TUGAS KELOMPOK

FITOKIMIA
ISOLASI NEOFLAVON DARI TANAMAN
DALBERGIA ODORIFERA

SEMESTER : GENAP 2016/2017

DOSEN : IKA MARUYA KUSUMA

DISUSUN OLEH :
1. ARDIAN SURYA DEWANTARA (14334010)
2. PRAYOGO PANGESTU (14334013)
3. TRI NANDA PUTRA (14334060)
4. LAZUARDI RIZALDI (14334083)

FAKULTAS FARMASI
INSTITUT SAINS DAN TEKNOLOGI NASIONAL
JAKARTA 2017
 KATA PENGANTAR

Puji syukur kehadirat Allah SWT atas berkat, kasih dan karuniaNya sehingga Makalah
Fitokimia yang berjudul Isolasi Neoflavon dari Dalbergia odorifera dapat selesai dengan
lancar. Maksud dari penulisan makalah ini adalah mengkaji lebih dalam tentang neoflafon
yang ada pada tanaman Dalbergia odorifera, dan kegunaannya dalam kehidupan.
Penulis juga tahu dan sadar bahwa makalah ini masih banyak kekurangan, oleh sebab
itu saran dan kritik yang membangun sangat diharapkan agar makalah ini dapat berkembang
dengan lebih baik. Penulis berharap agar makalah ini dapat bermanfaat dan diaplikasikan
dalam kehidupan kita sehari-hari.

Jakarta, Mei 2017

Penyusun

2
 DAFTAR ISI

KATA PENGANTAR.............................................................................................................ii

DAFTAR ISI.........................................................................................................................iii

BAB I.....................................................................................................................................1

PENDAHULUAN..................................................................................................................1

A. Latar Belakang...........................................................................................................1

B. Rumusan Masalah.....................................................................................................2

C. Tujuan Penulisan.......................................................................................................2

BAB II....................................................................................................................................3

TINJAUAN PUSTAKA.........................................................................................................3

BAB III...................................................................................................................................5

PEMBAHASAN....................................................................................................................5

A. Latifolin from MeOH Extract Dalbergia odorifera.................................................5

B. Instrumen dan bahan................................................................................................6

C. isolasi latifolin..........................................................................................................6

BAB IV..................................................................................................................................7

KESIMPULAN......................................................................................................................7

DAFTAR PUSTAKA.............................................................................................................8

Lampiran................................................................................................................................9

3
 BAB I

PENDAHULUAN

A. Latar Belakang

Sebagian besar senyawa organik bahan alam adalah senyawa-senyawa aromatic. Senyawa-
senyawa ini tersebar luas sebagai zat warna alam yang menyebabkan warna pada bunga, kayu
pohon tropis, bermacam-macam kapang dan lumut termasuk zat alizarin.

Senyawa aromatik ini mengandung cincin karboaromatik yaitu cincin aromatic yang hanya
terdiri dari atom karbon seperti benzene, naftalen dan antrasen. Cincin karboaromatik ini
biasanya tersubstitusi oleh satu atau lebih gugus hidroksil atau gugus lainnya yang ekivalen
ditinjau dari biogenetiknya. Oleh karena itu senyawa bahan alam aromatic ini sering disebut
sebagai senyawa-senyawa fenol walaupun sebagian diantaranya bersifat netral karena tidak
mengandung gugus fenol dalam keadaan bebas.

Flavonoid merupakan kelompok senyawa fenol alam dan suatu golongan metabolilt sekunder
yang tersebar merata di dalam tumbuhan. Flavonoid mempunyai kerangka dasar yang terdiri
dari 15 atom C, 2 cincin benzene ( C 6) terikat pada suatu rantai propan (C 3) yang dapat atau
tidak dapat membentuk cincin ketiga, sehingga membentuk suatu konfigurasi C 6-C3-C6,
susunan dari senyawa tersebut dapat menghasilkan 3 jenis struktur, yaitu:
- 1,3 diarilpropan (Flavonoid)
- 1,2 diarilpropan (isoflavonoid)
- 1,1 diarilpropan (neoflavonoid)
Senyawa flavonoid mempunyai kerangka 2 fenil kroman. Posisi orto dari cincin A dan atom
karbon yang terikat dari cincin B dari 1,3 diarilpropan dihubungkan oleh jembatan oksigen
sehingga membentuk suatu cincin heterosiklik yang baru (cincin C).

B. Rumusan Masalah
1. Apa yang dimaksud neoflavon ?

1
 2. Apa itu tanaman dalbergia odorifera ?
3. Bagaimanakah cara ekstrasi dan isolasinya ?

C. Tujuan Penulisan
1. Untuk mengetahui apa yang dimaksud neoflavon
2. Untuk mengetahui isolasi dan ekstraksi dari senyawa neoflavon.

2
 BAB II

TINJAUAN PUSTAKA

Flavonoid mempunyai kerangka dasar karbon yang terdiriu dari 15 atom karbon, dimana dua
cincin benzen (C6) terikat pada suatu rantai propana (C3) sehingga membentuk suatu susunan
C6-C3-C6. Susunan ini dapat menghasilkan tiga jenis struktur senyawa flavonoid. Susunan ini
dapat menghasilkan tiga jenis struktur, yaitu 1,3-diarilpropan atau flavonoid, 1,2-diarilpropan
atau isoflavon, dan 1,1-diarilpropan atau neoflavon.

Gambar 1. Struktur kerangka dasar senyawa flavonoid

3
 Gambar 2. Isoflavonoid

Gambar 3. Neoflavonoid

Senyawa-senyawa isoflavonoid dan neoflavonoid hanya ditemukan dalam beberapa jenis

tumbuhan, terutama pada suku Leguminose. Jenis-jenis senyawa yang termasuk isoflavonoid
adalah isoflavon, rotenoid, pterokorpan, dan kumestan. Sedangkan neoflavonoid meliputi
jenis-jenis 4-arilkumarin dan berbagai dalbergion.

4
5
 Gambar 4. Beberapa Senyawa Isoflavonoid dan Neoflavonoid

BAB III

PEMBAHASAN

A. Latifolin from MeOH Extract Dalbergia odorifera

Latifolin Species: odorifera
Family: Fabaceae Common Names: Dalergia, dalbergia,
Genus: Dalbergia lignum dalbergiae
PLANT DESCRIPTION Part Used: wood
Docu
mente
Di Korea dan Cina, d kayu batang Dalbergia
odorifera T. Chen Proper Analgesic, antihemorraghic, merupakan obat
tradisional yang ties hepatic, tonic penting. Digunakan
untuk mengobati & kelainan darah,
iskemia, Action pembengkakan, dan
nyeri epigastrik. s: Mengidentifikasi efek
Plant
Chemi 6
cals Dalbergin, Nordalbergin
Includ
e:
terapeutik yang ampuh dari latifolin, yang menjamin penyelidikan lebih lanjut sebagai
pengobatan potensial untuk penyakit inflamasi.

Kayu batang Dalbergia odorifera T. Chen, Bernama 'Jiangxiang' dalam pengobatan

tradisional Tiongkok (Leguminosae), adalah kayu Cina yang penting dan Obat tradisional
yang digunakan untuk mengobati kelainan darah, ischeMia, bengkak, dan nyeri epigastrik di
Korea dan China (Chan et al., 1998).
D. odorifera telah secara umum dalam berbagai sediaan herbal cina termasuk
Rebusan Qi-Shen-Yi-Qi, pil Guanxin-Danshen, dan Injeksi Danshen,
Studi fitokimia sebelumnya Melaporkan isolasi flavonoid, quinines, dan phe-konstituen nolic
dari D. odorifera (Goda et al., 1992).
Studi sebelumnya telah melaporkan bahwa latifolin memiliki anti-
rayap dan anti-jamur (Sekine et al., 2009) dan anti kegiatan karsinogenik (Yin et al., 2004),
tetapi tidak ada studi yang mempublikasikan mengenai mekanisme antiinflammatory
Efek latifolin

B. Instrumen dan bahan.

JEOL (Akishima, Tokyo, Jepang) untuk mencatat Spektrum NMR di CD 3 OD.
Eclipse 400MHz spektrometer (400MHz untuk 1 H dan 125MHz untuk 13 C), untuk
pergeseran kimia yang direferensikan Ke puncak pelarut residu masing-masing.
Kolom kromatography dilakukan pada silika gel 60 (70-230 mesh, Merck).

C. isolasi latifolin.
D. Odorifera Kayu ulin dibeli dari Daehak Hanyakguk, Iksan, Korea, pada Agustus
2011. Spesimen voucher (WK-2011-08-01) didepositkan di Herbarium PT College of
Pharmacy, Universitas Wonkwang (Korea).
Dikeringkan D. odorifera heartwood (1.2kg)
Diekstraksi dengan MeOH panas (2L) selama 2jam.
Ekstrak MeOH (173 g) dilarutkan dalam MeOH 60% berair (1 L) dan dipartisi dua
kali dengan n -hexane (800ml) dan CH 2 Cl 2 (800mL), berturut-turut.
Fraksi -soluble CH 2 Cl 2 (20 g) dipisahkan dengan kromatografi pada gel silica
Kolom (6,5 60cm) dengan menggunakan n -hexane / EtOAc (gradient) sebagai
eluen.
Hasilnya, enam fraksi diperoleh (P. DO-A1-A6).
DO-A2 (9g) dipisahkan oleh Kromatografi pada kolom silika gel (6,5 x 60 cm)
dengan menggunakan n -hexane / aseton (3: 1) sebagai eluen untuk memperoleh
empat Pecahan (Fr. DO-A21-A24).

7
 Selanjutnya, pecahan DO-A23 (7g) dipisahkan dengan kromatografi dengan silika gel
kolom (6,5 60 cm) dengan menggunakan n -hexane / aseton (3: 1) sebagai eluen
untuk mendapatkan latifolin (243,4 mg, 0,14 b / b %).

BAB IV

KESIMPULAN

1 Flavonoid mempunyai kerangka dasar karbon yang terdiriu dari 15 atom karbon,
dimana dua cincin benzen (C6) terikat pada suatu rantai propana (C3) sehingga
membentuk suatu susunan C6-C3-C6. Susunan ini dapat menghasilkan tiga jenis
struktur, yaitu :
1,3-diarilpropan atau flavonoid,
1,2-diarilpropan atau isoflavon,
dan 1,1-diarilpropan atau neoflavon.
2 Neoflavonoid meliputi jenis-jenis 4-arilkumarin dan berbagai dalbergoin
3 Senyawa neoflavon digunakan sebagai anti-inflamasi, anti-kanker, anti-virus, anti-
alergi, efek Estrogenik, anti-kolesterol, dan antioksidan
"Nama botani: Dalbergia odorifera
Nama farmasi: Lignum Dalbergiae.
Properti: tajam, hangat.
Saluran masuk: Hati, Limpa, Perut.
Fungsi dan penggunaan klinis: Disperses Congealed Blood dan berhenti
berdarah: digunakan untuk luka dalam dari trauma, patah tulang,
keseleo, atau kontusi.
4 Ekstrasi dengan cara maeserasi menggunakan MeOH
5 Fraksinasi dan isolasi dengan n hexane, CH 2 Cl 2 , kromatografi dengan silica gel
colom

8
 DAFTAR PUSTAKA
Achmad, S. A. 1986. Kimia Organik Bahan Alam. Jakarta: Universitas Terbuka.
Anonim. 2011. Metabolit Sekunder.
DONG SUNG LEE dkk 2014. The Neoflavonoid Latifolin Isolated from MeOH
Extract of Dalbergia odorifera Attenuates Inflammatory Responses by
Inhibiting NF-B
Activation via Nrf2-Mediated Heme Oxygenase-1. PHYTOTHERAPY
RESEARCH. Korea

http://www.rain-tree.com/dalbergia.htm

9
 Lampiran
PHYTOTHERAPY RESEARCH
Phytother. Res. 28: 12161223 (2014)
Published online 29 January 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5119

The Neoflavonoid Latifolin Isolated from MeOH

Extract of Dalbergia odorifera Attenuates Inflammatory
Responses by Inhibiting NF-B Activation via Nrf2-
Mediated Heme Oxygenase-1 Expression
1 2,3 2,3 1 2,3 4
Dong-Sung Lee, Kyoung-Su Kim, Wonmin Ko, Bin Li, Samell Keo, Gil-Saeng Jeong,
1,2,3 1,2,3
Hyuncheol Oh and Youn-Chul Kim *
1
Hanbang Body Fluid Research Center, Wonkwang University, Iksan 570-749, Korea
2
Standardized Material Bank for New Botanical Drugs, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea 3Institute of
Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea 4College of Pharmacy,
Keimyung University, 1095 Dalgubeol-daero, Dae-gu 704-701, Korea

In Korea and China, the heartwood of Dalbergia odorifera T. Chen is an important traditional medicine used to treat blood
disorders, ischemia, swelling, and epigastric pain. In this study, we investigated the in-hibitory effects of latifolin, a major
neoflavonoid component isolated from the MeOH extract of D. odorifera, on the inflammatory reaction of thioglycollate-
elicited peritoneal macrophages exposed to lipo-polysaccharide, with a particular focus on heme oxygenase-1 (HO-1)
expression and nuclear factor-B (NF-B) signaling. Latifolin significantly inhibited the protein and mRNA expression of
inducible nitric ox-ide synthase and COX-2, reduced NO, prostaglandins E2, tumor necrosis factor-, and interleukin-1
pro-duction in primary murine peritoneal macrophages exposed to lipopolysaccharide. Latifolin also suppressed inhibitor
B- levels, NF-B nuclear translocation, and NF-B DNA-binding activity. Furthermore, latifolin upregulated HO-1
expression via nuclear transcription factor-E2-related factor 2 (Nrf2) nuclear transloca-tion. In addition, using inhibitor tin
protoporphyrin IX (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of latifolin on the proinflammatory
mediators and NF-B DNA-binding activity were associated with the HO-1 expression. These results suggested that the
latifolin-mediated up-regulation of HO-1 expression played a critical role in anti-inflammatory effects in macrophages. This
study therefore identified potent therapeutic effects of latifolin, which warrants further investigation as a potential treat-ment
for inflammatory diseases. Copyright 2014 John Wiley & Sons, Ltd.

Keywords: latifolin; Dalbergia odorifera; macrophages; inflammation; heme oxygenase-1; nuclear factor-B.
INTRODUCTION
The heartwood of Dalbergia odorifera T. Chen,
named.Jiangxiang in Chinese traditional medicine
(Leguminosae), is an important Chinese wood and
tra-ditional medicine used to treat blood disorders,
ische-mia, swelling, and epigastric pain in Korea and
China (Chan et al., 1998). D. odorifera has been in
general use in various Chinese herbal preparations
including Qi-Shen-Yi-Qi decoction, Guanxin-
Danshen pills, and Danshen injection. Previous
phytochemical studies have reported the isolation of
flavonoids, quinines, and phe-nolic constituents from
D. odorifera (Goda et al., 1992). Previous study had
reported that latifolin had anti-termite and anti-
fungal (Sekine et al., 2009) and anti-carcinogenic
activities (Yin et al., 2004), but no study has
*Correspondence to: Youn-Chul Kim, Standardized Material Bank for
New Botanical Drugs; College of Pharmacy, Wonkwang University,
Iksan 570-749, Korea.

10
E-mail: yckim@wku.ac.kr

These authors contributed equally to this work.

yet been published on the mechanism of
antiinflammatory effects of latifolin.
Copyright 2014 John Wiley & Sons, Ltd.
Inflammation is a defense mechanism of the body
to various stimuli such as irradiation, physical
damage, and metabolic overload. However,
prolonged or deregulated inflammatory responses
can lead to a vari-ety of diseases including arthritis,
hepatitis, septic shock syndrome, neurodegenerative
disorders, and sepsis (Ferrero-Miliani et al., 2006).
Accordingly, recently, antiinflammatory studies
afford that search for develop-ment into drugs for the
treatment of various inflamma-tory-related diseases.
Macrophages play an important role in regulating
inflammatory responses via produc-tion of
proinflammatory mediators, such as nitric oxide
(NO) and prostaglandins E2 (PGE2), and cytokines
such as TNF- and interleukin-1 (IL-1). Nuclear
fac-tor-B (NF-B) activation is very important in
inflam-matory reactions. Lipopolysaccharide (LPS)
induces NF-B activation through phosphorylation
of inhibitor B (IB). This results in IB-
degradation, releases NF-B from IB-, and
translocation of NF-B to the nucleus (Klemm and
Ruland, 2006), where NF-B binds to promoters of
proinflammatory genes, such as
Received 30
May 2013
Revised 1
January 2014
Accepted 2
January 2014

11
ANTIINFLAMMATORY EFFECT OF LATIFOLIN VIA HEME OXYGENASE-1 EXPRESSION 1217

inducible nitric oxide synthase (iNOS) and COX-2

(Pahl, 1999). Recent studies have shown that heme
oxy-genase (HO)-1 has important Instruments and materials. NMR spectra were
immunomodulatory and antiinflammatory functions recorded in CD3OD by using a JEOL (Akishima,
(Ryter et al., 2002). The regu-lation of NF-B Tokyo, Japan) Eclipse 400 MHz spectrometer (400
1
signaling by HO-1, an enzyme that is es-sential for MHz for H and
heme degradation, is one of the important
mechanisms for cellular pathophysiological
conditions of inflammation (Otterbein et al., 2000).
Heme oxygenase-1 also decreased levels of
proinflammatory COX-2 and iNOS, resulting in
reduced COX-2-derived PGE2 and iNOS-derived
NO production via the NF-B activation (Suh et al.,
2006). Heme oxygenase-1 degrades heme to
generate carbon monoxide (CO), free iron, and
biliver-din, which is subsequently converted into
bilirubin (BR) by biliverdin reductase (Montellano,
2000). The degrada-tion of the proinflammatory free
heme, coupled with production of the
antiinflammatory BR and CO, influ-ence
inflammatory reactions (Gueler et al., 2007). In
addition, HO-1 induction is primarily regulated at
the transcriptional level, and its induction involves
the redox-dependent transcription factors such as
nuclear transcription factor-E2-related factor 2
(Nrf2). The Nrf2, which is a master regulator of the
antioxidant response and NF-B signaling, has been
shown to mediate HO-1 in-duction (Itoh et al.,
1997). Therefore, in the course of our study aiming
at the identification of antiinflammatory metabolites
from plants of traditional medicine, we iso-lated
latifolin, one of the major neoflavonoids, as an active
antiinflammatory metabolite presented in the MeOH
ex-tract of D. odorifera and investigated its
inhibitory mecha-nism on the inflammatory reaction
in LPS-stimulated murine peritoneal macrophages.

MATERIALS AND METHODS

Plant materials and isolation of latifolin. D. odorifera

heartwood was purchased from Daehak Hanyakguk,
Iksan, Korea, in August 2011. The voucher specimen
(WK-2011-08-01) was deposited at the Herbarium of
College of Pharmacy, Wonkwang University
(Korea). Dried D. odorifera heartwood (1.2 kg) was
extracted with hot MeOH (2 L) for 2 h. The MeOH
extract (173 g) was dissolved in 60% aqueous
MeOH (1 L) and partitioned twice with n-hexane
(800 mL) and CH2Cl2 (800 mL), successively. The
CH2Cl2-soluble fraction (20 g) was separated by
chromatography on a silica gel column (6.5 60 cm)
by using n-hexane/EtOAc (gradi-ent) as the eluent.
As a result, six fractions were obtained (Fr. DO-A1
A6). DO-A2 (9 g) was separated by chromatography
on a silica gel column (6.5 60 cm) by using n-
hexane/acetone (3:1) as the eluent to obtain four
fractions (Fr. DO-A21A24). Subsequently, fraction
DO-A23 (7 g) was separated by chromatography on
a silica gel column (6.5 60 cm) by using n-
hexane/acetone (3:1) as the eluent to obtain latifolin
(243.4 mg, 0.14 w/w %). Latifolin was deposited at
the Standardized Material Bank for New Botanical
Drugs, Wonkwang University (Republic of Korea)
(no. NNMBP026).

12
 13
125 MHz for C), and chemical shifts were
referenced to the respective residual solvent peaks.
Column chroma-tography was performed on silica
gel 60 (70230 mesh, Merck). Dulbeccos modified
Eagles medium, RPMI-1640 medium, fetal bovine
serum (FBS), and other tissue culture reagents were
purchased from Gibco BRL Life Technologies
(Grand Island, NY, USA). Tin protopor-phyrin IX
(SnPP), an inhibitor of HO activity, was obtained
from Porphyrin Products (Logan, UT, USA).
Thioglycollate (TG) was purchased from BD
Pharmingen (San Diego, CA, USA). All other
chemicals were obtained from Sigma Chemical Co.
(St. Louis, MO, USA) unless indicated otherwise.
All primary and second-ary antibodies for western
blot analysis were all purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
Primary peritoneal macrophage culture and viability
assay. C57BL/6 mice were purchased from Orient
Bio Co. (Sungnam, KyungKiDo, Korea).
Thioglycollate-elic-ited peritoneal macrophages
were harvested 4 days after intraperitoneal injection
of 3 mL of TG (Narumi et al., 1990). Macrophages
were maintained at 37 C in a humid-ified
atmosphere containing 5% CO2 and 95% air at 5
5 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1
10 cells/mL in Dulbeccos modified Eagles medium
supplemented with 10% heat-inactivated FBS,
penicillin G (100 units/mL), streptomycin (100
mg/mL), and L-gluta-mine (2 mM). The effects of
various experimental condi-tions on cell viability
were evaluated by determining mitochondrial
reductase function with an assay based on reduction
of the tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide, to formazan
crystals.
Copyright 2014 John Wiley & Sons, Ltd.
12161223 (2014)
13
Determination of nitrite production and PGE 2, TNF-
, and IL-1 assays. Production of nitrite in
conditioned media was determined using a method
based on the Griess reaction (Titheradge, 1998).
Levels of PGE2, TNF-, and IL-1 present in each
sample were deter-mined using a commercially
available kit from R&D Systems (Abingdon, UK).
The assay was performed according to the
manufacturers instructions.
Preparation of cytosolic and nuclear fractions. Cells
were collected and washed with phosphate-buffered
saline and suspended in 200 L of lysis buffer [10
mM HEPES (pH 7.9), 10 m M KCl, 0.1 mM EDTA,
0.1 mM ethylene glycol tetraacetic acid (EGTA), 1
mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonyl fluoride (PMSF), and a
protease in-hibitor cocktail]. The cells were allowed
to swell on ice for 15 min; then, 12.5 L of 10% NP-
40 was added. The tubes were agitated on a vortex
for 10 s and then centrifuged for 5 min. The resulting
supernatant represented the cytosolic extract. The
nuclear pellets were resuspended in 50 L of ice-
cold nuclear extraction buffer [20 m M HEPES (pH
mM DTT, 0.5 mM PMSF, and a protease inhibitor
cocktail] and incubated on ice for 1 h with
intermittent vortexing. This nu-clear extract was
centrifuged for 10 min at 15,000 g; the resulting
supernatant represented the nuclear fraction.
Western blot analysis. Western blot analysis was
performed by lysing cells in 20 mM TrisHCl buffer
Phytother. Res. 28:
1218 D.-S. LEE ET AL.
ATTC-3 (forward) and 5-GAT
(pH 7.4) containing protease TAG TAC TGTAGG GTTAAT
inhibitors (0.1 mM PMSF, 5 G-3 (reverse); and -actin: 5-
mg/mL aprotinin, 5 mg/mL TGT GAT GGT GGG AAT
GGG TCA G-3 (forward) and
pepstatin A, and 1 mg/mL 5-TTT GAT GTC ACG CAC
chymostatin). Protein GAT TTC C-3 (reverse).
concentration was determined
using a Lowry protein assay kit
(P5626; Sigma Chemical Co.). DNA-binding activity of NF-B.
An equal amount of protein for Macrophages were pretreated for
each sample was resolved by 12 h with the indicated
12% sodium dodecyl sulfate- concentrations of latifolin and
polyacryl-amide gel then stimulated for 1 h with LPS
electrophoresis and then (1 g/mL). The DNA-binding
electrophoretically transferred activity of NF-B in nuclear
onto a Hybond enhanced extracts was measured using the
chemilumines-cence TransAM kit (Active Motif,
nitrocellulose membrane (Bio- Carls-bad, CA, USA) according
Rad, Hercules, CA, USA). The to the manufacturers instructions.
membrane was blocked with 5%
skim milk and sequentially
incubated with primary antibody Plasmids, transfections, and
(Santa Cruz Biotechnology) and luciferase assays. To con-struct
horseradish peroxi-dase- the antioxidant response
conjugated secondary antibody, element (ARE)-luciferase
followed by en-hanced vector, tandem repeats of
double-stranded oligonucleo-
chemiluminescence detection tides spanning the Nrf2 binding
(Amersham Pharmacia Biotech, site (5-TGACTCA-GCA-3)
Piscataway, NJ, USA). were introduced into the
restriction sites of the pGL2
promoter plasmid (Madison,
WI, USA). All transfection
Reverse transcription- experiments were performed
polymerase chain reaction (RT- using the Lipofectamine reagent
PCR) analysis. Total cellular (Invitrogen) according to the
RNA was isolated using TRIzol manufacturers instructions. For
(Invitrogen, Carlsbad, CA,
USA) extraction according to luciferase assays, cell lysates
the manufacturers instructions. were first mixed with luciferase
Total RNA (1 g) was converted substrate solution (Promega,
to cDNA by using 200 units of Madison, Wisconsin, USA), and
reverse transcrip-tase and 500 luciferase ac-tivity was
ng of oligo-dT primer in 50 m M measured using a luminometer.
TrisHCl (pH 8.3), 75 mM KCl, For each
3 mM MgCl2, 10 mM DTT, and
1 mM dNTPs at 42 C for 1 h.
The reaction was stopped by
heating at 70 C for 15 min, and
3 L of the cDNA mixture was
used for PCR amplification.
PCR was performed in 50 mM
KCl, 10 mM TrisHCl (pH.8.3),
1.5 mM MgCl2, 0.2 mM dNTPs,
2.5 units Taq DNA polymerase,
and 0.1 M each of the specific
primers for iNOS, COX-2, and
-actin. Amplification
conditions included denatur-
ation at 94 C for 3 min for the
first cycle and for 45 s in each
subsequent cycle, followed by
an annealing step at 55 C for
40 s for iNOS, 65 C for 40 s
for COX-2, and 58 C for 45 s
for -actin for 35 cycles. A final
extension was performed at 72
C for 7 min. Primers used were
iNOS: 5-AGC CCA ACA ATA
CAA ATG ACC CTA-3
(forward) and 5-TTC CTG
TTG TTT CTATTT CCT TTGT-
3 (reverse); COX-2: 5-CAC
TCA GTT TGT TGA GTC
experiment, luciferase activity
was determined in tripli-cate
and normalized using -
galactosidase activity for each
sample.
Statistical analysis. Data are
expressed as mean SD for at
least three independent
experiments. To compare three
or more groups, one-way
analysis of variance followed by
the NewmanKeuls post hoc
test was used. Statistical anal-
ysis was performed using
GraphPad Prism software, ver-
sion 3.03 (GraphPad Software
Inc, San Diego, CA, USA).
RESULTS
Effects of latifolin on cell
viability in primary murine
peritoneal macrophages
The structure of latifolin (Fig.
1A) was identified by com-
parison of nuclear magnetic
resonance (Fig. S1) and mass
spectrometry (MS) data with
those of the literature (Sekine et
al., 2009), and the purity of
latifolin (>99%) was evaluated
by HPLC analysis (Fig. S2).
Figure 1B shows the effects of
latifolin on cell viability as
determined by 3-[4,5-
dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide
assay. Cell viability was not
significantly de-creased at
concentrations of latifolin up to
80 M. There-fore, for all
subsequent experiments, the
concentration range of latifolin
used was 10 to 80 M.
Effects of latifolin on
proinflammatory cytokines
Copyright 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 12161223 (2014)
ANTIINFLAMMATORY EFFECT OF LATIFOLIN VIA HEME OXYGENASE-1 EXPRESSION
and expression of iNOS and
COX-2 proteins in LPS-
stimulated primary murine
peritoneal macrophages
We investigated the effects of
latifolin from the MeOH extract
of D. odorifera on the
production of proin-flammatory
mediators and cytokines in
primary murine peritoneal
macrophages. Macrophages
were pretreated with latifolin at
different concentrations for 12 h
and then treated with LPS (1
g/mL) for 18 h. Latifolin de-
creased NO, PGE2, TNF-, and
IL-1 production in a dose-
dependent manner as measured
by enzyme immu-noassay in
primary murine peritoneal
macrophages (Fig. 1CF). In
addition, LPS treatment
significantly increased iNOS
and COX-2 mRNA and protein
expres-sion levels, but
pretreatment of the primary
murine peri-toneal macrophages
with latifolin inhibited iNOS
and COX-2 mRNA and protein
expression (Fig. 2AD).
Effects of latifolin on IB-
levels, NF-B nuclear
translocation, and NF-B
DNA-binding activity in
LPS-stimulated primary
murine peritoneal
macrophages
We attempted to determine
whether latifolin inhibited IB-
phosphorylation and
degradation, thus inhibiting NF-
B (p65) nuclear translocation.
As shown in Fig. 2E, IB- was
degraded after the exposure of
primary murine peritoneal
macrophages to LPS for 1 h.
However, 12-h latifolin
pretreatment (10 to 80 M)
markedly suppressed this LPS-
induced phosphorylation and
degradation of
1219
Figure 1. The chemical structure of latifolin (A) and its effects on viability (B),
nitrite (C), PGE2 (D), TNF- (E), and IL-1 (F) levels in primary murine peritoneal
macrophages. Cells were incubated for 48 h with 10100 M latifolin (B). Cells
were pretreated for 12 h with the indicated concentrations of latifolin and then
stimulated for 18 h with LPS (1 g/mL) (CF). Data represent the mean SD of
*
three experiments. p < 0.05 compared with the group treated with LPS.
3B). Furthermore, latifolin
IB- in a dose-dependent gradually in-creased ARE
activation in a dose-dependent
manner, inhibiting p65 translo- manner (Fig. 3C). We also
cation to the nucleus (Fig. 2E). investigated whether the direct
We also investigated the DNA- effects
binding activity of NF-B in
nuclear extracts from primary
murine peritoneal macrophages
stimulated with LPS for 1 h.
This treatment induced an
approximately four-fold
increase in NF-B DNA-
binding activity, which was
inhibited by latifolin in a dose-
dependent manner (Fig. 2F).

Effects of latifolin on HO-1

protein expression and
nuclear translocation of
Nrf2 in primary murine
peritoneal macrophages

Next, we examined the effects

of latifolin on the HO-1 ex-
pression in primary murine
peritoneal macrophages. Cells
were treated with 1080 M
latifolin for 12 h, which in-
duced HO-1 expression in a
dose-dependent manner (Fig.
3A). The highest level of HO-1
expression was found in cells
exposed to 80 M latifolin.
These cells showed HO-1
induction after 6 h, which
increased in a time-dependent
manner (Fig. 3A). Because
induction of HO-1 is mediated
via Nrf2 nuclear translocation,
we in-vestigated this in the
latifolin-treated cells.
Macrophages incubated with 80
M latifolin for 15120 min
showed in-creased nuclear Nrf2
levels and decreased
cytoplasmic Nrf2 levels (Fig.
of latifolin via Nrf2 signaling
pathway inhibited NF-B
activation and inflammatory
conditions. Macrophages were
pretreated with latifolin at
different concentrations for 2 h
and then treated with LPS (1
g/mL) for 18 h (Fig. S5AD)
and 1 h (Fig. S5E). Latifolin
decreased NO, PGE2, TNF-,
and IL-1 production and NF-
B DNA-binding activity in a
dose-dependent manner (Fig.
S5).
Effects of SnPP on the
inhibition of proinflammatory
mediator production and NF-B
signaling by latifolin in LPS-
stimulated primary murine
peritoneal macrophages
To investigate the role of
latifolin-induced HO-1 in
modulating proinflammatory
mediators, we used SnPP, a
competitive inhibitor of HO
Copyright 2014 John Wiley & Sons, Ltd.
Phytother. Res. 28: 12161223 (2014)
activity. Macrophages were
pretreated with 40 or 80 M
latifolin for 12 h in the presence
or absence of SnPP. The effects
of latifolin in suppressing LPS-
stimulated NO, PGE2, TNF-,
and IL-1 production were
partially reversed by SnPP (Fig.
4AD). We therefore
investigated whether SnPP also
reduced the inhibition of NF-B
translocation by latifolin.
Macrophages were pretreated
with latifolin (40 or 80 M) for
12 h in the presence or absence
of SnPP (50 M) and then
stimulated for 1 h with LPS (1
g/mL). The addition of SnPP
significantly attenuated the
inhibi-tory effect of latifolin on
IB- degradation, NF-B
trans-location (Fig. 4E), and the
DNA-binding activity of NF-
B (Fig. 4F).
1220 D.-S. LEE ET AL.

Figure 2. The effects of latifolin on iNOS (A and C) expression, COX-2 (B and D) expression, IB- phosphorylation and degradation, NF-B
activation (E), and NF-B DNA-binding activity (F) in primary murine peritoneal macrophages stimulated with LPS. Cells were pretreated for 12
h with the indicated concentrations of latifolin and then stimulated with LPS (1 g/mL) for 18 h (AD) and 1 h (E and F). Representative blots of
three independent experiments are shown. As described in the Materials and Methods section, RNA quantification (A and B) and western blot
analyses (C and D) were performed, western blot analyses of IB- and p-IB- in the cytoplasm and NF-B in the nucleus (E) were carried
*
out, and nuclear NF-B binding was investigated (F). The data represent the mean values of three experiments SD. p < 0.05 compared with
the group treated with LPS.
odorifera (Goda et al., 1992). Although latifolin had vari-
ous activities, the unique chemical structure of latifolin led
DISCUSSION us to evaluate its biological activities with various assay
systems in our laboratory. This study examined the
antiinflammatory effects of latifolin isolated from the
Natural products and traditional medicines afford sig- MeOH extract of D. odorifera in LPS-stimulated primary
nificant promise for the identification of bioactive murine macrophages obtained from C57BL/6 mice.
components and their development into drugs for the Inflammation is a complex process regulated by
treatment of various human disorders, including inflam- proinflammatory mediators and cytokines, such as NO,
matory diseases. Flavonoids are a major class of polyphe-
nolic compounds widely distributed throughout the plant PGE2, TNF-, and IL-1 in immune cells. It is important to
kingdom, which possess interesting antiinflammatory inhibit these mediators of inflammatory diseases. NO is a
effects (Bremner and Heinrich, 2005; Paul et al., 2006; free radical that is cytotoxic in inflammatory dis-eases.
Romano et al., 2013b). Latifolin, a member of less en- Lipopolysaccharide-induced iNOS can mediate several
countered open type of neoflavonoids, had been isolated injurious responses, including inflammation (Palmer et al.,
from the heartwood of D. odorifera (Leguminosae). The 1988). The excessive production of iNOS-derived NO has
heartwood of D. odorifera, T. Chen in Chinese traditional been shown to influence various inflammatory disorders.
medicine, was used in China and Korea for the treatment of TNF- and IL-1 are well-known proinflammatory
blood stagnation syndrome, ischemia, swelling, necro-sis, cytokines and play a crucial role in inflammation (Smith et
and rheumatic pain. Flavonoids isolated from D. odorifera al., 1997). Plant-derived compounds belonging to different
have shown various biological activities, includ-ing chemical classes, such as polysaccharides (Yu et al., 2013),
antiallergic, antitumor (Iwashita et al., 2000; Kim et al., terpenes (Ferrari et al., 2013), phenols (Romano et al.,
2001), antioxidant (Yu et al., 2007), and vasodilatory effects 2013a), lignans (Jin et al., 2012), saponins (Lai et al.,
(Yu et al., 1995). Other studies showed that several 2013), and flavo-noids (Chao et al., 2010) have been shown
constituents from D. odorifera had antiinflammatory to inhibit nitric oxide production in macrophages. In this
activity (Chan et al., 1998; Goda et al., 1992), but no study study, we ini-tially suggested that the MeOH extract of D.
has yet been published on the mechanism of anti- odorifera suppressed both NO and PGE2 production in
inflammatory active components from D. odorifera. Pre- RAW264.7 cells (Fig. S3). Latifolin also suppressed levels
vious phytochemical studies have reported the isolation of
flavonoids, quinines, and phenolic constituents from D.

Copyright 2014 John Wiley & Sons, Ltd. Phytother. Res. 28: 12161223 (2014)
ANTIINFLAMMATORY EFFECT OF LATIFOLIN VIA HEME OXYGENASE-1 EXPRESSION 1221
Figure 3. The effects of latifolin on HO-1 protein levels (A), Nrf2 nuclear translocation (B), and ARE activation (C) in primary murine perito-neal
macrophages. Cells were incubated with the latifolin in a concentration-dependent (10, 20, 40, and 80 M) for 12 h and time-dependent (0, 3, 6,
12, 18, and 24 h) manner with 80 M latifolin of HO-1 protein expression (A). Cells were treated with 80 M latifolin for 0120 min
(B) and treated with latifolin for 120 min (C). Nrf2 protein was detected by western blot analysis, and representative blots of three indepen-dent
experiments are shown. Quiescent cells transiently transfected with ARE-luciferase or control vector were incubated for 1 h with the indicated
concentrations of latifolin in the presence of 5% FBS. Fold induction of luciferase activity in each cell lysate was calculated by normalizing for
*
transfection efficiency and dividing by the control value. The data represent the mean SD of three experiments. p < 0.05 compared with the
control group.

Figure 4. The effects of SnPP on nitrite (A), PGE 2 (B), TNF- (C), and IL-1 (D) levels, and IB- degradation, NF-B translocation (E), and
NF-B DNA-binding activity (F) in latifolin-pretreated LPS-stimulated primary murine peritoneal macrophages. Macrophages were pretreated
for 12 h with latifolin (40 or 80 M) in the presence or absence of SnPP (50 M) and then stimulated for 18 h (A, B, C and D) and 1 h (E and F)
with LPS (1 g/mL). The representative blots of three independent experiments are shown. The data represent the mean SD of three
* ** #
experiments. p < 0.05 compared with the control group; p < 0.05 compared with the group treated with LPS alone; p < 0.05 compared with
the group treated with latifolin and LPS.

Copyright 2014 John Wiley & Sons, Ltd. Phytother. Res. 28: 12161223 (2014)
1222 D.-S. LEE ET AL.
can activate Nrf2 by directly
of proinflammatory mediators, binding to Keap1, leading to the
cytokines, and enzymes on LPS- induction of HO-1 expression.
induced inflammatory Nuclear transcription factor-E2-
conditions in RAW264.7 related factor 2 subsequently
macrophages (Fig. S4). translocates to the nucleus
Therefore, we experimented where it forms heterodimers
whether latifolin, a major with small Maf proteins and
neoflavonoid component of D. alters gene expression via
odorifera, inhibited the interaction with an ARE (Kaspar
production of proinflammatory et al., 2009). The
antiinflammatory action of HO-
mediators, cytokines, and 1 is mediated by the inhibition
enzymes on LPS-induced in- of the production of
flammatory conditions in proinflammatory cy-tokines and
murine macrophages, and then,
all subsequent experiments were chemokines, such as NO, PGE2,
therefore carried out in primary TNF-, IL-1, and IL-6,
murine peritoneal macrophages through NF-B pathways in
activated macro-phages. In
instead of RAW264.7 addition, the redox-dependent
macrophages. transcription fac-tors such as
Lipopolysaccharide exerts its Nrf2, which is a master
inflammatory effects via the regulator of the NF-B
activation of NF-B, a tran- signaling, have been shown to
scription factor that regulates mediate HO-1 induction (Itoh et
the expression of various genes al., 1997). Therefore, we next
including iNOS, COX-2, TNF- examined whether latifolin
, and IL-1. Under normal induced HO-1 expression via
conditions, the NF-B dimers, Nrf2 translocation in murine
p50 and p65, are bound to IB- peritoneal macrophages. In the
in the cytoplasm (Magnani et present study, HO-1 levels were
al., 2000). However, in the increased by latifolin in a dose-
presence of proinflammatory dependent and time-dependent
stimuli, IB- is phosphorylated manner (Fig. 3A) through the
and degraded, allowing NF-B
translocation to the nucleus,
where it binds to target sites and
induces expression of
proinflammatory mediators (Li
and Verma, 2002). We examined
the effects of latifolin on IB-
phosphorylation and
degradation, and NF-B p65
subunit translocation. Following
treatment with latifolin, LPS-
induced IB- degradation, NF-
B ac-tivation, and NF-B
DNA-binding activity were
suppressed in primary murine
macrophages (Fig. 2).
Heme oxygenase-1 has been
recognized for its major im-
munomodulatory and
antiinflammatory properties in a
number of inflammation
models. Heme oxygenase-1
activation has various effects,
including the degradation of the
prooxidant heme, generation of
antioxidant bili-verdin and BR,
and release of antiinflammatory
CO (Kapturczak et al., 2004).
Nuclear transcription factor-E2-
related factor 2, a regulator of
the antioxidant and
antiinflammatory response, is
inactivated in the cytoplasm
through binding to Kelch-like
ECH-associated protein
(Keap1). Nuclear transcription
factor-E2-related factor 2 is well
known that modulates the
transcriptional activation of HO-
1 gene expression (Choi and
Alam, 1996). Recent studies
have shown that phytochemicals
 activation of Nrf2 (Fig. 3B).
Furthermore, we found that
latifolin induced Nrf2
translocation into the nucleus,
where it was involved in
increasing ARE transcriptional
activity (Fig. 3C). The inhibition
of HO-1 by SnPP, an HO activity
inhibitor, partially reversed the
inhibitory effects of latifolin on
LPS-induced NO, PGE2, TNF-,
and IL-1 levels (Fig. 4AD).
Moreover, treatment with latifolin
inhibited LPS-induced IkB-
degradation and NF-B nuclear
trans-location, and these effects
were markedly reduced in the
presence of SnPP (Fig. 4E and F).
These results indicated that the
antiinflammatory effects of
latifolin were associated with HO-
1 expression and activity. Nuclear
transcription factor-E2-related
factor 2 plays a critical part in
basal activ-ity and coordinated
induction of genes encoding
numerous antioxidant and phase
II detoxifying enzymes and
related proteins, such as HO-1,
glutathione, catalase, superoxide
dismutase, glutathione-S-
transferase, -glutamyl cysteine
li-gase, UDP-
glucuronosyltransferase,
NAD(P)H:quinone
oxidoreductase-1, glutamate
cysteine ligase, glutathione
peroxidase, glutathione reductase,
and thioredoxin (Choi et al.,
2007; Dinkova-Kostova et al.,
2002). These phase II detoxifying
enzymes and related proteins play
important roles in protecting cells
from free radical and oxidative
stress imposed by reactive oxygen
species and NO. There-fore, in
our Fig. 4, SnPP partly attenuated
the LPS-induced cell responses,
meaning that HO-1 expression as
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Conflict of Interest
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