6-Shogaol, a ginger product, modulates neuroinflammation: a new approach to neuroprotection.
ABSTRACT Inflammatory processes in the central nervous system play an important role in a number of neurodegenerative diseases mediated by microglial activation, which results in neuronal cell death. Microglia act in immune surveillance and host defense while resting. When activated, they can be deleterious to neurons, even resulting in neurodegeneration. Therefore, the inhibition of microglial activation is considered a useful strategy in searching for neuroprotective agents. In this study, we investigated the effects of 6-shogaol, a pungent agent from Zingiber officinale Roscoe, on microglia activation in BV-2 and primary microglial cell cultures. 6-Shogaol significantly inhibited the release of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). The effect was better than that of 6-gingerol, wogonin, or N-monomethyl-l-arginine, agents previously reported to inhibit nitric oxide. 6-Shogaol exerted its anti-inflammatory effects by inhibiting the production of prostaglandin E(2) (PGE(2)) and proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and by downregulating cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) expression. In addition, 6-shogaol suppressed the microglial activation induced by LPS both in primary cortical neuron-glia culture and in an in vivo neuroinflammatory model. Moreover, 6-shogaol showed significant neuroprotective effects in vivo in transient global ischemia via the inhibition of microglia. These results suggest that 6-shogaol is an effective therapeutic agent for treating neurodegenerative diseases.
- [show abstract] [hide abstract]
ABSTRACT: 6-Shogaol has been shown to possess many antitumor properties including inhibition of cancer cell growth, inhibition of cancer metastasis, induction of apoptosis in cancer cells and induction of cancer cell differentiation. Despite its prominent antitumor effects, the direct molecular target of 6-shogaol has remained elusive. To identify the direct targets of 6-shogaol, a comprehensive antitumor profile of 6-shogaol (NSC752389) was tested in the NCI-60 cell line in an in vitro screen. The results show that 6-shogaol is COMPARE negative suggesting that it functions via a mechanism of action distinct from existing classes of therapeutic agents. Further analysis using microarray gene profiling and Connectivity Map analysis showed that MCF-7 cells treated with 6-shogaol display gene expression signatures characteristic of peroxisome proliferator activated receptor γ (PPARγ) agonists, suggesting that 6-shogaol may activate the PPARγ signaling pathway for its antitumor effects. Indeed, treatment of MCF-7 and HT29 cells with 6-shogaol induced PPARγ transcriptional activity, suppressed NFκB activity, and induced apoptosis in breast and colon cancer cells in a PPARγ-dependent manner. Furthermore, 6-shogaol is capable of binding to PPARγ with a binding affinity comparable to 15-delta prostaglandin J2, a natural ligand for PPARγ. Together, our findings suggest that the antitumor effects of 6-shogaol are mediated through activation of PPARγ and imply that activation of PPARγ might be beneficial for breast and colon cancer treatment.Cancer letters 04/2013; · 4.86 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells. TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with a TRPV1 agonist capsaicin (CAP), a TRPA1 agonist allyl-isothiocyanate (AITC), or TU-100 and jejunum IBF was evaluated using a laser Doppler blood flowmetry. All 3 compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1-antagonist but not by a TRPV1-antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient -shogaol (6SG) increased ADM release dose-dependently and the effects were abrogated by TRPA1-antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.AJP Gastrointestinal and Liver Physiology 12/2012; · 3.65 Impact Factor
6-Shogaol, a ginger product, modulates neuroinflammation: A new approach
Sang Keun Haa,b, Eunjung Moona, Mi Sun Juc, Dong Hyun Kimc,d, Jong Hoon Ryuc,d,
Myung Sook Ohc,d, Sun Yeou Kima,*
aGraduate School of East-West Medical Science, Kyung Hee University Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyeonggi-do 446-701,
Republic of Korea
bKorea Food Research Institute, 516 Baekhyun-dong, Bundang-gu, Sungnam, Gyeonggi-do 463-746, Republic of Korea
cDepartment of Oriental Pharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University,
#1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
dDepartment of Life and Nanopharmaceutical Sciences, Kyung Hee University, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
Received 7 July 2011
Received in revised form
13 February 2012
Accepted 15 March 2012
a b s t r a c t
Inflammatory processes in the central nervous system play an important role in a number of neurode-
generative diseases mediated by microglial activation, which results in neuronal cell death. Microglia act
in immune surveillance and host defense while resting. When activated, they can be deleterious to
neurons, even resulting in neurodegeneration. Therefore, the inhibition of microglial activation is
considered a useful strategy in searching for neuroprotective agents. In this study, we investigated the
effects of 6-shogaol, a pungent agent from Zingiber officinale Roscoe, on microglia activation in BV-2 and
primary microglial cell cultures. 6-Shogaol significantly inhibited the release of nitric oxide (NO) and the
expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). The effect was
better than that of 6-gingerol, wogonin, or N-monomethyl-L-arginine, agents previously reported to
inhibit nitric oxide. 6-Shogaol exerted its anti-inflammatory effects by inhibiting the production of
prostaglandin E2 (PGE2) and proinflammatory cytokines, such as interleukin-1b (IL-1b) and tumor
necrosis factor-a (TNF-a), and by downregulating cyclooxygenase-2 (COX-2), p38 mitogen-activated
protein kinase (MAPK), and nuclear factor kappa B (NF-kB) expression. In addition, 6-shogaol sup-
pressed the microglial activation induced by LPS both in primary cortical neuron-glia culture and in an
in vivo neuroinflammatory model. Moreover, 6-shogaol showed significant neuroprotective effects in vivo
in transient global ischemia via the inhibition of microglia. These results suggest that 6-shogaol is an
effective therapeutic agent for treating neurodegenerative diseases.
? 2012 Elsevier Ltd. All rights reserved.
Inflammation in the brain is closely associated with the path-
ogenesis of numerous neurodegenerative diseases, including Par-
kinson’s disease (PD), Alzheimer’s disease, and cerebral ischemia
(Kim and Joh, 2006; McGeer and McGeer, 1995; Stoll et al., 1998).
Neuroinflammation results primarily from the activation of
astrocytes and microglia, which are the resident immune cells of
the brain. Under normal conditions, microglia act in immune
surveillance, and astrocytes serve to maintain neuron survival by
secreting nerve growth factors and buffering the actions of
neurotransmitters (Aloisi, 1999; Kreutzberg, 1996). Activated
microglia secrete a variety of proinflammatory and cytotoxic
factors, such as nitric oxide (NO), tumor necrosis factor-a (TNF-a),
interleukin-1b (IL-1b), arachidonic acid, eicosanoids, and reactive
oxygen species (McGeer and McGeer, 1995; Minghetti and Levi,
1998). The accumulation of proinflammatory and cytotoxic
factors is deleterious to neurons in vitro, and these factors are
thought to participate actively in the progression of neurodegen-
erative diseases in vivo (Banati et al., 1993; Boje and Arora, 1992;
Bronstein et al., 1995; Jeohn et al., 1998; Kreutzberg, 1996; Raine,
1994). Therefore, the inhibition of proinflammatory mediators
secreted from activated microglia would be an effective thera-
peutic approach to regulate the progression of neurodegenerative
* Corresponding author. College of Pharmacy, Gachon University, #534-2 Yeonsu-
dong, Yeonsu-gu, Incheon 406-799, Republic of Korea. Tel.: þ82 32 820 4821; fax:
þ82 32 899 6591.
E-mail address: email@example.com (S.Y. Kim).
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ e see front matter ? 2012 Elsevier Ltd. All rights reserved.
Neuropharmacology 63 (2012) 211e223
Lipopolysaccharide (LPS), a major component of the outer
membrane of Gram-negative bacteria, is a potent activator of
microglia. LPS induces intracellular signaling pathways involving
nuclear factor kappa B (NF-kB) and mitogen-activated protein
kinases (MAPKs), such as p38, extracellular signal-regulated kinase
(ERK), and c-Jun N-terminal kinase (JNK). Both the NF-kB and MAPK
pathways are capable of regulating the expression of numerous
immune response genes, including proinflammatory cytokines and
chemokines (Hanada and Yoshimura, 2002).
Ginger, the rhizome of the plant Zingiber officinale Roscoe in the
family Zingiberaceae, has long been used widely as a spice for
cooking and as a medicinal herb in traditional herbal medicine.
Ginger is reported to have antioxidative, anti-inflammatory, anti-
microbial, and anticarcinogenic properties (Ali et al., 2008; Shukla
and Singh, 2007). Ginger contains 1.0e3.0% volatile oils and
a numberof pungent compounds (Chrubasik et al., 2005). Gingerols
are the most abundant pungent compounds in fresh roots, and
ginger contains several gingerols of various chain lengths (n6 to
n10), with 6-gingerol being the most abundant. Shogaols, the
dehydrated form of gingerols, are found in only small quantities in
the fresh root and mainly in dried and thermally treated roots, with
6-shogaol being the most abundant (Jolad et al., 2004). 6-Gingerol
and 6-shogaol have a number of pharmacological activities,
including anti-inflammatory, antipyretic, analgesic, antitussive, and
hypotensive effects (Pan et al., 2008; Suekawa et al., 1984).
Although ginger is commonly used in foods and folk medicines and
various activities of its constituents have been revealed, it is still not
known whether the neuroprotective effects of it are derived from
the inhibition of microglia or not.
This study evaluated the anti-inflammatory effects of 6-shogaol
in primary microglia cells and in an in vivo systemic inflammatory
model induced by LPS. Based on the anti-inflammatory activities of
6-shogaol in vitro and in vivo, we investigated the effects of 6-
a primary cortical neuron-glia culture, in a transient global
2. Materials and methods
Dulbecco’s modified Eagle medium (DMEM), minimum essential medium
(MEM), MEM-a, fetal bovine serum (FBS), horse serum (HS), and penicillin-
streptomycin (PS) were purchased from Invitrogen (Carlsbad, CA, USA). LPS, N-
monomethyl-L-arginine (NMMA), sodium
polycytidylic acid [poly(I:C)] were obtained from Sigma Chemical Company (St.
Louis, MO, USA). A synthetic lipopeptide (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-
PamCSK) was obtained from Calbiochem (San Diego, CA, USA). 6-Gingerol and 6-
shogaol were purchased from Wako Pure Chemical (Osaka, Japan).
Animal maintenance and treatment were carried out in accordance with the
Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and
the Animal Care and Use guidelines of Kyung Hee University, Seoul, Korea. Sprague-
Dawley (SD) rats were purchased from Orient Bio (Kyunggido, Korea). Male C57BL/6
mice (7 weeks) were purchased from the Dae Han Biolink Company (Eumseong,
Korea). Animals were housed 5 or 6 per cage at an ambient temperature of 23 ?1?C
and a relative humidity of 60 ? 10% under a 12 h light/dark cycle, with free access to
water and food.
2.3. Cell culture
The BV-2 mouse microglial cell line, originally developed by Dr. V. Bocchini at
the University of Perugia (Perugia, Italy), has both the phenotypic and functional
properties of reactive microglia cells (Blasi et al., 1990). BV-2 cells were generously
provided by Dr. E. Choi at Korea University (Seoul, Korea). BV-2 cells were main-
tained in 10 ml of DMEM supplemented with 5% FBS and 1% PS. Primary microglia
cells were cultured from the cerebral cortices of neonatal SD rats (1-day-old) which
were purchased from Orient Bio (Kyunggido, Korea). Cortices were triturated into
single cells in MEM-a containing 10% FBS and plated into a 75 cm2T-flask for 2
weeks. The microglia were detached by mild shaking and applied to a nylon mesh
(70 mm, Spectrum, California, USA) to remove cell clumps. The purity of the
microglial cultures was over 95%, as judged by immunostaining with anti-OX-42
antibody (Chemicon, Temecula, CA).
2.4. Measurement of NO production and cell viability
Nitrite, a soluble oxidation product of NO, was measured in the culture media
using the Griess reaction. The supernatant (50 ml) was harvested and mixed with an
equal volume of Griess reagent (1% sulfanilamine, 0.1% naphthylethylene diamine
dihydrochloride, 2% phosphoric acid). After 10 min, the absorbance at 540 nm was
measured using a microplate reader. Sodium nitrite was used as a standard to
calculate the NO2
dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay.
?concentration. Cell viability was assessed by a 3-[4, 5-
2.5. NO radical scavenging assay
NO generated fromsodium nitroprusside (SNP) was measured using the method
of Marcocci et al. (1994). Briefly, the reaction mixture (5.0 ml) containing SNP
(5 mM) in phosphate buffered saline (PBS, pH 7.3), with or without 6-shogaol at
different concentrations, was incubated at 25?C for 180 min. The supernatant (50ml)
was harvested and mixed with an equal volume of Griess reagent. The absorbance at
540 nm was measured using a microplate reader.
2.6. iNOS activity assay
iNOS activity assay was performed using the method of Chen et al. (2001). BV-2
cells were cultured in a 100-mm plate and activatedwith LPS (1 mg/ml) for 12 h. Cells
were collected and washed twice with PBS to remove LPS. Cells were plated at
a concentration of 5 ?104cells/ml into 96-well plates, and indicated 6-shogaol was
added. NMMA, a classical inhibitor of iNOS enzyme activity, was used as a positive
control. After 12 h, the amount of nitrite was measured by the Griess reaction as
2.7. Measurement of prostaglandin E2(PGE2), IL-1b and TNF-a
Media was collected and centrifuged 24 h after treatment with LPS (100 ng/ml)
in the presence or absence of 6-shogaol. PGE2, IL-1b and TNF-a were measured by
a competitive enzyme immunoassay kit (PGE2,Cayman Chemical, Ann Arbor, MI,
USA), specific ELISA kit (IL-1b and TNF-a, R&D Systems, Minneapolis, MN) according
to the manufacturer’s protocol.
2.8. NF-kB assay
Nuclear extracts from treated microglia were prepared using the Nuclear Extract
Kit (Active Motif, Carlsbad, CA). NF-kB activity was measured by a NF-kB p65 assay
kit (Active Motif) according to the manufacturer’s protocol.
2.9. Western blot analysis
BV-2 and primary microglia cells were seeded in a 6-well plate and exposed to
LPS (100 ng/ml) in the presence or absence of 6-shogaol for various times. Protein
samples from the cell extracts were separated by 8% or 10% SDS-PAGE and trans-
ferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Buck-
inghamshire, UK). The membrane was blocked with 5% skim milk and incubated
with primary antibodies against iNOS (BD Transduction Laboratories, San Diego, CA,
USA), cyclooxygenase (COX)-2 (Santa Cruz Biotechnology, CA, USA), p38, ERK, JNK,
phospho-p38, phospho-ERK, phospho-JNK, inhibitory kappa B (IkB), and phospho-
IkB (Cell Signaling, Beverly, MA, USA). After washing with TBST, HRP-conjugated
secondary antibodies (goat anti-rabbit IgG, Amersham Pharmacia Biotech; donkey
anti-goat IgG, Santa Cruz Biotechnology) were applied. The blots were developed
using ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech).
Densitometry analysis of bands was performed with the ImageMaster? 2D Elite
software, version 3.1 (Amersham Pharmacia Biotech).
2.10. Cortical neuron-glia culture
Neuron-glia cultures were prepared from the cerebral cortices of embryonic day
16 (E16) SD rats according toa previouslyreported method with slight modifications
(Qin et al., 2002). Briefly, the meninges were removed and dissociated by trituration
in HBSS media. After trypsinization, cells were harvested and seeded at a density of
5 ?105cells in 24-well plates coated with 20mg/ml of poly-D-lysine (Sigma, St. Louis,
MO, USA). The culture medium consisted of MEM supplemented with 10% FBS, 10%
HS, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential
amino acids, and 0.5% PS. Cultures were maintained in a humidified 5% CO2incu-
bator at 37?C. Seven-day-old cultures were used for the experiment. The compo-
sition of cortical neuron-glia cultures was determined by immunostaining with
antibodies against MAP-2, Neu-N, and OX-42. Cortical neuron-glia cultures
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
contained 60% Neu-N-immunoreactive (IR) neurons and 3% OX-42-IR microglia. The
remaining cells were presumed to be astrocytes.
cell bodies and neuritis, and with an antibody against Neu-N, a marker for neuronal
cell bodies. Microglial cells were visualized by staining with an OX-42 antibody.
Briefly, cells were fixed for 20 min in 3.7% paraformaldehyde in phosphate buffered
saline (PBS). After washing twice with PBS, the cultures were incubated with 1%
normal serum followed by incubation overnight at 4?C with the primary antibody.
Then, cells were washed three times for 10 min in PBS. Afterward, biotinylated anti-
for 10 min and incubated in Vectastain ABC reagent (Vector Laboratory, Piscataway,
NJ, USA) for 1 h. The color was developed with 3,30-diaminobenzidine (DAB). Images
were recorded using a Zeiss inverted microscope connected to a digital CCD camera
(Axiocam, Zeiss, Oberko, Germany). For cell counting, nine representative areas per
well were counted under the microscope at ?200 magnification.
2.12. Inflammatory model in mice
6-Shogaol and NMMA (positive control) were dissolved in 10% DMSO and
administered orally at two doses of 5 mg/kg and 20 mg/kg once per day for 3 days
beforeLPStreatment.Threehoursafterthe last drug administration, LPS dissolvedin
normal saline was injected intraperitoneally at a dose of 5 mg/kg. An equal volume
of vehicle was given to the control and LPS groups. Three hours after LPS injection,
the mice were prepared for the histological analysis.
Fig. 1. Effect of 6-shogaol on LPS-induced NO production and iNOS expression in microglia cells. (A) Effect of 6-gingerol and 6-shogaol on LPS-induced NO production in primary
microglia cells. (B) Effects of 6-shogaol, wogonin, and NMMA on LPS-induced NO production in primary microglia cells. (C) Effect of 6-shogaol on cell viability in LPS-stimulated
primary microglia cells. (D) Effect of 6-gingerol and 6-shogaol on LPS-induced iNOS expression in microglia cells. (E) Effect of 6-shogaol on NO scavenging. (F) Effect of 6-shogaol on
iNOS activity in BV-2 cells. (G) Effect of 6-shogaol post-treatment on NO production in LPS-stimulated primary microglia cells. All data are presented as the mean ? S.E.M of three
independent experiments. #p < 0.05 indicates statistically significant difference between the control and LPS alone-treated groups. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate
statistically significant differences compared to treatment with LPS alone.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
2.13. Transient global ischemia by bilateral common carotid arteries occlusion
Mice were anesthetized in a chamber with a mixture of N2O and O2(70:30)
containing 2% isoflurane. Bilateral common carotid arteries occlusion (2VO)
was induced as described elsewhere (Cho et al., 2007), with minor modifica-
tions. Briefly, after making a median incision in the neck skin of a mouse, both
common carotid arteries were exposed and occluded with aneurysm clips for
25 min. Body temperature was maintained at 37 ? 0.5?C throughout surgery
by a heating pad (Biomed S.L., Alicante, Spain). Circulation was restored by
removing the clips. The mice which received the same surgical operation
without clipping of the carotid arteries served as sham-operated controls.
Right after 2VO induction, drug treatments were done once a day for three
consecutive days. 6-Shogaol and NMMA were dissolved in 10% Tween 80
solution and administered orally. Seven days after reperfusion, the mice were
prepared for the histological analysis.
2.14. Preparation brain tissues for the histology
The mice were anesthetized with pentobarbital sodium (60 mg/kg, i.p.) and
perfused transcardially with 0.1 M phosphate buffer (pH 7.4) followed by ice-cold
4% paraformaldehyde. Brains were removed, post-fixed in the same fixative
solution overnight, and then immersed in 30% sucrose solution (in 0.05 M PBS) for
the cryoprotection at 4?C until sectioned. Frozen brains were sectioned coronally
into 30 mm sections on a cryostat (CM3000; Leica, Wetzlar, Germany) and then
stored in storing solution containing glycerine, ethylene glycol, and phosphate
buffer at 4?C.
2.15. Cresyl violet staining and immunohistochemistry
For the cresyl violet staining, brain sections were mounted onto gelatin-coated
slides, stained with 0.5% cresyl violet, dehydrated through graded alcohols (70, 80,
90, and 100% ? 2), placed in xylene, and coverslipped using Histomount medium.
For the immunohistochemistry, free floating sections were rinsed in PBS at room
temperature and pretreated with 1% hydrogen peroxide for 15 min. Then they were
incubated overnight with anti-CD11b antibody (Mac-1), anti-IL-1b and anti-TNF-
a antibody (each 1:1000 dilution, Santa Cruz Biotechnology) at 4?C. Sections were
then incubated for 90 min with biotinylated secondary antibody (1:200 dilution),
treated with avidin-biotin-peroxidase complex (1:100 dilution) for 1 h at room
temperature, and reacted with 0.02% DAB and 0.01% H2O2for about 3 min. Aftereach
incubation step, sections were washed three times with PBS for 5 min. Finally,
sections were mounted on gelatin-coated slides, dehydrated in an ascending alcohol
series, and cleared in xylene.
2.16. Histological analyses
For the analysis in the inflammatory mice model, quantification of the microglia
cells was performed by counting the number of these cells in the cortex and
hippocampus at ?100 magnification using a Stereo Investigator (MicroBrightField,
Williston, USA) and the images were photographed at ?200 magnification using
a AxioSkop 2 microscope (CarlZeiss Inc., Göttingen, Germany); these values are
presented as a percent of the control group values. Results were averaged for three
sections per mouse for five or six mice per group.
For the analysis in the mouse model of transient global ischemia by 2VO, cell
counts in CA1 were performed using a computerized image analysis system (Leica
Fig. 2. Effect of 6-shogaol on PGE2production, COX-2 expression, IL-1b and TNF-a in LPS-treated microglia cells. (A) PGE2was assessed by using a competitive enzyme immu-
noassay kit after treatment with LPS (100 ng/ml) for 6 h in the presence or absence of 6-shogaol (1, 5, and 10 mM). (B, C) Expression of COX-2 was assessed by western blot analysis
using COX-2 antibody. (D, E) Levels of IL-1b and TNF-a in the culture supernatants were determined by ELISA analysis. All data are presented as the mean ? S.E.M of three
independent experiments. #p < 0.05 indicates statistically significant difference between the control and LPS alone-treated groups. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate
statistically significant differences compared to treatment with LPS alone.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
Microsystems AG, Wetzlar, Germany). Cells in the hippocampal CA1 region were
counted in six sections per mouse for four mice per group by one person unaware of
the treatment history. Cell counts were performed using a computerized image
analysis system (Leica Microsystems AG).
2.17. Statistical analysis
The data were analyzed using Statistical Analysis System software (PRISM). All
the data are expressed as mean ? S.E.M. Statistical comparisons between the
different treatments were performed using one-way ANOVA with Tukey’s multiple
3.1. Effect of 6-shogaol on NO production and iNOS regulation in
The effects of pretreatment with 6-gingerol and shoagol on NO
production were tested in BV-2 and primary microglia cells. 6-
Shogaol effectively decreased LPS-induced NO production in
primary microglia cells, while 6-gingerol did not inhibit NO
wogonin, potent well-known anti-inflammatory agent from Scu-
tellaria baicalensis, and NMMA (the iNOS inhibitor) on NO
production were evaluated in primary microglia cells. Among these
compounds, 6-shogaol showed the most potent NO inhibitory
activity (Fig. 1B). The results of the MTT assay showed that the
concentrations of 6-shogaol used in this study did not affect cell
NO production in microglia is regulated primarily by the iNOS
enzyme. We performed western blot to determine whether the NO
inhibitory effect of 6-shogaol is related to the regulation of the
expression of iNOS. As shown in Fig. 1D, pretreatment of cells with
6-shogaol led to a significant decrease in iNOS protein level at
10 mM. Similar to what was observed in BV-2 cells, 6-shogaol
inhibited expression of iNOS protein in primary cultured micro-
glia. In contrast, 6-gingerol did not have an inhibitory effect on LPS-
induced iNOS expression.
To investigate the precise mechanisms of 6-shogaol on NO
regulation, we also performed NO radical scavenging assay and
iNOS activity assay. As shown in Fig. 1E and F, 6-shogaol did not
influence on the accumulation of nitrite upon decomposition of
NO$ doner, sodium nitroprusside. However, 6-shogaol (5 and
10 mM) significantly reduced iNOS activity.
The effect of post-treatment with 6-shogaol on NO production in
primary microglia was examined. As shown in Fig.1G, while treatment
Fig. 3. Effect of 6-shogaol on LPS-induced NF-kB and MAPKs activation in primary cultured microglia cells. (A) Nuclear extracts were prepared by using a nuclear extract kit. NF-kB
activity was measured using an ELISA kit. (B) Protein levels of IkB and p-IkB were evaluated by western blot analysis. (CeE) Activation of MAPKs was evaluated by western blot
analysis using antibodies that recognize the phosphorylated or unphosphorylated forms of p38, JNK, and ERK. The data are expressed relative to percentage of control (IkB) or LPS
stimulation (p-IkB, p-p38, p-JNK, p-ERK) and are presented as the mean ? S.E.M of three independent experiments. #p < 0.05 indicates statistically significant difference between
the control and LPS alone-treated groups. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences compared to treatment with LPS alone.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
of microglia cells with LPS for 1, 3, 6, and 12 h increased NO production
severely, post-treatment with 10 mM of 6-shogaol had a significant
3.2. Effect of 6-shogaol on PGE2production and COX-2 expression
Pretreatment of primary microglia cells with 6-shogaol has
decreased LPS-induced PGE2production significantly in a dose-
dependent manner (Fig. 2A).
It is known that COX-2 mediates PGE2production in response to
proinflammatory stimulation. Therefore, the effect of 6-shogaol on
the expression of the COX-2, a key enzyme responsible for PGE2
production, was determined using western blot analysis in BV-2
and primary microglia cells. 6-Shogaol reduced LPS-induced
expression of COX-2 significantly in a concentration-dependent
manner (Fig. 2B and C). Furthermore, 6-shogaol at 10 mM simi-
larly decreased COX-2 expression in primary microglia cells. These
results indicate that 6-shogaol suppresses LPS-induced PGE2
synthesis through downregulation of COX-2.
3.3. Effect of 6-shogaol on proinflammatory cytokines in microglia
To investigate whether 6-shogaol inhibits the production of
proinflammatory cytokines such as IL-1b and TNF-a, primary
microglia cells were treated with LPS alone or with 6-shogaol (1, 5,
and 10 mM) for 24 h. Stimulation of microglia cells with LPS led to
the increased production of IL-1b and TNF-a. The production of
these cytokines was significantly decreased in a dose-dependent
manner by pretreatment with 6-shogaol (Fig. 2D and F).
3.4. Effect of 6-shogaol on NF-kB activation in microglia
Because NF-kB is an important upstream modulator of proin-
flammatory cytokines, iNOS, and COX-2 expression (Baeuerle and
Henkel, 1994; Nomura, 2001), the effects of 6-shogaol on NF-kB
activity were investigated using an NF-kB ELISA kit and western
blot analysis. As shown in Fig. 3A, LPS significantly enhanced the
DNA binding activity of nuclear NF-kB p65 in primary microglia.
The increase in NF-kB activity was decreased significantly by pre-
treating cells with 10 mM 6-shogaol.
NF-kB is inactivated in the cytosol by binding to IkB, and
becomes active through translocation to the nucleus preceded by
Baltimore, 1988; Zandi et al., 1997). As shown in Fig. 3B, IkB was
phosphorylated and degraded 1 h after LPS treatment. Pretreat-
ment of primary microglia cells with 6-shogaol (10 mM) decreased
to phosphorylation and degradation of IkB in response to LPS,
indicating that the subsequent NF-kB inactivation induced by 6-
Fig. 4. Effect of 6-shogaol on NO production induced by TLRs agonist in primary microglia cells. (A) Primary microglia cells were stimulated with PamCSK (synthetic lipopeptide,
TLR2 agonist, 0.01, 0.1, and 1 mg/ml) or poly(I:C) (double-stranded RNA, TLR3 agonist, 0.25, 2.5, and 25 mg/ml) for 24 h. (B) Primary microglia cells were pretreated with 6-shogaol
(10 mM) for 30 min and then stimulated with PamCSK (1 mg/ml) or poly(I:C) (25 mg/ml) for another 24 h. The culture medium was then collected for a nitrite assay. Nitrate was
measured using a Griess reaction. All data are presented as the mean ? S.E.M of three independent experiments. #p < 0.05 indicates statistically significant difference between the
control and TLR agonists alone-treated groups. ***p < 0.001 indicates a statistically significant difference compared to treatment with LPS alone.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
3.5. Effect of 6-shogaol on LPS-induced MAPKs activation
We evaluated the effect of 6-shogaol on MAPK signaling which
plays an important role in the regulation of inflammatory
responses and coordinates the induction of many genes encoding
inflammatory mediators (Kaminska, 2005). As shown in Fig. 3CeE,
treatment with LPS (100 ng/ml) for 30 min stimulated the phos-
phorylation of p38, ERK, and JNK in primary microglia cells. The
amount of non-phosphorylated p38, ERK, and JNK was unaffected
by LPS or 6-shogaol treatment. p38 and MAPK phosphorylation in
response to LPS was strongly suppressed by 6-shogaol. Further-
more, 6-shogaol inhibited the activation of JNK, but not that of ERK.
3.6. Effect of 6-shogaol on NO production induced by TLRs agonist
in primary microglia cells
We investigated the effect of 6-shogaol on NO production by
TLRs agonist in microglia cells. While exposure to PamCSK (a
synthetic lipopeptide TLR2 agonist) and poly (I:C) (a double-
stranded RNA TLR3 agonist) stimulated NO production in a dose-
Fig. 5. Effect of 6-shogaol on microglial activation in mice models of LPS-induced neuroinflammation. (AeF) Representative photomicrographs of mac-1-IR microglia in the cortex
(left column) and hippocampus (right column) of each group at magnification of ?200. The scale bar ¼ 100 mm. Quantification of the microglia cells was performed by counting the
number of microglial cells in (G) cortex and (H) hippocampus at ?100 magnification using a Stereo Investigator (MicroBrightField, U.S.A.) and was presented as a percent of the
control group values. (A) control group; (B) LPS group; (C) LPS þ NMMA 5 mg/kg group; (D) LPS þ NMMA 20 mg/kg group; (E) LPS þ 6-shogaol 5 mg/kg group; (F) LPS þ 6-shogaol
20 mg/kg group. The data are represented as the mean ? S.E.M. #p < 0.05 indicates statistically significant difference between the control and LPS alone-treated groups. *p < 0.05,
**p < 0.01 and ***p < 0.001 indicate statistically significant differences compared with the LPS-only treated group.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
dependent manner (Fig. 4A), pretreatment with 6-shogaol for
30 min inhibited it significantly (Fig. 4B). TLR2 activated only the
MyD88-dependent pathway, whereas TLR3 led to activation of the
Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-b (TRIF)-
dependent pathway and not the MyD88-dependent pathway.
These results suggest that 6-shogaol inhibits NO production
induced by both the MyD88-dependent and TRIF-dependent
pathways in primary microglia cells.
3.7. Effect of 6-shogaol on microglial activation in the
neuroinflammatory mouse model
To evaluate the potential anti-inflammatory effect of 6-shogaol
in vivo, neuroinflammation was induced by systemic administra-
tion of LPS (5.0 mg/kg, i.p.). 6-Shogaol was administered orally at
5.0 mg/kg and 20.0 mg/kg once per day for 3 days before LPS
treatment (Fig. 5). Pretreatment of mice with 6-shogaol signifi-
cantly reduced to microglial activation in the brain cortex by 43.9%
and 66.8% at 5.0 mg/kg and 20.0 mg/kg, respectively. 6-Shogaol
suppressed microglial activation in the hippocampus by 65.9%
and 65.5% at 5.0 mg/kg and 20.0 mg/kg, respectively. NMMA, an
iNOS inhibitor, was used as the positive control. These results
suggest that 6-shogaol can regulate inflammation due to microglial
activation in vivo as well as in vitro.
3.8. Effect of 6-shogaol on LPS-induced neurotoxicity and microglial
activation in primary cortical neuron-glia culture
As shown in Fig. 6A, pretreatment with 10 mM 6-shogaol
number of neurons (34.7%). The addition of 6-shogaol alone did
not result in any significant changes in the number of Neu-N
positive neurons compared to the control group. Also, it is repor-
ted that LPS did not cause neuronal cell death in the absence of
microglia (Chao et al., 1992; Xie et al., 2004, 2002). These results
suggest that 6-shogaol can protect cortical neurons from LPS-
To examine the effects of 6-shogaol on LPS-induced microglial
activation, cortical neuron-glia cultures were pretreated with 6-
shogaol followed by treatment with LPS. Pretreatment with 6-
shogaol (10 mM) have suppressed LPS-induced microglial activa-
tion by 41% (Fig. 6B). The effect of 6-shogaol on LPS-induced NO
production was also evaluated. As shown in Fig. 6C, treatment of
cortical neuron-glia cultures with 100 ng/ml LPS increased the
production of NO markedly. 6-Shogaol inhibited the NO production
induced by LPS at 12, 24 and, 48 h time points by 40.6%, 32.9%, and
3.9. Effect of 6-shogaol on neuronal cell death induced by the
ischemic injury mouse model
To determine the neuroprotective effect of 6-shgaol in vivo,
transient global ischemia was induced in mice by 2VO. Compared
with vehicle-treated ischemic animals, oral administration of 6-
shogaol at a dose of 10.0 mg/kg showed 30.0% inhibition of CA1
cell death (Fig. 7A and B). Moreover, treatment of 6-shogaol
(10.0 mg/kg) suppressed active caspase-3-positive cells in the
hippocampal CA1 region after 2VO (Fig. 7C and D).
Based on the anti-inflammatory effects of 6-shgaol in vitro and
in vivo, we hypothesize that its neuroprotective effect is due to
inhibition of neuroinflammation. Microglia plays an active role in
brain inflammation. Thus, the effect of 6-shogaol on microglial
activation was assessed through immunohistochemistry using an
antibody against Mac-1. Ischemic injury strongly induced micro-
glial activation, and the number of Mac-1 positive microglia was
significantly reduced by 6-shogaol (Fig. 8A and B). 6-Shogaol at
10.0 mg/kg had inhibited microglial activation by 48.0%. More-
over, we found the expression of neuroinflammatory markers
including IL-1b (Fig. 8C and D) and TNF-a (Fig. 8E and F) in the
hippocampus. These markers were attenuated by 6-shogaol
Fig. 6. Effect of 6-shogaol on LPS-induced neurotoxicity and -microglial activation in cortical neuron-glia cultures. (A) Cells were fixed and stained for MAP-2 as described in the
methods section. After cell treatment, the number of Neu-N positive cortical neurons was counted. (B) Cells were stained for OX-42 as described in the methods section. Quan-
tification of the microglia cells was performed by counting the number of microglial cells in cortical neuron-glia cultures. (C) Nitrate was measured using a Griess reaction. The data
is represented as a mean ? S.E.M of the three independent experiments. The scale bar is 50 mm #p < 0.05 indicates statistically significant difference between the control and LPS
alone-treated groups. **p < 0.01 and ***p < 0.001 indicate a statistically significant difference from treatment with LPS alone.
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
treatment, too. These results indicate that the neuroprotective
effects of 6-shogaol are likely due to their anti-inflammatory
This study focused on the anti-inflammatory and neuro-
protective effects of 6-shogaol, a main active ingredient of ginger,
at the molecular, cellular, and in vivo levels. Ginger is used in Asian
traditional medicine to treat many inflammatory conditions and
associated pain (Grzanna et al., 2005). In 2009, Jung et al. reported
that ginger extract inhibited the production of NO and proin-
flammatory cytokines in LPS-stimulated BV-2 microglial cells via
the NF-kB pathway (Jung et al., 2009). 6-Gingerol had an inhibi-
tory effect on the production of proinflammatory cytokines in
murineperitoneal macrophages (Tripathiet al., 2007).
Furthermore, 6-shogaol has been shown to inhibit LPS-induced
iNOS and COX gene expression in macrophages (Pan et al.,
2008). Thus, we hypothesized that 6-gingerol and 6-shogaol
might regulate neuroinflammation by inhibiting microglial acti-
vation in brain. We found that 6-shogaol had anti-inflammatory
effects, while 10 mM of 6-gingerol did not show significant
inhibitory effects on NO production and iNOS expression in
microglia cells. 6-Shogaol did down-regulate the microglial acti-
vation both in cortical neuron-glia culture and in a systemic
inflammatory model. Moreover, 6-shogaol showed significant
neuroprotective effects in the cortical neuron-glia culture, in the
ischemia model. It might be due to anti-inflammatory effects via
inhibition of microglial activation.
Inflammatory processes in the central nervous system (CNS) are
believed to play an important role in neuronal cell death in
neurodegenerative diseases. Brain inflammation itself does not
Fig. 7. Effect of 6-shogaol on neuronal cell death induced by ischemic injury. (A) Representative photomicrographs of cresyl violet-stained hippocampal regions of each group.
Boxed regions in a, b, c, and d (?100) are shown in e, f, g, and h (?400), respectively. Scale bar in a and e is 200 and 50 mm, respectively. (B) Neuronal cell density in hippocampus
was measured by cresyl violet staining. Viable cells in CA1 region of hippocampus were numbered at ?400 magnification. (C) Representative photomicrographs showed cleaved
caspase-3-positive cells in CA1 region of hippocampus of each group (?400, Scale bar ¼ 50 mm). (D) Quantification of active caspase-3-positive cells was performed by counting the
number of hippocampus at ?400 magnification. The data are represented as the mean ? S.E.M of three independent experiments. #p < 0.05 indicates statistically significant
difference between the sham-operated and ischemia-induced groups. *p < 0.05 indicates statistically significant differences compared to vehicle-treated ischemic group. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
Fig. 8. Effectof6-shogaol onmicroglialactivation inducedbyischemic injury.(A)Representativephotomicrographs showedmac-1-IRmicrogliainhippocampus of each group.Boxed
regionsina, b, c, andd (?100) areshownine, f, g,and h (?400), respectively.Scale bar in a ande is200 and50mm, respectively.(B) Quantification of the microgliacells was performed
by counting the number of hippocampus at ?400 magnification. (C and E) Representative photomicrographs showed IL-1b (C) and TNF-a positive cells (E) in CA1 region of hippo-
campus of each group (?400, Scale bar ¼ 50 mm). (D and F) Quantification of IL-1b (D) and TNF-a positive cells (F) was performed by counting the number of hippocampus at ?400
magnification. The dataarerepresentedasthemean?S.E.M of threeindependentexperiments.#p<0.05indicatesstatisticallysignificantdifferencebetweenthe sham-operatedand
ischemia-induced groups. *p < 0.05 indicates statistically significant differences compared to vehicle-treated ischemic group.
cause neuronal cell death. When uncontrolled, however, it may
lead to potentially damaging consequences as seen in several
inflammatory diseases. Inflammation in the brain is mediated
primarily by activated microglia. Chronic neurodegeneration is
accompanied by an inflammatory response characterized by the
selective activation of the microglial cells in the CNS (Campbell,
2004; Liu and Hong, 2003; McGeer and McGeer, 2004). Activation
of microglia has been observed during the development of neuro-
degenerative diseases such as Alzheimer’s and PD (Dickson et al.,
1993; McGeer et al., 1988).
Several studies have suggested that inhibition of microglial
activation is a promising therapeutic strategy for neurodegenera-
tive diseases. Minocycline, a derivative of tetracycline, has shown
neuroprotective effects in several neurodegenerative disease
models by inhibiting microglial activation (Fan et al., 2007; Henry
et al., 2008; Yrjanheikki et al., 1998, 1999). Recently, natural prod-
ucts such as non-steroidal anti-inflammatory drugs (NSAID) have
received significant attention due to their ability to regulate the
inflammatory response. A number of studies have provided scien-
tific proof that anti-inflammatory herbal medicine and its constit-
uents are effective at slowing-down the neurodegenerative
process. For example, curcumin exerted a neuroprotective effect by
reducing microglial activation in neuron-glia cultures (Lee et al.,
2007; Yang et al., 2008). In the previous studies in our lab, we
showed that apigenin and wogoninsuppress activation of microglia
which results in neuroprotection (Ha et al., 2008; Lee et al., 2003).
These results suggest that regulation of microglial activation is
a promising treatment strategy for neurodegenerative diseases.
Thus, anti-neuroinflammatory effect of 6-shogaol in vitro and
in vivo was evaluated.
It has been reported that iNOS and COX-2 were highly
expressed in CNS-related diseases (Hunot et al., 1996; Teismann
et al., 2003), even though iNOS is not expressed in the normal
brain. However, inflammatory mediators such as LPS and cyto-
kines induce expression of iNOS in microglia and astrocytes
(Murphy, 2000), and possibly in neurons (Heneka and Feinstein,
2001). iNOS continuously produces high levels of NO. Whereas
NO at low concentrations functions as a signaling molecule
(Murphy, 2000), at high levels it induces neuronal cell death.
Neuronal stress, such as ischemia and excitotoxicity, is associated
with strong upregulation of neuronal COX-2 expression, which
suggests that COX-2 is involved in neurotoxic mechanisms (Planas
et al., 1995; Tocco et al., 1997). COX-2, a rate-limiting enzyme for
PGE2synthesis, is induced during inflammation and participates in
inflammation-mediated cytotoxicity. Furthermore, PGE2 is an
important mediator of inflammation. Recently, it has been repor-
ted that selective COX-2 inhibitors such as celecoxib and rofecoxib
can slow-down the development of some neurological diseases
(Aisen et al., 2003; Candelario-Jalil et al., 2003; Klivenyi et al.,
2003). In this study, 6-shogaol inhibited the release of NO and
PGE2by suppressing iNOS and COX-2 protein expression, respec-
tively. This study also shows that 6-shogaol suppresses the acti-
vation of NF-kB and MAPK.
Additional experiments were carried out to examine the effect
of 6-shogaol on the activation of NF-kB. It has been reported that
NF-kB is related to inflammatory responses and other chronic
diseases (Karin et al., 2004). The transcription factor NF-kB plays an
important role in the production of proinflammatory cytokines and
is believed to be a promising target for the treatment of inflam-
matory diseases. Recently, 6-shogaol was found to inhibit iNOS and
COX-2 expression by blocking LPS-induced NF-kB activation in
macrophages(Panet al., 2008). The effectof 6-shogaol on theNF-kB
pathway was also evaluated. After activating NF-kB in primary
microglia, 6-shogaol reduced activation of NF-kB by blocking
phosphorylation of IkB and the subsequent degradation of IkB.
Therefore, 6-shogaol may exert anti-neuroinflammatory effects by
inhibiting NF-kB in primary microglia.
Upon exposure to LPS, multiple signaling pathways are known
to be activated in microglia. Especially, MAPKs are known to play
important roles in inflammatory processes. Several studies have
shown that MAPKs are required for NF-kB-dependent gene
expression (Carter et al., 1999; Meyer et al., 1996). Furthermore,
a previous study reported that 6-shogaol inhibited ERK and Akt
activation in LPS-stimulated macrophages, but did not affect
activation of p38 MAPK (Pan et al., 2008). In the current study, the
effects of 6-shogaol on MAPKs (p38, ERK, and JNK) were investi-
gated. Exposure to LPS in primary microglia cells strongly acti-
vated all MAPKs. The phosphorylation of p38 and JNK in response
to LPS was reduced by 6-shogaol treatment. However, 6-shogaol
had no effect on ERK. Our results are consistent with a previous
report that p38 MAPK mediated inflammatory responses in
microglia (Bhat et al., 1998) and that inhibition of JNK reduced the
induction of several genes regulated by AP-1, including COX-2,
TNF-a, and IL-6 in LPS-stimulated primary microglia (Waetzig
et al., 2005). Our study indicates that 6-shogaol has anti-
inflammatory effects through inhibition of p38 MAPK and JNK in
LPS triggers innate immune responses through TLR4, a member
of the TLR family that participates in pathogen recognition. Cellular
response to LPS occurs through the interaction of LPS with
a circulating LPS-binding protein and CD14, and subsequently
activates TLR4. LPS-induced dimerization of TLR4 is required for the
activation of downstream signaling pathways including NF-kB. Ahn
et al. reported 6-shogaol inhibited LPS-induced TLR4 dimerization
(Ahn et al., 2009). 6-Shogaol may regulate TLR activity via modu-
lation of receptor dimerization. It can lead to decrease inflamma-
tory gene expression.
Post-ischemic inflammation and the formation of oxygen-
derived free radicals are thought to be pivotal for reperfusion-
induced delayed neurodegeneration (Giulian and Vaca, 1993;
Kitagawa et al., 1990; Yamamoto et al., 1997). Cerebral ischemia
evokes secondary inflammation in the brain that contributes to
ischemic insults (Barone and Feuerstein,1999). During the delayed
progression of ischemic stroke, post-ischemic inflammation may
playan important role in brain damage (Dirnagl et al.,1999). iNOS is
inpart responsible for ischemic injury (del Zoppo et al., 2000). COX-
2 inhibitionprevents delayed death of CA1 hippocampal neurons in
global ischemia (Nakayama et al.,1998; Nogawa et al.,1997). In the
present study, 6-shogaol significantly inhibited the delayed death
of CA1 hippocampal neurons and the activation of microglia in
transient global ischemia model. Therefore, the anti-inflammatory
effects of 6-shogaol may provide neuroprotection against ischemic
Ginger is widely used in foods as a spice all over the world and
it has been one of the most frequently used medicinal plants, for
a wide array of unrelated diseases for a long time. And so
numerous research and review articles have reported various
pharmacological actions of ginger (Ali et al., 2008). In this study,
we discovered the neuroprotective effects of 6-shogaol, one of the
major components of ginger, which were mediated by microglia
inactivation related with regulations of various pathways. Natural
products are still proving to be the source that leads most
consistently to successful development of new drugs (Rollinger
et al., 2006), and in the future, they will continue to play
a major role as active substances, and model molecules for the
discovery and validation of drug targets. 6-Shogaol is a promising
ingredient for new drugs because of its strengths in the simplicity
of structure and richness in resources. Thus, 6-shogaol could be
a good candidate for neurodegenerative disease which related
S.K. Ha et al. / Neuropharmacology 63 (2012) 211e223
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