Natural Stilbenoids Have Anti-Inflammatory Properties in Vivo and Down-Regulate the Production of Inflammatory Mediators NO, IL6, and MCP1 Possibly in a PI3K/Akt-Dependent Manner

Article abstract of DOI:10.1021/acs.jnatprod.7b00384

Stilbenoids are a group of polyphenolic compounds found in plants, trees, berries, and nuts. Stilbenoids have been shown to serve an antimicrobial and antifungal function in plants. There is also evidence that as a part of the human diet, stilbenoids play

Full text of DOI:10.1021/acs.jnatprod.7b00384

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Natural Stilbenoids Have Anti-Inflammatory Properties in Vivo and
Down-Regulate the Production of Inflammatory Mediators NO, IL6,
and MCP1 Possibly in a PI3K/Akt-Dependent Manner
Heikki Erasalo,† Mari Hamalainen,† Tiina Leppanen,† Ilari Maki-Opas,† Mirka Laavola,†
̈
̈
̈
̈
̈
̈
Raisa Haavikko,‡ Jari Yli-Kauhaluoma,‡ and Eeva Moilanen*,†
†The Immunopharmacology Research Group, Faculty of Medicine and Life Sciences, University of Tampere and Tampere University
Hospital, FI-33014 Tampere, Finland
‡Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki,
Finland
ABSTRACT: Stilbenoids are a group of polyphenolic compounds found in plants, trees, berries,
and nuts. Stilbenoids have been shown to serve an antimicrobial and antifungal function in plants.
There is also evidence that as a part of the human diet, stilbenoids play an important role as
antioxidants and may have anti-inflammatory effects. The PI3K/Akt pathway is a well-characterized
signaling pathway controlling cellular functions involved in growth and cell cycle and in metab-
olism. There is also increasing evidence to show the involvement of this pathway in the regulation
of inflammatory responses. In the present study, an attempt was made to investigate the anti-
inflammatory properties of the naturally occurring stilbenoids pinosylvin (1), monomethylpino-
sylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5), and rhapontigenin (6). Glycosylated
derivatives of piceatannol and rhapontigenin, namely, astringin (7) and rhaponticin (8), respec-
tively, were also investigated. In addition to the natural stilbenoids, pinosylvin derivatives (9−13)
were synthesized and subjected to the testing of their effects on the PI3K/Akt pathway in inflam-
matory conditions. The investigated natural stilbenoids (except the glycosylated derivatives) were
found to down-regulate Akt phosphorylation, which is a well-acknowledged marker for PI3K
activity. It was also found that all of the studied natural stilbenoids had anti-inflammatory effects in vitro. The three most potent
stilbenoids, piceatannol, pinosylvin, and pterostilbene, were selected for in vivo testing and were found to suppress inflammatory
edema and to down-regulate the production of inflammatory mediators IL6 and MCP1 in carrageenan-induced paw inflam-
mation in mice. When compared to the commercial PI3K inhibitor LY294002, the anti-inflammatory effects appeared to be quite
similar. The results reveal hitherto unknown anti-inflammatory effects of natural stilbenoids and suggest that those effects may be
mediated via inhibition of the PI3K/Akt pathway.
tilbenoids are a group of polyphenolic compounds found in
nature and particularly in plants. Stilbenoids have been
readily found in pine knots11 and also in pine nuts.12 There
are fragmentary data on the health-promoting effects of those
stilbenoids, sometimes resembling the effects of resveratrol13
including some anti-inflammatory effects.14−16
S
shown to have antimicrobial and antifungal properties, and it is
likely that their role in plants is to serve as defensive com-
pounds against microbial pathogens.1 They are synthesized in
trees as secondary metabolites during heartwood biogenesis,
and they are also actively produced as a response to infection or
injury. Stilbenoids can therefore be also classified as phyto-
alexins due to their antimicrobial properties.2,3
Due to being abundant in plants, stilbenoids are also present
in human nutrition. Their health effects and nutritional signif-
icance have also been studied. Resveratrol, probably the most
well-known and investigated stilbenoid, is present in grapes,
different berries, nuts, and also in red wine.4 Resveratrol has
been reported to have anti-inflammatory, anticarcinogenic, anti-
oxidative, and cardioprotective properties.5 However, in addi-
tion to resveratrol, several other stilbenoid compounds are present
in human nutrition. For instance, pterostilbenoid, rhapontigenin,
and piceatannol can all be found in different berries, fruits, nuts,
and other edible plants.6−10 Structurally very closely related
stilbenoids pinosylvin and monomethylpinosylvin are also
The PI3K/Akt pathway is a cellular signaling pathway involved
in various cellular processes, such as cell proliferation, migration,
and survival as well as glucose metabolism. It is also considered
to have a remarkable role in inflammatory responses,17 and its
role in cancer is also vastly studied.18 The PI3K/Akt pathway
consists of phosphatidylinositol 3-kinases, which catalyze the phos-
phorylation of phosphatidylinositols, a class of structurally impor-
tant lipid molecules in cell membranes. The phosphatidylinositol-
phosphates (PIPs) generated in the process then serve as a
docking platform for Akt kinase (also known as PKB). Akt
kinase is a serine/threonine kinase that has a wide range of down-
stream targets regulating different processes, such as cell pro-
liferation, glucose metabolism, neuronal development, and
Received: May 2, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Molecular structures of the studied natural and semisynthetic stilbenoids.
inflammation.19−21 It has been shown that when the PI3K/Akt
pathway is inhibited with pharmacological compounds, the
cellular inflammatory responses are attenuated due to down-
regulation of inflammatory genes.22−24
possibly via a PI3K/Akt-dependent mechanism. An anti-
inflammatory effect was also observed in the in vivo model
and was further elucidated with ex vivo studies.
The aim of the present study was to investigate natural
stilbenoids (Figure 1), including pinosylvin (1), monomethyl-
pinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5),
and rhapontigenin (6), and test their effects on the PI3K/Akt
pathway in activated macrophages. Additionally, the glycoside
derivatives of piceatannol and rhapontigenin, namely, astringin (7)
and rhaponticin (8), respectively, were investigated. In addition
to the natural stilbenoids, pinosylvin derivatives (9−13) were
synthesized and subjected to anti-inflammatory testing. Another
aim was to investigate the effects of the aforementioned stil-
benoids on the production of the inflammatory mediators inter-
leukin 6 (IL6), monocyte chemotactic protein 1 (MCP1), and
nitric oxide (NO). Further, the anti-inflammatory effects of three
potent stilbenoids (piceatannol, pinosylvin, and pterostibene)
were also investigated in vivo in the carrageenan-induced paw
inflammation model in mice. Additionally the in vivo studies
were backed up with ex vivo studies by measuring inflammatory
cytokines in inflamed paw tissue. The results reveal that all of
the investigated natural stilbenoids (excluding the glycoside
derivatives) have an inhibitory effect on the activation of the
PI3K/Akt pathway. It was also found that these compounds
down-regulate the production of inflammatory mediators,
RESULTS
■
Synthesis of Pinosylvin Derivatives. A panel of pinosylvin
derivatives bearing different alkyloxy and allyloxy moieties
(compounds 9−13) was designed (Figure 1). The compounds
were synthesized by a nucleophilic substitution reaction between
the commercially available pinosylvin and different alkyl (Et, n-Pr,
Bn, and i-Bu) and allyl bromides in the presence of potassium
carbonate as a base in acetone. The novel pinosylvin ethers
were produced in low to moderate yields.25 Their chemical struc-
tures were confirmed by H and 13C NMR spectroscopy.
1
Natural Stilbenoids Inhibited PI3K/Akt Activation in a
Concentration-Dependent Manner in J774 Macrophages.
Stimulation of macrophages through a TLR4-mediated mech-
anism by exposing the cells to bacterial endotoxin lip-
opolysaccharide (LPS) (10 ng/mL)-induced PI3K-mediated
phosphorylation of Akt protein on amino acid residue S473 and
treating the cells with the commercial PI3K inhibitor LY294002
inhibited Akt phosphorylation (Figure 2). Next, the effects of
natural stilbenoids pinosylvin (1), monomethylpinosylvin (2),
resveratrol (3), pterostilbene (4), piceatannol (5), and rhaponti-
genin (6) on the phosphorylation of Akt in activated macrophages
were investigated. The results showed that all of the investigated
B
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
stilbenoids 1−8 on the production of inflammatory mediators
NO, IL6, and MCP1 in activated macrophages, the J774 mac-
rophage cells were stimulated with LPS in the presence of
increasing concentrations of the stilbenoids for 24 h prior to
cell culture media collection. It was found that the investigated
stilbenoids had a concentration-dependent inhibitory effect on
the production of the aforementioned inflammatory mediators
that was comparable to the effects obtained with the known
PI3K inhibitor LY294002 (Figures 5−7).
The control compound LY294002 inhibited NO production
by 50% at a 8.9 μM concentration. Closely comparable values
were obtained for resveratrol (3, 12.15 μM), pterostilbene
(4, 16.7 μM), and pinosylvin (1, 16.6 μM). Clearly higher val-
ues were obtained for monomethylpinosylvin (2, 37.0 μM) and
rhapontigenin (6, 102.8 μM). For piceatannol (5) the value was
the lowest (2.6 μM) (Figure 5).
LY294002 inhibited IL6 expression half-maximally at a 5.1 μM
concentration. Comparable to this were resveratrol (3, 6.8 μM),
pterostilbene (4, 8.7 μM), and piceatannol (5, 13.0 μM).
Higher values were detected with pinosylvin (1, 32.1 μM), mono-
methylpinosylvin (2, 30.2 μM), and rhapontigenin (6, 99.1 μM)
(Figure 6).
For MCP1 production, LY294002 had a 50% inhibitory effect at
3.7 μM. The only stilbenoid comparable to that was piceatannol
(5, 4.0 μM) and perhaps pterostilbene (4, 10.5 μM). Clearly
higher values were found for pinosylvin (1, 38.7 μM), mono-
methylpinosylvin (2, 23.7 μM), resveratrol (3, 13.6 μM), and
rhapontigenin (6, 19.4 μM) (Figure 7).
Figure 2. LPS-stimulated phosphorylation of Akt in activated
macrophages. J774 macrophages were stimulated with bacterial
lipopolysaccharide (LPS, 10 ng/mL) in the absence or in the presence
of 10 μM LY294002 for 4 h. Thereafter cellular proteins were
extracted and the level of Akt phosphorylation was measured by
Western blotting with an antibody specific toward phosphorylated
S473 residue. Values are mean + SEM, n = 4, **p < 0.01.
In contrast, the glycoside derivatives of piceatannol and rhap-
ontigenin, i.e., astringin (7) and rhaponticin (8), respectively,
appeared to have lost their activity when compared to the
corresponding nonglycosylated stilbenoid (Figures 5−7).
Natural Stilbenoids Showed Anti-Inflammatory Ef-
fects Both in Vivo and Ex Vivo. The carrageenan-induced
paw edema mouse model was used as an in vivo model of inflam-
mation to investigate the anti-inflammatory effects of natural
stilbenoids. Pinosylvin (1), pterostilbene (4), and piceatannol (5)
were selected for the in vivo studies due to their having similar
effects and relatively similar inhibition percentages in the in vitro
studies to the PI3K inhibitor LY294002. Also, based on the
literature search pterostilbene (4) and piceatannol (5) appeared
also to be the least investigated stilbenoids, with very little
published data from in vivo experiments. A widely used anti-
inflammatory drug, dexamethasone, was used as the control
compound. It was found that all of the investigated natural stil-
benoids, i.e., 1, 4, and 5 (when given at the dose of 30 mg/kg),
had an anti-inflammatory effect in vivo comparable to that of
LY294002 (15 mg/kg) and dexamethasone (2 mg/kg) (Figure 8).
In addition to the in vivo studies, ex vivo studies were con-
ducted by measuring the levels of inflammatory cytokines IL6
and MCP1 in the mouse paw tissue after carrageenan injection.
It was found that the inflamed paw tissue from mice treated
with either stilbenoids or the control compound LY294002 or
dexamethasone showed a diminished inflammatory response,
which was seen as lower expression of the inflammatory cyto-
kines IL6 and MCP1 (Figure 9).
stilbenoids inhibited Akt phosphorylation in a concentration-
dependent manner (Figure 3A−F). Piceatannol (5) was the most
potent of the tested stilbenoids followed by monomethylpino-
sylvin (2) and pinosylvin (1), which all inhibited Akt
phosphorylation in a statistically significant manner already at
3 μM concentrations, while rhapontigenin (6) appeared to be
the least potent in the series of natural stilbenoids. The glyco-
side derivatives of piceatannol and rhapontigenin, i.e., astringin
(7) and rhaponticin (8), respectively, were also investigated. The
glycosides had no effect on Akt phosphorylation (Figure 3).
Semisynthetic Pinosylvin Derivatives Exhibited a
Structure-Related Loss of Effect on Akt Phosphorylation
When Compared to Natural Stilbenoids. In order to com-
pare the effects of natural stilbenoids versus synthetic pinosylvin
derivatives, Akt phosphorylation was stimulated with LPS in
J774 macrophages treated with the pinosylvin derivatives 9−13.
Pinosylvin (1) was used as a reference compound in the
experiments. It was found that although some of the synthetic
stilbenoid derivatives inhibited Akt phosphorylation, the effect
was not comparable in magnitude to the effect of the natural
stilbenoid pinosylvin (Figure 4): Monoethylpinosylvin (9) and
monoallyl pinosylvin (11) reduced Akt phosphorylation in a
statistically significant manner when used at 30 μM concen-
trations. Monoisobutylpinosylvin (12) and monobenzylpino-
sylvin (13) had no effect at 30 μM concentrations, while
monopropylpinosylvin (10) appeared to have a small inhibitory
effect, but it did not reach statistical significance. The loss of
effect appeared to correlate with the length of the carbon side
chain on the hydroxy group connected to the R1 position in the
phenyl ring.
DISCUSSION
■
In the present study, our aim was to investigate anti-
inflammatory properties of natural stilbenoids pinosylvin (1),
monomethylpinosylvin (2), resveratrol (3), pterostilbene (4), pice-
atannol (5), rhapontigenin (6), astringin (7), and rhaponticin (8).
The hypothesis was that stilbenoids would down-regulate
Natural Stilbenoids Down-Regulated the Production
of Inflammatory Mediators NO, IL6, and MCP1 in J774
Macrophages. In order to investigate the effects of the natural
C
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 3. Concentration-dependent effect of natural stilbenoids and stilbenoid glycosides on Akt phosphorylation in activated macrophages. J774
macrophages were stimulated with bacterial lipopolysaccharide (LPS, 10 ng/mL) for 4 h in the presence of increasing concentrations of
(A) pinosylvin (1), (B) monomethylpinosylvin (2), (C) resveratrol (3), (D) pterostilbene (4), (E) piceatannol (5), and astringin (7), and
(F) rhapontigenin (6) and rhaponticin (8). Thereafter cellular proteins were extracted and the level of Akt phosphorylation was investigated by
Western blotting with an antibody specific toward phosphorylated S473 residue. Values are mean + SEM, n = 4−6, *p < 0.05 and **p < 0.01.
inflammatory gene expression and the activity of the PI3K/Akt
pathway in relation to inflammation. The results obtained
in this study support the idea that natural stilbenoids have
anti-inflammatory properties and the effect is possibly mediated
via inhibition of the PI3K/Akt pathway. Further, it was found
that the investigated semisynthetic pinosylvin derivatives showed
D
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 4. Effect of semisynthetic pinosylvin derivatives on Akt phosphorylation in activated macrophages. J774 macrophages were stimulated with
bacterial lipopolysaccharide (LPS, 10 ng/mL) for 4 h in the presence of the semisynthetic stilbenoids 9−13 (30 μM). Pinosylvin and LY294002 were
used as reference compounds. After incubation cellular proteins were extracted and Akt phosphorylation was investigated by Western blotting with
an antibody specific toward phosphorylated S473 residue. Values are mean + SEM, n = 4, **p < 0.01.
weaker effects on the PI3K/Akt pathway in inflammatory con-
ditions when compared to their naturally occurring counter-
parts.
particularly class I PI3K subunits PI3Kδ and PI3Kγ, which are
considered to regulate cytokine signaling and chemotaxis of
immune cells.22,28
The health-beneficial properties of the stilbenoid resveratrol
and red wine have been a popular research topic ever since the
concept of the French paradox was formulated in the 1980s.26
Reports have shown that resveratrol has anti-inflammatory,
anticarcinogenic, antioxidative, and cardioprotective properties5
as well as beneficial effects on aging, diabetes, and neurological
dysfunction.27 The in vitro anti-inflammatory effects of res-
veratrol were also reproduced in the present study. However,
there exists a plethora of foodstuffs containing stilbenoids other
than resveratrol, such as different berries, nuts, and other edible
plants that are a natural part of human nutrition worldwide.
Therefore, in this study we focused mainly on other stilbenoids
besides resveratrol, namely, pinosylvin (1), monomethylpino-
sylvin (2), pterostilbene (4), piceatannol (5), and rhapontige-
nin (6) as well as five synthetic derivatives of pinosylvin.
The PI3K/Akt signaling pathway controls an array of cellular
functions, some of which are also involved in inflammation.
Regarding inflammation, the PI3K/Akt pathway is documented
to regulate survival, motility, differentiation, and proliferation
of leukocytes via cytokine and chemokine signaling as well as
NF-κB-mediated transcription.21 Among three different classes
of PI3Ks there also exist several different PI3K isoforms. The
class I PI3Ks are considered to be involved in inflammation and
Low-grade chronic inflammation has been documented to be
increasingly prevalent especially in Western countries. Western
lifestyle and diet have been suggested as one of the main causes
of low-grade inflammation, and the prevalence of metabolic
syndrome and type 2 diabetes is considered to be a direct effect
caused by low-grade inflammation.29 Obesity is also known to
up-regulate inflammation, and it has been reported that macro-
phages located in the adipose tissue produce significant amounts
of IL6 and MCP1 as well as other inflammatory mediators.30
It has also been documented that a healthy diet containing
phytonutrients from fruits and vegetables modulates low-grade
inflammation toward a more healthy level.31 A recent study also
documented that the PI3K/Akt pathway is activated in low-
grade inflammation and that the inflammation can be atten-
uated with PI3K inhibition.32 On the basis of our present find-
ings we hypothesize that a diet containing natural stilbenoids
could possibly also attenuate low-grade inflammation via a
PI3K-dependent mechanism.
In this study we used a commercially available PI3K inhib-
itor, LY294002, as a reference compound for PI3K inhibition.
LY294002 is a well-characterized class I PI3K inhibitor.33
The phosphorylation of Akt is often considered as a marker for
PI3K activity. Upon binding the phosphatidylinositols produced
E
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 5. Effects of natural stilbenoids on NO production in activated macrophages. J774 macrophages were stimulated with bacterial
lipopolysaccharide (LPS, 10 ng/mL) in the presence of increasing concentrations of (A) pinosylvin (1), (B) monomethylpinosylvin (2),
(C) resveratrol (3), (D) pterostilbene (4), (E) piceatannol (5), and (F) rhapontigenin (6) for 24 h. After incubation the cell culture media were
collected. NO production was investigated by measuring the accumulation of its stable metabolite nitrite in the cell culture media with the Griess
method. In addition, the effects of stilbenoid glycosides astringin (7) and rhaponticin (8) on NO production were investigated and compared to
their aglycones (G). LY29004 was used as a reference compound (H). Values are mean + SEM, n = 4, *p < 0.05 and **p < 0.01.
by PI3Ks, Akt is docked to the cell membrane, which allows
PDK1 and MTOR to phosphorylate it.20 Our results show that
the control compound PI3K inhibitor LY294002 used in this
study inhibits Akt phosphorylation upon LPS stimulation also
in activated macrophages and inhibits the production of
inflammatory cytokines IL6 and MCP1 in vitro and in vivo as
expected.
Based on our findings, it appears that the investigated stil-
benoids also exhibit similar inhibitory effects to the PI3K inhib-
itor LY294002 on the phosphorylation of Akt indicative of the
inhibition of the activity of the PI3K/Akt pathway. These data
are supported by the few studies that have focused on the effects
of stilbenoids on the PI3K/Akt pathway in other cell types and
experimental settings.14,34,35 However, the findings reported in
F
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 6. Effects of natural stilbenoids on IL6 expression in activated macrophages. J774 macrophages were stimulated with bacterial
lipopolysaccharide (LPS, 10 ng/mL) in the presence of increasing concentrations of (A) pinosylvin (1), (B) monomethylpinosylvin (2),
(C) resveratrol (3), (D) pterostilbene (4), (E) piceatannol (5), and (F) rhapontigenin (6) for 24 h. After incubation the cell culture media were
collected and IL6 concentrations were measured with ELISA. In addition, the effects of stilbenoid glycosides astringin (7) and rhaponticin (8) on
IL6 production were measured and compared to their aglycones (G). LY29004 was used as a reference compound (H). Values are mean + SEM,
n = 4, *p < 0.05 and **p < 0.01.
this study suggest that among the investigated stilbenoids there
are differences regarding the PI3K/Akt inhibition potency.
Piceatannol (compound 5) appeared to be the most potent of
the tested natural stilbenoids, followed by pinosylvin (com-
pound 1) and monomethylpinosylvin (compound 2), while
rhapontigenin (compound 6) was the least potent. A very similar
pattern of activity was also seen in their inhibitory effects on the
production of the inflammatory factors nitric oxide, IL6, and
MCP1; see below.
The semisynthetic pinosylvin derivatives were less potent
than the natural stilbenoid pinosylvin, and the loss of activity
seemed to associate with the length of the side chain on the R1
position in the left-hand-side phenyl ring. When the pinosylvin
derivatives were compared to the natural pinosylvin, it appeared
that a small substituent such as methyl on the R1 carbon did
not significantly effect the potency of PI3K/Akt inhibition, as
was documented with monomethylpinosylvin (compound 2).
However, when the substituent on R1 became longer, such as
G
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 7. Effects of natural stilbenoids on MCP1 expression in activated J774 macrophages were stimulated with bacterial lipopolysaccharide (LPS,
10 ng/mL) in the presence of increasing concentrations of (A) pinosylvin (1), (B) monomethylpinosylvin (2), (C) resveratrol (3), (D) ptero-
stilbene (4), (E) piceatannol (5), and (F) rhapontigenin (6) for 24 h. After incubation the cell culture media were collected and the concentrations
of MCP1 were measured with ELISA. In addition, the effects of stilbenoid glycosides astringin (7) and rhaponticin (8) on MCP1 production were
measured and compared to their aglycones (G). LY29004 was used as a reference compound (H). Values are mean + SEM, n = 4, **p < 0.01.
the ethyl, propyl, or isobutyl ether group on compounds 9, 10,
and 12, respectively, or bulkier such as the allyl or benzyl ether
groups on compounds 11 and 13, respectively, the effect observed
on PI3K/Akt inhibtion was clearly weakened. In fact, com-
pounds 12 and 13 had no effect at 30 μM concentrations, and
compound 10 had a minor effect that did not reach statistical
significance. This is possibly due to the fact that longer or
bulkier substituents are more likely to cause steric hindrance in
the interaction mechanisms between these molecules.
In addition, the glycosidic derivatives of piceatannol and
rhapontigenin, namely, astringin and rhaponticin, respectively,
did not appear to have an effect on the PI3K/Akt pathway. This
is possibly due to the same mechanism described above and/or
related to the fact that glycoside derivatives are not readily
transported into the cells but rather stay in the cell culture
medium, where they remain inactive. To further elucidate this,
it would be meaningful to investigate other different glycosidic
derivatives (e.g., glycosides with galactose or fructose conjugates)
H
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 8. Effects of natural stilbenoids in the carrageenan-induced paw inflammation model in mice. The investigated stilbenoids pterostilbene (4),
pinosylvin (1), and piceatannol (5) (all 30 mg/kg) or the PI3K inhibitor LY294002 (15 mg/kg) or dexamethasone (2 mg/kg) was administered to
the animals intraperitoneally 2 h prior to carrageenan (1.5% in saline) injection into the paw, and saline was injected into the contralateral paw to
serve as a control. Paw edema was measured before and 3 and 6 h after carrageenan injection. Edema is expressed as the difference between the
volume changes of the carrageenan-treated paw and the control paw (injected with saline). Values are mean + SEM, n = 6 (stilbenoids) or 7
(dexamethasone and LY294002), *p < 0.05 and **p < 0.01.
Figure 9. Effects of natural stilbenoids on the expression of the inflammatory cytokines IL6 and MCP1 in the carrageenan-induced paw inflammation
model in mice. The investigated stilbenoids pterostilbene (4), pinosylvin (1), and piceatannol (5) (all 30 mg/kg) or the PI3K inhibitor LY294002
(15 mg/kg) or dexamethasone (2 mg/kg) was administered to the animals intraperitoneally 2 h prior to carrageenan (1.5% in saline) injection into
the paw, and saline was injected into the contralateral paw to serve as a control. Six hours after carrageenan exposure the animals were sacrificed and
the paw tissue was extracted. The expression of IL6 and MCP1 in the tissue was measured with ELISA. Values are mean + SEM, n = 6 (stilbenoids)
or 7 (dexamethasone and LY294002), *p < 0.05 and **p < 0.01.
because both of the tested compounds were O-beta-D-glycoside
derivatives of the stilbenoids in question.
piceatannol (5) has hydroxy groups in positions R1, R3, R4,
and R5, which may contribute to their anti-inflammatory activity.
The glycosylated stilbenoids remained ineffective possibly because
they are not readily transported into the cells, which is necessary
for their effects on the production of inflammatory mediators.
The production of IL6, MCP1, and nitric oxide is activated
by various inflammatory stimuli, and also different transcription
factors are documented to be involved. The cellular effects of
PI3K are occasionally reported to be mediated via the NF-κB
transcription factor, and the three measured inflammatory medi-
ators are also reported to be NF-κB responsive.36−39 Our findings
document that inhibition of the PI3K/Akt pathway with
LY294002 down-regulates the expression of these inflammatory
mediators, which in turn suggests that these inflammatory
Nitric oxide produced by inducible nitric oxide synthase
(iNOS) as well as the cytokines IL6 and MCP1 are well-known
inflammatory mediators. Piceatannol (compound 5) was the
most potent of the tested stilbenoids in decreasing nitric oxide
production, while rhapontigenin (compound 6) was the least
potent of the nonglycosylated stilbenoids; the potency of the
other nonglycosylated stilbenoids was between those two. The
glycosylated stilbenoids astringin (compound 7) and rhaponticin
(compound 8) did not have any effect. A very similar pattern of
activity was also seen in their inhibitory effects on Akt phos-
phorylation and IL6 and MCP1 production. Rhapontigenin (6)
is the only aglycone that has a methyl group at position C4, while
I
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Commercially available reagents were used without further
purification: all solvents were anhydrous and HPLC grade, purchased
from Sigma-Aldrich (St. Gallen, Switzerland). All reactions were
performed in oven-dried glassware under an inert atmosphere of dry
argon. Thin-layer chromatography (TLC) was performed on E. Merck
silica gel 60 aluminum packed plates, visualization was accomplished by
UV illumination, and staining was done with H2SO4 (5% v/v) in MeOH.
1H NMR spectra were recorded on a Varian Mercury-VX 300 MHz
with chemical shifts reported as parts per million (in acetone-d6 at 23 °C,
solvent peak at 2.05 ppm as an internal standard). 13C NMR spectra
were obtained on a Varian Mercury-VX 75 MHz spectrometer with
chemical shifts reported as parts per million (acetone-d6 at 23 °C,
solvent peaks at 29.84, and 206.26 ppm as an internal standard).
Synthesis of Pinosylvin Monoethers. General Procedure.
A mixture of pinosylvin (0.050 g, 0.24 mmol), potassium carbonate
(0.050 g, 0.36 mmol), and alkyl or allyl bromide (0.36 mmol) in
acetone (3−5 mL) was refluxed under argon for 24 h. The reaction
mixture was poured into water and extracted with diethyl ether. The
organic layer was washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The crude products were purified with a
silica column eluting with 10−30% hexanes/EtOAc to give mono-
ethers in 8−28% yields.
mediators are PI3K/Akt responsive. It was also found that the
natural stilbenoid compounds investigated in the present study
down-regulated the expression of these inflammatory media-
tors. When these data are considered alongside our findings
that the stilbenoids also inhibited PI3K/Akt activation, it is
plausible that the down-regulated inflammatory response is
mediated via PI3K inhibition.
Carrageenan-induced paw edema is a well-characterized in vivo
model of acute inflammation. It has been widely reported that
the inflammatory factors mediating carrageenan-induced edema
include IL6, MCP1, and NO.40−42 The PI3K/Akt pathway is
also considered to be one of the signaling pathways involved in
acute inflammation such as the inflammatory edema caused by
exposure to carrageenan. We and others have earlier documented
that inhibition of the PI3K/Akt pathway leads to reduced edema
and decreased inflammatory cell migration upon carrageenan
irritation.21,24 These data support the idea that PI3K/Akt
signaling is involved in mediating the formation of the inflam-
matory response induced by exposure to carrageenan. In the
present study, we found that the investigated natural stilbenoids
also reduced the carrageenan-induced edema and suppressed
the expression of the inflammatory cytokines IL6 and MCP1 in
the inflamed paw tissue. According to our knowledge, this is a
novel in vivo effect for pterostilbene (4) and piceatannol (5)
that has not been previously reported. The effect appears to be
comparable to the effect obtained with the commercial PI3K
inhibitor LY294002. This in turn suggests that the investigated
stilbenoids would have anti-inflammatory properties in vivo and
that the mechanism of the effect possibly includes an inhibitory
interaction with the PI3K/Akt pathway.
In this study we document our findings regarding several
naturally occurring stilbenoid compounds, i.e., pinosylvin, mono-
methylpinosylvin, resveratrol, pterostilbene, piceatannol, and
rhapontigenin, which are all known to be included in a normal
healthy human diet. It was found that these stilbenoids have
anti-inflammatory properties when investigated in vitro and
in vivo along with their inhibitory action on the PI3K/Akt
signaling pathway. The effects were comparable to the effects
obtained with the known PI3K/Akt inhibitor LY294002.
Additionally, we found that the tested glycoside derivatives of
these stilbenoids did not have a significant effect in the inflam-
matory models. Based on these findings it appears that the nat-
urally occurring stilbenoids included in human nutrition have
anti-inflammatory properties, and it also appears that these
effects could possibly be mediated via the PI3K/Akt pathway.
This may be a significant finding that could be exploited to
alleviate inflammatory conditions by a stilbenoids-containing die-
tary intervention, but further studies are needed to understand the
mechanisms of action and to prove bioavailability and actual
clinical efficacy in humans.
Pinosylvin monoethyl ether (9): off-white crystals (10.7 mg, 29%);
1
Rf = 0.76 (n-hexane/EtOAc, 1:1); H NMR (300 MHz, acetone-d6)
δ 8.32 (s, 1H), 7.58 (m, 2H), 7.27 (m, 1H), 7.36 (m, 2H), 7.15 (d, J =
4.5 Hz, 2H), 6.68 (m, 2H), 6.35 (t, J = 2.2 Hz, 1H), 4.04 (q, J =
7 Hz, 2H), 1.35 (t, J = 7 Hz, 3H).
Pinosylvin monopropyl ether (10): starting from 69 mg (0.33 mmol)
of pinosylvin as well as using 44 μL (0.49 mmol, 1.5 equiv) of
1-bromopropane and 0.22 g of K2CO3 (1.6 mmol): white crystals
(9.8 mg, 12%); Rf = 0.70 (n-hexane/EtOAc, 1:1); 1H NMR
(300 MHz, acetone-d6) δ 8.31 (s, 1H), 7.59 (m, 2H), 7.36 (m, 2H),
7.25 (m, 1H), 7.15 (d, J = 4.2 Hz, 2H), 6.68 (m, 2H), 6.36 (t, J =
2.2 Hz, 1H), 3.95 (t, J = 6.5 Hz 2H), 1.78 (m, 2H), 1.03 (t, J =
7.4 Hz, 3H); 13C NMR (75 MHz, acetone-d6) δ 161.0, 158.9, 139.7,
137.7, 129.0, 128.8, 128.8, 127.8, 127.7, 126.7, 106.3, 104.3, 101.7,
69.3, 22.6, 10.1.
Pinosylvin monoallyl ether (11): white crystals (17 mg, 28%); Rf =
0.82 (n-hexane/EtOAc, 1:1); 1H NMR (300 MHz, acetone-d6) δ 8.34
(s, 1H), 7.58 (m, 2H), 7.37 (m, 2H), 7.25 (m, 1H), 7.15 (d, J =
4.7 Hz, 2H), 6.70 (m, 2H), 6.39 (t, J = 2.2 Hz, 1H), 6.06 (m, 1H),
5.43 (dq, J = 17.3, 1.5 Hz, 1H), 5.25 (dq, J = 10.6, 1.5 Hz, 1H), 4.57
(dt, J = 5.2, 1.6 Hz, 3H); 13C NMR (75 MHz, acetone-d6) δ 160.5,
158.9, 139.7, 137.7, 134.2, 129.0, 128.9, 128.8, 127.8, 126.7, 116.6,
106.6, 104.5, 102.0, 68.6.
Pinosylvin monoisobutyl ether (12): white crystals (4.8 mg, 7.5%);
1
Rf = 0.77 (n-hexane/EtOAc, 1:1); H NMR (300 MHz, acetone-d6)
δ 8.29 (s, 1H), 7.58 (m, 2H), 7.37 (m, 2H), 7.25 (m, 1H), 7.15 (d, J =
4.5 Hz, 2H), 6.69 (m, 2H), 6.37 (t, J = 2.2 Hz, 1H), 3.77 (d, J =
6.5 Hz, 2H), 2.06 (m, 1H), 1.03 (d, J = 6.5 Hz, 6H); 13C NMR
(75 MHz, acetone-d6) δ 161.1, 158.9, 139.7, 137.7, 129.0, 128.8, 128.8,
127.7, 126.7, 106.3, 104.4, 101.8, 74.3, 18.8.
Pinosylvin monobenzyl ether (13): white crystals (13 mg, 18%);
1
Rf = 0.75 (n-hexane/EtOAc, 1:1); H NMR (300 MHz, acetone-d6)
δ 8.37 (s, 1H), 7.62−7.54 (m, 2H), 7.53−7.47 (m, 2H), 7.45−7.31
(m, 5H), 7.30−7.19 (m, 1H), 7.16 (d, J = 4.7 Hz, 2H), 6.82 (dd, J =
7.2, 5.5 Hz, 1H), 6.76−6.65 (m, 1H), 6.45 (t, J = 2.2 Hz, 1H), 5.22−
5.03 (m, 2H); 13C NMR (75 MHz, acetone-d6) δ 161.3, 159.6, 144.6,
140.5, 138.5, 138.3, 132.1, 129.6, 129.5, 129.3, 128.6, 128.4, 127.4,
124.8, 107.4, 105.3, 102.8, 70.5.
Anti-Inflammatory Testing. Cell Culture. Murine J774 macro-
phages (American Type Culture Collection, Manassas, VA, USA) were
cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified
Eagle’s medium with Ultraglutamine 1 (Sigma-Aldrich, MO, USA)
supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL
penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B
(all from Invitrogen, Paisley, UK). Cells were cultured on 24-well plates
for Western blot, ELISA, and nitrite measurements and on
96-well plates for cytotoxicity testing. Cells were grown for 72 h to
confluence prior to the experiments.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The reagents used in the
study were obtained as follows: pAkt S473 and Akt antibodies from
Cell Signaling Technology, Danvers, MA, USA; β-actin antibodies and
HRP-conjugated goat polyclonal anti-rabbit IgG antibodies from Santa
Cruz Biotechnology, Santa Cruz, CA, USA; PI3K inhibitor LY294002
from Merck Millipore, Billerica, MA, USA; pinosylvin, monomethylpi-
nosylvin, and resveratrol from Sequoia Research Products, Pan-
gbourne, UK; pterostilbene, piceatannol, and dexamethasone from
Orion Pharma, Finland; rhapontigenin from Cayman Chemical, MI,
USA; astringin from Polyphenols Laboratories, Norway; and
rhaponticin from Sigma-Aldrich, MO, USA.
J
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Cytotoxicity of tested compounds was ruled out by measuring cell
viability using Cell Proliferation Kit II (Roche Diagnostics, Mannheim,
Germany).
AUTHOR INFORMATION
Corresponding Author
*E-mail: eeva.moilanen@uta.fi.
■
Nitrite Assays. The effects of the tested compounds on cellular NO
production was investigated by measuring the accumulation of nitrite
ORCID
−
(NO2 , a stable metabolite of NO) in the cell culture medium with the
Heikki Erasalo: 0000-0003-3333-8094
Jari Yli-Kauhaluoma: 0000-0003-0370-7653
Notes
̈
Griess test.43
IL6 and MCP1 ELISA. IL6 and MCP1 concentrations in the cell
culture medium and paw tissue extracts were measured by enzyme
linked immunosorbent assay (ELISA) using reagents from R&D
Systems Europe Ltd. (Abingdon, UK).
The authors declare no competing financial interest.
Western Blotting. At indicated time points, cells were rapidly
washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) and
lysed with a buffer containing 10 mM Tris-base (pH 7.4), 5 mM
EDTA, 50 mM NaCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl
fluoride, 1 mM sodium orthovanadate, 20 μg/mL leupeptin, 50 μg/mL
aprotinin, 5 mM NaF, 2 mM sodium pyrophosphate, and 10 μM
n-octyl-β-D-glucopyranoside. After incubation for 15 min on ice, cell
lysates were centrifuged (13000g, 4 °C, 10 min), and supernatants
were collected and stored in −20 °C in SDS sample buffer containing
0.5 M Tris (pH 6.8), glycerol (10% v/v), SDS (2% v/v), bromophenol
blue (0.25‰ v/v), and 2-mercaptoethanol (5% v/v). An aliquot of the
supernatant was used to determine protein concentration with the
Bradford protein assay.44
Protein samples (20 μg of total protein from lysates) were analyzed
according to standard Western blotting protocol as described pre-
viously.45 The membrane was incubated with the primary antibody in
the blocking solution at 4 °C overnight and with the secondary
antibody in the blocking solution for 1 h at room temperature. Bound
antibody was detected using Super Signal West Pico or Dura chem-
iluminescent substrate (Pierce, Rockford, IL, USA) and an Image
Quant LAS 4000 imaging system (GE Healthcare Bio-Sciences,
Buckinghamshire UK). The quantitation of the chemiluminescent
signal was carried out with the use of Image Quant TL software
(GE Healthcare).
Carrageenan-Induced Paw Edema in Mice. The in vivo anti-
inflammatory effects of PI3K inhibitor LY294002 and different stil-
benoids were studied by measuring carrageenan-induced paw edema in
male C57BL/6 mice. The study was carried out in accordance with the
legislation for the protection of animals used for scientific purposes
(directive 2010/63/EU) and approved by the National Animal
Experiment Board (approval number ESAVI/5019/04.10.03/2012,
granted September 3, 2012). Paw edema was induced under anes-
thesia, and all efforts were made to minimize suffering.
Animals were housed under conditions of optimum light, tem-
perature, and humidity (12:12 h light:dark cycle, 22 1 °C, 50−60%
humidity) with food and water provided ad libitum. One hour prior to
carrageenan injection the investigated compounds were delivered i.p.
dissolved in a vehicle containing 10% DMSO in PBS (pH 7.4). The
mice were anesthetised by intraperitoneal injection of 0.5 mg/kg
of medetomide (Domitor 1 mg/mL, Orion Oyj, Espoo, Finland) and
75 mg/kg of ketamine (Ketalar 10 mg/mL, Pfizer Oy Animal Health,
Helsinki, Finland), and thereafter the mice received a 50 μL intra-
dermal injection in one hindpaw of normal saline containing λ-carrageenan
(1.5% w/v). The contralateral paw received 50 μL of saline only, and
it was used as a control. Edema was measured before and 3 and
6 h after carrageenan injection with a plethysmometer (Ugo Basile,
Comerio, Italy).
ACKNOWLEDGMENTS
■
This study was funded by the Finnish Bioeconomy Cluster
(FIBIC Ltd), the Finnish Funding Agency for Technology and
Innovation (TEKES), and the Medical Research Fund of
Tampere University Hospital. We wish to thank M. Kukkonen,
S. Hietakangas, and T. Salonen for excellent technical assistance
and H. Maatta for skillful secretarial help.
̈
̈
̈
REFERENCES
■
(1) Jeandet, P.; Delaunois, B.; Conreux, A.; Donnez, D.; Nuzzo, V.;
Cordelier, S.; Clement, C.; Courot, E. Biofactors 2010, 36, 331−341.
(2) Chiron, H.; Drouet, A.; Lieutier, F.; Payer, H. D.; Ernst, D.;
Sanderman, H. Plant Physiol. 2000, 124, 865−872.
(3) Keylor, M. H.; Matsuura, B. S.; Stephenson, C. R. J. Chem. Rev.
2015, 115, 8976−9027.
(4) Catalgol, B.; Batirel, S.; Taga, Y.; Ozer, N. K. Front. Pharmacol.
2012, 3, 141.
(5) Patel, K. R.; Scott, E.; Brown, V. A.; Gescher, A. J.; Steward, W.
P.; Brown, K. Ann. N. Y. Acad. Sci. 2011, 1215, 161−169.
(6) Piotrowska, H.; Kucinska, M.; Murias, M. Mutat. Res., Rev. Mutat.
Res. 2012, 750, 60−82.
(7) McCormack, D.; McFadden, D. J. J. Surg. Res. 2012, 173, e53−61.
(8) Misiti, F.; Sampaolese, B.; Mezzogori, D.; Orsini, F.; Pezzotti, M.;
Giardina, B.; Clementi, M. E. Brain Res. Bull. 2006, 71, 29−36.
(9) Matsui, Y.; Sugiyama, K.; Kamei, M.; Takahashi, T.; Suzuki, T.;
Katagata, Y.; Ito, Y. J. Agric. Food Chem. 2010, 58, 11112−11118.
(10) Rimando, A. M.; Kalt, W.; Magee, J. B.; Dewey, J.; Ballington, J.
R. J. J. Agric. Food Chem. 2004, 52, 4713−4719.
(11) Holmbom, B.; Willfoer, S.; Hemming, J.; Pietarinen, S.; Nisula,
L.; Eklund, P.; Sjoeholm, R. Materials, Chemicals, and Energy from
Forest Biomass; ACS Symposium Series 954; American Chemical
Society: Washington, DC, 2007; pp 350−36210.1021/bk-2007-
0954.ch022.
(12) Lee, S. K.; Lee, H. J.; Min, H. Y.; Park, E. J.; Lee, K. M.; Ahn, Y.
H.; Cho, Y. J.; Pyee, J. H. Fitoterapia 2005, 76, 258−260.
(13) Roupe, K. A.; Remsberg, C. M.; Yanez, J. A.; Davies, N. M. Curr.
Clin. Pharmacol. 2006, 1, 81−101.
(14) Ko, Y. J.; Kim, H. H.; Kim, E. J.; Katakura, Y.; Lee, W. S.; Kim,
G. S.; Ryu, C. H. Int. J. Mol. Med. 2013, 31, 951−958.
(15) Matsuda, H.; Kageura, T.; Morikawa, T.; Toguchida, I.; Harima,
S.; Yoshikawa, M. Bioorg. Med. Chem. Lett. 2000, 10, 323−327.
(16) Cheong, H.; Ryu, S. Y.; Kim, K. M. Planta Med. 1999, 65, 266−
268.
(17) Hawkins, P. T.; Stephens, L. R. Biochim. Biophys. Acta, Mol. Cell
Biol. Lipids 2015, 1851, 882−897.
(18) Fruman, D. A.; Rommel, C. Nat. Rev. Drug Discovery 2014, 13,
140−156.
(19) Yang, Z. Z.; Tschopp, O.; Baudry, A.; Dummler, B.; Hynx, D.;
Hemmings, B. A. Biochem. Soc. Trans. 2004, 32, 350−354.
(20) Hers, I.; Vincent, E. E.; Tavare, J. M. Cell. Signalling 2011, 23,
1515−1527.
(21) Di Lorenzo, A.; Fernandez-Hernando, C.; Cirino, G.; Sessa, W.
C. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 14552−14557.
(22) Rommel, C.; Camps, M.; Ji, H. Nat. Rev. Immunol. 2007, 7,
191−201.
(23) Khan, S.; Shehzad, O.; Jin, H. G.; Woo, E. R.; Kang, S. S.; Baek,
S. W.; Kim, J.; Kim, Y. S. J. J. Nat. Prod. 2012, 75, 67−71.
At 6 h the mice were sacrificed, and the paw tissue injected with
carrageenan and the contralateral tissue injected with the vehicle were
collected for analysis into a buffer containing Tris (50 mM, pH 7.4),
NaCl (150 mM), and protease and phosphatase inhibitors phenyl-
methylsulfonyl fluoride (0.5 mM), sodium orthovanadate (2 mM),
leupeptin (0.10 μg/mL), aprotinin (0.25 μg/mL), and NaF (1.25 mM).
The tissue was minced and centrifuged (10 min, 10000g), and the
supernatant was collected for ELISA measurements.
Statistics. Results are expressed as mean + standard error of mean
(SEM). Statistical significance of the results was calculated by one-way
ANOVA with Dunnett’s or Bonferroni’s post-test. Differences were
considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.
K
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(24) Erasalo, H.; Laavola, M.; Hamalainen, M.; Leppanen, T.;
Nieminen, R.; Moilanen, E. Basic Clin. Pharmacol. Toxicol. 2015, 116,
53−61.
(25) Ali, M. A.; Kondo, K.; Tsuda, Y. Chem. Pharm. Bull. 1992, 40,
1130−1136.
(26) Ferrieres, J. Br. Heart J. 2004, 90, 107−111.
(27) Park, E. J.; Pezzuto, J. M. Biochim. Biophys. Acta, Mol. Basis Dis.
2015, 1852, 1071−1113.
(28) Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.;
Bilanges, B. Nat. Rev. Mol. Cell Biol. 2010, 11, 329−341.
(29) Minihane, A. M.; Vinoy, S.; Russell, W. R.; Baka, A.; Roche, H.
M.; Tuohy, K. M.; Teeling, J. L.; Blaak, E. E.; Fenech, M.; Vauzour, D.;
McArdle, H. J.; Kremer, B. H.; Sterkman, L.; Vafeiadou, K.; Benedetti,
M. M.; Williams, C. M.; Calder, P. C. Br. J. Nutr. 2015, 114, 999−
1012.
(30) Fain, J. N. Vitam. Horm. 2006, 74, 443−477.
(31) Calder, P. C.; Ahluwalia, N.; Brouns, F.; Buetler, T.; Clement,
K.; Cunningham, K.; Esposito, K.; Jonsson, L. S.; Kolb, H.; Lansink,
M.; Marcos, A.; Margioris, A.; Matusheski, N.; Nordmann, H.;
O’Brien, J.; Pugliese, G.; Rizkalla, S.; Schalkwijk, C.; Tuomilehto, J.;
Warnberg, J.; Watzl, B.; Winklhofer-Roob, B. M. Br. J. Nutr.. 2011,
106, Suppl 3, S5−78.10.1017/S0007114511005460.
(32) Wymann, M. P.; Solinas, G. Ann. N. Y. Acad. Sci. 2013, 1280,
44−47.
(33) Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. J. Biol.
Chem. 1994, 269, 5241−5248.
(34) Kim, J. S.; Kang, C. G.; Kim, S. H.; Lee, E. O. J. J. Nat. Prod.
2014, 77, 1135−1139.
(35) Pan, M. H.; Chiou, Y. S.; Chen, W. J.; Wang, J. M.; Badmaev, V.;
Ho, C. T. Carcinogenesis 2009, 30, 1234−1242.
(36) Aktan, F. Life Sci. 2004, 75, 639−653.
(37) Collart, M. A.; Baeuerle, P.; Vassalli, P. Mol. Cell. Biol. 1990, 10,
1498−1506.
(38) Hershko, D. D.; Robb, B. W.; Luo, G.; Hasselgren, P. O. Am. J.
Physiol. Regul. Integr. Comp. Physiol 2002, 283, R1140−8.
(39) Hildebrand, D. G.; Alexander, E.; Horber, S.; Lehle, S.;
Obermayer, K.; Munck, N. A.; Rothfuss, O.; Frick, J. S.; Morimatsu,
̈
M.; Schmitz, I.; Roth, J.; Ehrchen, J. M.; Essmann, F.; Schulze-Osthoff,
K. J. Immunol. 2013, 190, 4812−4820.
(40) Fulgenzi, A.; Dell’Antonio, G.; Foglieni, C.; Dal Cin, E.; Ticozzi,
P.; Franzone, J. S.; Ferrero, M. E. BMC Immunol. 2005, 6, 18.
(41) Loram, L. C.; Fuller, A.; Fick, L. G.; Cartmell, T.; Poole, S.;
Mitchell, D. J. J. Pain 2007, 8, 127−136.
(42) Salvemini, D.; Wang, Z. Q.; Wyatt, P. S.; Bourdon, D. M.;
Marino, M. H.; Manning, P. T.; Currie, M. G. Br. J. Pharmacol. 1996,
118, 829−838.
(43) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.;
Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131−138.
(44) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254.
(45) Leppanen, T.; Korhonen, R.; Laavola, M.; Nieminen, R.;
̈
Tuominen, R. K.; Moilanen, E. PLoS One 2013, 8, e52741.
L
DOI: 10.1021/acs.jnatprod.7b00384
J. Nat. Prod. XXXX, XXX, XXX−XXX