to 3) is shared by sulforaphane and
3,5-dihydroxy-4-methoxybenzyl alcohol, suggesting potential value of flazin and
flazin-rich foods for prevention of oxidation-related health disorders.
Keywords: Keap1; Nrf2; hepatocyte; phase II enzyme; flazin; ORAC
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1. INTRODUCTION
The balance of reactive oxygen species (ROS) is maintained by a cytoprotection
system whose components can be divided into enzymatic components (superoxide
dismutase [SOD], catalase [CAT], etc.) and non-enzymatic components (glutathione
1
[GSH], vitamins, etc.) . Non-compensated overproduction of ROS damages cells,
causing ageing 2 and various pathological conditions, such as neoplasia 3, inflammation
4, atherosclerosis 5, and neurodegenerative diseases 6. Ingestion of phenolic
antioxidants in foods is crucially important for health and for the maintenance of
physiological conditions resistant to ROS 7.
Dinkova-Kostova and Talalay proposed classifying antioxidants into two groups:
8
direct and indirect antioxidants . Direct antioxidants scavenge ROS through redox
reactions. During this process, the direct antioxidants are chemically oxidized and in
some cases regenerated 9,10. The antioxidant potential of direct antioxidants can be
evaluated through in vitro chemical analyses, such as 2,2-diphenyl-1-picrylhydrazyl
(DPPH) assays or oxygen radical absorbance capacity (ORAC) assays. On the other
hand, indirect antioxidants induce
a
series of antioxidant enzymes or
low-molecular-weight substances by activating the Kelch-like ECH-associated protein 1
(Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and thus ultimately
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biologically catalyse ROS breakdown in cells. Indirect antioxidants may be redox active,
but this characteristic is not necessary for their indirect antioxidant activity 8.
The role of the Keap1-Nrf2 pathway to pathological conditions has been studied in
human patients and Nrf2-deficient and/or Keap1-deficient mice. These studies reported
that Nrf2 activation alleviated (or exacerbated) the pathological conditions including
neurodevelopmental disorder, chronic kidney disease, neurodegenerative disorders,
pulmonary hypertension, emphysema and cancer 11,12. In human patients, the
Nrf2-activators, sulforaphane and bardoxolone methyl (CDDO-Me), alleviated the
13
14
symptom of the neurodevelopmental disorder
and chronic kidney disease
,
respectively. Using both Nrf2-deficient and Keap1-deficient mice, oltipraz (Nrf2
activator) attenuated pulmonary hypertension 15. In Nrf2-deficient mice, smoking
increased inflammation in the lung and induced emphysema 16. In cancer, Nrf2
activation was beneficial or deleterious for cancer-bearing host depending on the time
and the place of body 11. Using Nrf2-deficient mice, Nrf2 activator of oltipraz reduced the
number of neoplasms of the forestomach 17. However, activation of Nrf2 mediated by
dipeptidyl peptidase-4 inhibitors (Sax and Sit) enhanced tumor metastasis 18.
Flazin is a -carboline-derived alkaloid found in Japanese fermented foods such as
Japanese rice wine lees, rice vinegar, soy sauce, and soy paste 19; it has also been
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found in a marine bacterium 20 and in a fungus from China 21. Flazin has been reported
to induce NAD(P)H: quinone oxidoreductase (NQO1) in a murine hepatoma cell line 22.
Since NQO1 belongs to the family of Nrf2-dependent phase II antioxidant enzymes, we
aimed to uncover the possible relationship of flazin and its analogues with the
Keap1-Nrf2 pathway in this study.
To this end, we chemically synthesized flazin, four analogues 21 and decarboxylated
derivertive perlolyrine in the present study. Furthermore, we compared these
compounds with chlorogenic acid and curcumin, which are representative direct and
indirect antioxidants, respectively 23, in assessments of cytotoxicity, ORAC, Keap1-Nrf2
pathway activation, and cytoprotection against oxidative stress.
In addition, we here propose a model for predicting the usefulness of antioxidants in
which log P and cytoprotective effects against oxidative stress serve as determinant
factors. Log P, or the partition coefficient 24, is common in drug design in the
pharmaceutical and biotechnology industries as a measure of a solute's hydrophobicity
and as a proxy for its membrane permeability; however, this parameter has been
uncommon in the food industry. Our proposed model reveals a unique position of flazin
among antioxidants, indicating its remarkable potential with regard to the prevention
and/or alleviation of health disorders involving oxidation processes.
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2. MATERIALS AND METHODS
2.1. Chemicals. The chemicals used in this study were described in the Supporting
Materials and Methods.
2.2. n-Octanol/water partition coefficients (log P values). The n-octanol/water
partition coefficients (log Pow or log P values) of flazin and perlolyrine were obtained
from
the
PubChem
Open
Chemistry
Database
(https://pubchem.ncbi.nlm.nih.gov/search/) maintained by the National Institutes of
Health (NIH). The log P values for the flazin analogues, chlorogenic acid (8), and
curcumin (7) were obtained from the Virtual Computational Chemistry Laboratory
(http://www.vcclab.org/lab/alogps/) and the literature 25-26
.
2.3. Chemical synthesis
2.3.1. General. The chemicals and procedures in chemical synthesis were described in
the Supporting Materials and Methods.
2.3.2. Synthesis of flazin and its analogues. The structures of the compounds
synthesized in this study as well as those of chlorogenic acid (8) and curcumin (7) are
shown in Fig. 1. Flazin (1) and its analogues (3-6) were synthesized as reported by
Tang et al. 21, and each synthesized compound was confirmed to be identical to the
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previous report on the basis of NMR chemical shifts and the molecular weights
determined by mass spectrometry.
2.3.3. Synthesis of perlolyrine
2.3.3.1. Synthesis of compound 10. The synthesis of compound 10 and data of
proton and carbon NMR were described in the Supporting Materials and Methods.
2.3.3.2. Synthesis of compound 11. The synthesis of compound 11 and data of
proton and carbon NMR were described in the Supporting Materials and Methods.
2.3.3.3. Synthesis of perlolyrine (2). The synthesis of perlolyrine (2) and data of
proton and carbon NMR were described in the Supporting Materials and Methods.
2.4. Cell culture. Human hepatoma-derived cells (C3A) were purchased from the
ATCC. The cells were maintained in MEM supplemented with 10% (v/v) FBS and the
PSN antibiotic mixture under an atmosphere of 5% CO2 and 95% air in an incubator
with controlled humidity at 37°C.
2.5. Cytotoxicity assay. All procedures were performed according to a previously
described protocol, with minor modifications 27. Cytotoxicity assay in details was
performed as indicated in the Supporting Materials and Methods.
2.6. Reporter gene assay. Reporter gene assay in details was performed as indicated
in the Supporting Materials and Methods. Chlorogenic acid (8) and curcumin (7) were
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used as negative and positive controls, respectively, in the reporter gene assay since
we previously reported that curcumin significantly increases relative luciferase activity,
while chlorogenic acid does not significantly affect it 23.
2.7. Cytoprotection assay. Cytoprotection assay in details was performed as indicated
in the Supporting Materials and Methods.
2.8. First-strand cDNA synthesis and real-time PCR. Total RNA extracts, first-strand
cDNA synthesis and real-time PCR were performed as indicated in the Supporting
Materials and Methods.
2.9. Western blot analysis. Protein expression induced by flazin (1) in C3A cells was
examined using Western blotting. All procedures were performed according to a
previously described protocol, with minor modifications 23. Western blot analysis in
details was performed as indicated in the Supporting Materials and Methods.
2.10. Nuclear protein extraction and Western blot analysis. The effect of flazin (1)
on the translocation of the Nrf2 protein from the cytoplasm to the nucleus was examined
using nuclear protein extraction and Western blotting. Nuclear protein extraction and
Western blot analysis in details was performed as indicated in the Supporting Materials
and Methods.
2.11. Measurement of glutathione (GSH). The effect of flazin (1) on GSH levels in
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C3A cells was examined. All procedures were performed according to a previously
described protocol, with minor modifications 23. Measurement of glutathione in details
was performed as indicated in the Supporting Materials and Methods.
2.12. Oxygen radical absorbance capacity (ORAC) assay. The free
radical-scavenging activity of the antioxidants was examined using ORAC assays. All
procedures were performed according to a previously described protocol 28-29. The
relative ORAC value (TE) was calculated as follows: relative ORAC value = molarity of
Trolox/molarity of the samples.
2.13. Statistical analysis. The data from the reporter gene assay, cell viability assay,
and ORAC assay and the data from the nuclear protein, Nrf2 target gene and protein
expression analyses are expressed as the mean ± SD and were analysed by one-way
ANOVA using Prism 6.03. The significance level was set at p < 0.05.
3. RESULTS
3.1. Cytotoxicity. The half-maximal inhibitory concentration (IC50) values for synthetic
flazin (1), perlolyrine (2) and the flazin analogues are shown in Supplemental Fig. 1 and
Table 1. The IC50 values of flazin (1) and chlorogenic acid (8) were more than 500 M.
In contrast, those of perlolyrine (2), curcumin (7), flazin analogue I (3), flazin analogue
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II (4), flazin analogue III (5), and flazin analogue IV (6) were low at 11.0, 109, 51.5,
88.2, 12.0, and 22.4 M, respectively.
3.2. ORAC assay. The relative ORAC values of the antioxidants are shown in Table 1.
The relative ORAC values of flazin (1), perlolyrine (2), flazin analogue II (4), and flazin
analogue III (5) were 1.43 ± 0.112, 1.60 ± 0.230, 0.587 ± 0.124, and 0.886 ± 0.0735
mol of Trolox equivalents (TE)/mol, respectively. In contrast, those of flazin analogue
I (3) and flazin analogue IV (6) were relatively high (3.65 ± 0.392 and 2.50 ± 0.287 mol
of TE/mol, respectively). Curcumin (7) and secondarily chlorogenic acid (8) showed
the highest ORAC values: 8.12 ± 0.831 and 4.57 ± 0.303 mol of TE/mol, respectively.
3.3. Reporter gene assay. The results of the reporter gene assay for the studied
compounds at non-cytotoxic concentrations are shown in Fig. 3A and Table 1.
Compared to the control treatment (0 M), flazin (1) treatment at concentrations of
0-500 M dose-dependently increased relative luciferase activity by 23.4 ± 0.861-fold;
these effects were comparable to those of curcumin (7)(23.8 ± 7.30-fold increase at
concentrations of 20-40 M). Perlolyrine (2) increased relative luciferase activity by 4.64
± 0.812-fold at concentrations of 4-8 M. Flazin analogue II (4) at concentrations of
10-20 M increased the luciferase activity by 10.50 ± 4.5-fold. However, chlorogenic
acid (8), flazin analogue I (3), flazin analogue III (5), and flazin analogue IV (6) did not
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significantly affect the relative luciferase activity at any concentration.
3.4. Cytoprotection against pro-oxidants. The effects of the studied compounds at
non-cytotoxic concentrations on the viability of C3A cells exposed to the pro-oxidant
(AAPH) are shown in Fig. 3B and Table 1. Flazin (1) significantly and dose-dependently
increased cell viability by 5.21 ± 0.24-fold at concentrations of 0-500 M. Curcumin (7)
also significantly and dose-dependently increased viability by 2.66 ± 0.253-fold at
concentrations of 15-60 M, and flazin analogue II (4) increased viability by 2.00 ±
0.176-fold at concentrations of 10-20 M. Perlolyrine (2), chlorogenic acid (8), flazin
analogue I (3), flazin analogue III (5), and flazin analogue IV (6) did not significantly
affect cell viability at any concentration.
3.5. Effect of flazin on Nrf2 nuclear translocation and the expression of Nrf2 and
Keap1. The effect of flazin (1) on Nrf2 nuclear translocation in C3A cells is shown in
Fig. 4A and 4B. Compared to the control treatment (0 M), flazin (1) treatment at
concentrations of 0-500 M significantly and dose-dependently increased the protein
levels of Nrf2 in the nucleus fraction by approximately 1.5-fold. And flazin decreased the
levels of Nrf2 in the cytoplasmic fraction at concentrations of 250-500 M by
approximately one-half. Also flazin increased the protein and gene expression in total
lysates Nrf2 levels in a dose-dependent manner (Fig. 4C and D). Flazin decreased the
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protein expression of Keap1 levels at 500 M (Fig. 4E).
3.6. Effect of flazin on the expression of Nrf2 target genes. The Nrf2 target genes
induced by flazin in C3A cells are shown in Fig. 5A. Flazin (1) significantly induced the
expression of the Nrf2 target genes NQO1, glutamate-cysteine ligase modulatory
subunit (GCLM), glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione
S-transferase pi (GSTP) by more than 10-fold in a dose-dependent manner. More
moderate but statistically significant induction was observed for HO-1, SOD1, GR, GPx,
CAT, and GSTT.
3.7. Effect of flazin on the expression of Nrf2 target gene products. We paid
attention to the Nrf2 target genes, NQO1, GSTP, GCLM and GCLC whether they were
induced by flazin significantly (more than 10-fold) and the change in their protein
expressions. The effects of flazin (1) on the expression of three Nrf2 target gene
products, NQO1, GSTP and GSH, which is synthesized by composed of GCLC and
GCLM, are shown in Fig. 5B. Flazin (1) increased the protein expression of NQO1 and
GSH in a dose-dependent manner. In addition, flazin (1) significantly increased GSTP
expression levels at 500 M.
3.8. Cytoprotection and log P. The log P values of the studied compounds are shown
in Table 1. The highest log P was observed for curcumin (3.29), while the lowest was
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observed for chlorogenic acid (0.69). The log P values of flazin (2.1), perlolyrine (2.2),
and the flazin analogues (1.72-2.80) were in the middle range. For eight of the natural
antioxidants (a - h), there was a strong correlation between cytoprotection and the log P
2
value (y = 0.400x + 0.880, r = 0.766, p = 0.004) (Fig. 7). For another group of four
natural antioxidants (f - i), there was also a strong correlation between cytoprotection
and the log P value (y = 0.059x + 1.974, r 2 = 0.750, p = 0.333) (Fig. 7). The antioxidants
flazin, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and sulforaphane did not fit the
correlation lines and were distributed in division I.
4. DISCUSSION
Flazin has been reported to be present in traditional Japanese fermented foods,
such as sake (rice wine) lees, rice vinegar, soy sauce (fermented soybean liquid), and
miso (fermented soybean paste) 19, as well as in the fruiting bodies of Suillus granulatus
21
(a fungus) and in the marine-derived bacteria Streptomyces spp. 20. With regard to
22
biological function, flazin exhibits anti-HIV-1 activity 21, NQO1-inducing activity
,
leukaemic cell (HL-60) proliferation-inhibiting activity 30, and xanthine oxidase-inhibiting
31
activity . In our present study, flazin was found to activate the Nrf2 pathway (Fig. 3A
and 4A) and to promote the expression of a battery of Nrf2 target genes and products,
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including NQO1 (Fig. 5A and B). Flazin exhibit a strong cytoprotective effect against
AAPH despite its relatively low ORAC, suggesting that flazin essentially serves as an
indirect antioxidant. The action mechanisms of flazin as an indirect antioxidant are
summarized in Fig. 6.
The relationships between the chemical structures and anti-HIV activity of flazin and
21
its 45 synthetic analogues have been reported previously . In this study, the highest
anti-HIV activity was observed for flazin analogue III (5), in which the carboxylic acid at
carbon 3 of flazin has been converted to an amide 21 (Fig. 1). Flazin itself was found not
to exhibit anti-HIV-1 activity; thus, the activity was dramatically enhanced by the
introduction of an appropriate substituent at carbon 3. In contrast to anti-HIV-1 activity,
indirect antioxidant activity requires the carboxylic at position 3. Naturally occurring
flazin showed higher cytotoxicity, stronger Nrf2-activating ability, and greater
cytoprotective effects than the flazin analogues or perlolyrine (Supplemental Fig. 1, Fig.
3 and Table 1). Conversely, flazin analogue III (5) showed the lowest performance in
cytoprotection.
The ORAC assay is one of the most common assays for measuring peroxyl
radical-scavenging capacity in vitro, although its reaction mechanism remains unknown
32. In this study, the ORAC values of flazin analogues I (3) and IV (6) (3.65 ± 0.392 and
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2.50 ± 0.287 μmol of TE/μmol, respectively) were higher than that of flazin (1) (1.43 ±
0.112 μmol of TE/μmol) (Table 1). In flazin analogues I (3) and IV (6), the carboxylic
acid at carbon 3 of flazin has been replaced with a hydroxamic acid and aliphatic
alcohol, respectively (Fig. 1). We hypothesize that the hydroxamic acid and aliphatic
alcohol of the flazin analogues might easily form R-C(O)-NH-O ● and R-C-O ● ,
respectively, to enable these analogues to scavenge peroxyl radicals in the ORAC
assay more easily than flazin.
When exposed to oxidative stress or Nrf2 activators, several highly reactive
cysteine residues of Keap1 are modified to prevent Nrf2 from proteasome degradation,
resulting in rapid nuclear translocation of Nrf2 11. Resveratrol-treated HaCat cells
(spontaneously immortalized human keratinocytes) have been investigated on the Nrf2
and Keap1 levels in total cell lysates, and on the Nrf2 levels in nuclear and cytoplasmic
fractions 33. Resveratrol increased the Nrf2 levels in the nuclear fraction, but decreased
them in the cytoplasmic fraction. These results are in agreement with the present study
(Fig. 4A and 4B), where flazin-induced nuclear translocation of Nrf2 was confirmed. In
flazin-treated C3A cells, upregulation of Nrf2 levels and downregulation of Keap1 levels
in the total cell lysate were indicated (Fig. 4C-E). Flazin might have stabilized Nrf2
protein in the cytosol and accelerated its nuclear translocation. Similarly, resveratrol has
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been reported to increase Nrf2 levels in the total cell lysate with significant reduction in
the levels of Keap1 33.
The potent cytoprotective effect of flazin can be attributed at least partly to the
induction of a battery of antioxidant enzymes by flazin through activation of the Nrf2
pathway. Flazin induced the expression of Nrf2 protein and of the Nrf2 target genes
HO-1, GCLM, GCLC, NQO1, SOD1, GR, GPx, and GSTP (Fig. 5A and 5B). Of these,
NQO1, GCLM, GCLC, and GSTP and their products showed substantially increased
expression (Fig. 5A and 5B). The catalytic activity of NQO1 is directed towards the
reduction and detoxication of highly reactive quinones and their derivatives. NQO1 is
expressed in many tissues, and its expression is co-regulated by the Keap1-Nrf2
pathway 34. GSTP is a type of cytosolic GST and plays an important role in the classic
GSH-dependent enzymatic detoxification pathway of electrophilic metabolites and
xenobiotics 35. Nrf2 is one of the most important transcription factors recognized to
stimulate GSTP 36. GSH is present at micromolar levels in all eukaryotic cells and
provides protection against ROS 37. GSH is synthesized by glutamate-cysteine ligase
(GCL), which is composed of GCLC and GCLM, and GSH synthetase. GCL catalyses
the rate-limiting step in GSH synthesis and is the site of feedback inhibition by GSH
itself 38. Both human GCLC and GCLM are regulated by the Keap1-Nrf2 pathway 37. In
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this study, the increased expression of GCLC and GCLM due to flazin-induced Nrf2
activation was associated with an increase in GSH (Fig. 5C). The increase in GSH,
however, might be too small (2.6-fold) to fully account for the observed increases in
GCLM and GCLC expression of 12.7- and 42.5-fold, respectively. We propose that
synthesized GSH inhibits the catalytic activity of GCL.
In our previous study, we examined log P in ten natural antioxidants 23. In a scatter
plot showing the cytoprotective effect plotted against the log P for each antioxidant,
most antioxidants were distributed along a single curve. However, sulforaphane and
DHMBA showed unique distributions. A new scatter plot including the present data is
shown in Fig. 7, in which 4 divisions (I-IV) have been made on the basis of
cytoprotection (dividing line at 3-fold greater than control values) and log P (dividing line
at 3). Flazin is distributed in division I (upper left) together with sulforaphane and
DHMBA; moreover, flazin occupies the highest position among them in terms of its
cytoprotection. In contrast to flazin, the flazin analogues, perlolyrine and other
antioxidants are distributed along a curve in division III. With regard to sulforaphane
and DHMBA, studies have reported their usefulness in preventing and/or alleviating
health disorders; for instance, anticancer, anti-diabetes, and anti-inflammatory effects
have been reported for sulforaphane 39-40, and anti-non-alcoholic steatohepatitis (NASH)
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effects have been reported for DHMBA 41. On the basis of the above evidence, flazin
and flazin-rich foods may also have a promising future as functional foods.
As shown in the scatter plot, log P appears to increase the cytoprotective ability of
the antioxidants in division III (Fig. 7). However, the effect of log P reaches a plateau in
division IV. This can be explained by the association of higher log P values with higher
cytotoxicity (Table 1). It is a general rule that hydrophobic compounds (with higher log P
values) are more toxic in the body than hydrophilic compounds (with lower log P values)
24. Flazin and perlolyrine (e in Fig. 7) exhibited large differences in cytoprotection
despite their similar log P values, indicating that log P is not the only determinant of
cytoprotective ability. This difference must be due to the Nrf2-activating ability of flazin,
since divisions I and III are clearly separated at the cytoprotection level of
approximately 3-fold (Fig. 7). Since only a few antioxidants (sulforaphane, j; DHMBA, k;
and flazin) have been specified as belonging to division I so far, further surveys of novel
indirect antioxidants belonging to division I are warranted.
This study indicates that flazin is a potentially useful functional food for antioxidants
via activating Keap1-Nrf2 pathway. However, possible adverse effect of flazin remains
to be investigated. Flazin belongs to -carboline derivatives, which is supposed to
generate through the Maillard reaction as previously reported 42,43. The Maillard reaction
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products have both positive and negative effects on health such as antioxidants,
bacterucidals, antiallegenic, prooxidant, and carcinogens 44-47. The utility of flazin as a
functional food needs to be examined by animal model experiments and human trials in
future.
5. CONCLUSION
Flazin activates the Nrf2 pathway and protects human hepatocytes from oxidative
stress without showing any significant cytotoxicity. The potential for flazin to be used as
a functional food must be investigated in future animal model experiments and
subsequent human trials. Since log P values are accessible in chemical databases and
are useful for predicting the utility of individual antioxidants, a log P-based search for
indirect antioxidant foods is recommended. The limitation of this study is the lack of in
vivo confirmation of the effect and the safety of flazin. These issues remain to be
studied in the future.
COMPETING INTERESTS.
This study was partially funded by Watanabe Oyster Laboratory Co. Ltd., and
Hirotoshi Fuda works in a research laboratory donated by them. The funder had no role
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in the study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
ACKNOWLEGEMENTS.
The anti-Nrf2 antibodies were kindly donated by Dr. Masaharu Sakai, professor
emeritus, Hokkaido University. This work was financially supported by a Grant-in-Aid for
Scientific Research from the JSPS (16K07863), by the Regional Innovation Strategy
Support Program of the MEXT and by Watanabe Oyster Laboratory Co. Ltd. The
English proofreading by Dr. Darwish W. S. was very useful.
ABBREVIATIONS.
AAPH,
2,2’-azobis(2-amidinopropane)
dihydrochloride;
DHMBA,
3,5-dihydroxy-4-methoxybenzyl alcohol; ORAC, oxygen radical absorbance capacity.
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Journal of Agricultural and Food Chemistry
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