TRAF6

Journal of Functional Foods 17 (2015) 476–490

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Boerhavia diffusa L. ethanol extract suppresses inflammatory responses via inhibition of Src/Syk/TRAF6 Ha Van Thai a,1, Eunji Kim a,1, Seung Cheol Kim b,1, Deok Jeong a, Sungjae Yang a, Kwang-Soo Baek a, Yong Kim a, Zubair Ahmed Ratan a, Kee Dong Yoon c, Jong-Hoon Kim d,*, Jae Youl Cho a,** a

Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, South Korea Division of Gynecologic Oncology Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital College of Medicine, Ewha Womans University, Seoul 158-710, South Korea c College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, South Korea d Department of Veterinary Physiology, College of Veterinary Medicine, Biosafety Research Institute, Chonbuk National University, Jeonju 561-756, South Korea b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Boerhavia diffusa L. is a green vegetable used as an herbal medicine in several Asian coun-

Received 19 January 2015

tries for the treatment of hepatitis, kidney stones, liver disorders, nephritis, urinary disorders,

Received in revised form 4 June

and urinary retention. In this study, the anti-inflammatory activity and the molecular in-

2015

hibitory mechanisms of B. diffusa L. ethanol extract (Bd-EE) in vitro and in vivo were evaluated.

Accepted 5 June 2015

Lipopolysaccharide (LPS)-activated peritoneal macrophage RAW264.7 cells and a mouse gas-

Available online

tritis model induced by HCl/EtOH treatment were chosen to determine the in vitro and in vivo anti-inflammatory activities of Bd-EE. This extract (100 and 200 µg/ml) significantly (P < 0.01) inhibited nitric oxide (NO) and tumour necrosis factor (TNF)-α production in peritoneal macrophages and in RAW264.7 cells during LPS exposure at both. The inducible nitric oxide synthase (iNOS) and TNF-α mRNA expression levels were decreased. In addition, nuclear

* Corresponding author. Department of Veterinary Physiology, College of Veterinary Medicine, Biosafety Research Institute, Chonbuk National University, Jeonju 561-756, South Korea. Tel.: +82 63 270 2563; fax: +82 63 270 3780. E-mail address: [email protected] (J.-H. Kim). ** Corresponding author. Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Tel: +82 31 290 7868; fax: +82 31 290 7870. E-mail address: [email protected] (J.Y. Cho). 1 These authors contributed equally to this work. Abbreviations: Bd-EE, Boerhavia diffusa L. ethanol extract; HPLC, high performance liquid chromatography; PGE2, prostaglandin E2; NO, nitric oxide; COX, cyclooxygenase; iNOS, inducible NO synthase; TLR, Toll-like receptor (TLR); NF-κB, nuclear factor-κB; AP-1, Activator protein-1; IKK, IκBα kinase; MyD88, myeloid differentiation primary response protein-88; TRIF, TIR domain containing adapter inducing interferon-β; Syk, spleen tyrosine kinase; EIA, enzyme immunoassay; MTT, (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LPS, lipopolysaccharide; RT-PCR, reverse transcriptase-polymerase chain reaction; CMC, carboxymethyl-cellulose; MAPK, mitogen activated protein kinase; ERK, extracellular signal-related kinase; MKK3/6, MAP kinase kinase 3/6; IRAK1, interleukin-1 receptor-associated kinase 1; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositide 3-kinases; TAK1, TGF-beta activated kinase 1; TRAF, TNF receptorassociated factor; L-NAME, Nω-Nitro-L-arginine methyl ester hydrochloride http://dx.doi.org/10.1016/j.jff.2015.06.004 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 17 (2015) 476–490

Keywords: Boerhavia diffusa L. (Phyllanthaceae) Anti-inflammatory effect Src

levels of p65, p50, c-Fos, and c-Jun transcription factors were reduced by Bd-EE treatment; moreover, it inhibited NF-κB upstream signalling via IκBα, PI3K, Syk, and Src. Furthermore, analysis of AP-1 upstream signalling revealed that the AP-1 activation pathway consisting of TRAF6, TAK1, MKK4/7, and MKK3/6 was also predominantly inhibited by Bd-EE. By HPLC analysis, luteolin was identified as a major ingredient displaying NO inhibitory activity.

Syk

The data provided here strongly suggest that the anti-inflammatory property of Bd-EE is

TRAF6

linked to the suppression of Syk, Src, and TRAF6.

Luteolin

1.

477

© 2015 Elsevier Ltd. All rights reserved.

Introduction

Inflammation plays a critical role in attacking and removing infected pathogens. The activation of inflammatory cells by infected pathogens produces inflammatory mediators such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), and proinflammatory cytokines such as tumour necrosis factor (TNF)-α (Roberts-Thomson, Fon, Uylaki, Cummins, & Barry, 2011). Inflammation is divided into two different stages, the acute and chronic phases. Acute inflammation is the early stage involved in immediately protecting and healing wounds and infections; however, chronic inflammation is induced by prolonged inflammatory responses, leading to a loss of function in various organs. Such chronic inflammatory processes sometimes cause various serious diseases such as cancer, diabetes, atherosclerosis, and arthritis (McGeer & McGeer, 2008). Recently, great efforts have been made to screen potent antiinflammatory drugs for use in the treatment of chronic inflammation. Of many candidates, traditional herbal plants are considered as a good source of anti-inflammatory remedies showing effectiveness and higher safety (Lukhoba, Simmonds, & Paton, 2006). Numerous studies have increased our understanding of Toll like receptor (TLR) signalling pathways (Kurt-Jones et al., 2000). Upon lipopolysaccharide (LPS) treatment, TLR4 is activated to transduce the activation signal into the cytoplasm by recruiting two different adaptor proteins, the myeloid differentiation primary response protein-88 (MyD88) and the Toll/interleukin-1 receptor homology (TIR) domain containing adapter inducing interferon-β(TRIF) protein, through interaction between their TIR domains (Poltorak et al., 1998). Concomitantly, MyD88 or TRIF interact with various signalling proteins such as tyrosine kinases (e.g. Syk), IL-1 receptor-associated kinase family protein kinases (IRAK1 and IRAK4), and TANK-binding kinase 1 (TBK1) (Kobayashi et al., 2002). After the activation of these proteins, complicated signalling cascades composed of phosphatideinositol-3-kinase (PI3K), TNF receptor-associated factor (TRAF6), and transforming growth factor-β-activated kinase 1 (TAK1) trigger the NF-κB activation pathway linked to inhibitor of κB (IκBα) kinase (IKK) and IκBα (Sato et al., 2005), the AP-1 activation pathway linked to mitogen activated protein kinases (MAPKs) [extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK)] (Karin, Liu, & Zandi, 1997), and the interferon regulatory factor 3 (IRF)-3 activation pathway linked to IKKε (Yu et al., 2012a). These three pathways trigger macrophages to release proinflammatory cytokines, other mediators, and type I interferons (IFNs) such as NO, TNF-α, PGE2, and IFN-α.

Boerhavia diffusa L. (Nyctaginaceae) is a green vegetable that is used as herbal medicine and functional food in many countries of the world such as Vietnam, Republic of the Philippines, Brazil, and India for the treatment of hepatitis, kidney stones, liver disorders, nephritis, diabetes, cancer, urinary disorders, and urinary retention (Agrawal, Das, & Pandey, 2011; Mishra, Aeri, Gaur, & Jachak, 2014; Pari & Amarnath Satheesh, 2004; Sreeja & Sreeja, 2009). Various phytochemical studies found that flavonoid glycosides, isoflavonoids (rotenoids), steroids (ecdysteroid), alkaloids, and phenolic and lignan glycosides are major active ingredients in this plant (Mishra et al., 2014). Even though the plant has long been used as functional food and herbal medicine (Prathapan, Vineetha, Abhilash, & Raghu, 2013; Smith, Clegg, Keen, & Grivetti, 1996), there is no extensive study on the anti-inflammatory potential of this herb or its molecular pharmacological mechanisms. Therefore, in this study, we aimed to demonstrate the anti-inflammatory potential and molecular inhibitory mechanisms of B. diffusa L. ethanol extracts using in vitro and in vivo inflammatory models.

2.

Materials and methods

2.1.

Materials

The leaves and twigs of B. diffusa L. were collected at the Popa Mountain National Park, Mandalay Prov., Myanmar, in August 2013. Prof. Yong Dong Kim (Hallym University, Chuncheon, Korea) identified the plant. A voucher specimen (number: Cho S.H. et al. MM408) was deposited in the herbariums of Hallym University (Chuncheon, Korea) and the National Institute of Biological Resources (Incheon, Korea). Standard compounds (kaempferol, luteolin, and quercetin) for HPLC analysis, NGnitro-L-arginine methyl ester (L-NAME), prednisolone, 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), carboxymethylcellulose (CMC), dimethyl sulphate (DMSO), ranitidine (a H2 receptor blocker), and lipopolysaccharide (LPS, E. coli 0111:B4) were from Sigma Chemical Co. (St. Louis, MO, USA). Luciferase constructs containing the binding promoter for NF-κB and AP-1 were used as previously reported (Liang et al., 2013; Shen et al., 2013). Foetal bovine serum (FBS) and RPMI1640 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). RAW264.7 and HEK293 cells were purchased from ATCC (Rockville, MD, USA). All the chemicals were of Sigma grade. The phospho-specific or total antibodies recognizing p50, p65, c-Fos, c-Jun, lamin A/C, IκBα, β-actin, Src, Syk, HA, IKKα/β, p85/PI3K, AKT, JNK, p38, ERK, MKK3/6, MKK3, MKK4/ 7, IRAK1, IRAK4, TAK1, and TRAF6 were obtained from Cell

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Journal of Functional Foods 17 (2015) 476–490

Signaling (Beverly, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2.

Preparation of the ethanolic extract

The leaves and twigs of B. diffusa L. were dried at room temperature and then cut and pulverized. The pulverized plant tissues (125 g) were extracted with 95% ethanol (1500 ml) in an ultrasonic bath, and the extract was evaporated to dryness under reduced pressure to give a 95% ethanol extract (Bd-EE) of B. diffusa L. (3.7 g).

2.3.

Animals

Male BALB/c, 7-week-old mice weighing 19–22 g, were purchased from Daehan Biolink (Eumseong, Korea) and maintained in plastic cages under conventional conditions. Water and the pelleted diet (Samyang, Daejeon, Korea) were supplied ad libitum. All animal studies followed the guidelines established by the Sungkyunkwan University Institutional Animal Care and Use Committee (published in 2009, Approval ID: SKKUBBI 13-6-4).

2.4.

Preparation of the Bd-EE solution

Bd-EE was dissolved in 100% dimethyl sulphate (DMSO) to prepare stock solution (100 mg/ml). The working concentrations were diluted with culture medium into 100 and 200 µg/ ml concentrations for the in vitro assays. For the in vivo study, Bd-EE was suspended in 1.5% CMC.

2.5.

TNF-α production was determined by analysing the NO or TNF-α levels using Griess reagent or ELISA, respectively, as previously described (Green et al., 1982). The OD values at 550 nm (OD550) for NO assay or at 450 nm (OD450) for TNF-α assay were measured using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.8.

The cytotoxic effects of Bd-EE (0–200 µg/ml) incubated for 24 h in RAW264.7 or HEK293 cells were evaluated by a conventional MTT assay, as reported previously (Gerlier & Thomasset, 1986; Kim et al., 2013a). The reaction was stopped by the addition of 15% sodium dodecyl sulphate, as reported previously (Kim & Cho, 2013).

2.9. mRNA analysis by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) To evaluate mRNA expression of iNOS and TNF-α genes, RAW264.7 cells preincubated with Bd-EE for 30 min were additionally treated with LPS (1 µg/ml) for 6 h. Then, total RNA from the cells was isolated with TRIzol Reagent (Gibco BRL, Grand Island, NY, USA), according to the manufacturer’s instructions. Semi-quantitative RT reactions were performed as reported previously (Byeon et al., 2013). Data were expressed as the ratio of the optimal density relative to GAPDH. The primers (Bioneer, Seoul, Korea) used are indicated in Table 1.

Preparation of peritoneal macrophages 2.10.

Peritoneal exudates were prepared from male BALB/c mice by lavage 4 days after an intraperitoneal injection of 1 ml sterile 4% thioglycollate broth (Difco Laboratories, Detroit, MI, USA), according to the previous method (Yoon et al., 2013). After washing the peritoneal macrophage-rich fraction, the primary macrophages (1 × 10 6 cells/ml) were seeded in 100 mm tissue culture dishes for 4 h at 37 °C in a 5% CO2 humidified atmosphere.

2.6.

Cell culture

RAW264.7 and HEK263 cells were cultured in DMEM or RPMI1640 medium supplemented with 10% heat-inactivated FBS, glutamine, and antibiotics (penicillin and streptomycin) at 37 °C under 5% CO2. RAW264.7 and HEK293 cells were sub-cultured every 48 h in RPMI1640 cells and DMEM, respectively, at 37 °C in a 5% CO2 atmosphere. For each experiment, cells were detached using a cell scraper. Under our experimental cell density (2 × 106 cells/ml), the proportion of dead cells was less than 1% according to Trypan blue dye exclusion tests.

2.7.

Cell viability test

NO and TNF-α production

After pre-incubation of RAW264.7 cells or peritoneal macrophages (1 × 106 cells/ml) for 18 h, cells were pre-treated with Bd-EE (0–200 µg/ml) for 30 min and further incubated with LPS (1 µg/ml) for 6 or 24 h, showing maximum production levels of TNF-α and NO. The inhibitory effect of Bd-EE on NO and

Luciferase reporter gene activity assay

HEK293 cells (1 × 106 cells/ml) were co-transfected in 24-well plates with 0.25 µg plasmids containing either NF-κB- or AP1-Luc constructs with the adaptor protein plasmids (MyD88 and TRIF) or plasmid Syk as well as 0.1 µg of β-galactosidase using the polyethylenimine (PEI) method, as reported previously (Shen et al., 2011). After 48 h, the transfected cells were further incubated with Bd-EE for additional 8 h. Luciferase assays were performed using the Luciferase Assay System (Promega, Madison, WI, USA), as reported previously (Dung et al., 2014).

2.11. Preparation of total lysates and nuclear extracts, immunoblot analysis, and immunoprecipitation Tissue or cell lysates from stomach tissues or RAW264.7 cells were prepared, as reported previously (Yang et al., 2014a). Briefly,

Table 1 – Primer sequences used in this study for RTPCR analysis. Name

Direction

Sequence (5′–3′)

TNF-α

F R F R F R

5-TTGACCTCAGCGCTGAGTTG-3 5-CCTGTAGCCCACGTCGTAGC-3 5-CCCTTCCGAAGTTTCTGGCAGCAGC-3 5-GGCTGTCAGAGCCTCGTGGCTTTGG-3 5-CAATGAATACGGCTACAGCA-3 5-AGGGAGATGCTCAGTGTTGG-3

iNOS GAPDH

Journal of Functional Foods 17 (2015) 476–490

the tissues or RAW264.7 cells (5 × 106 cells/ml) were lysed using a sonicator (Thermo Fisher Scientific, Waltham, MA, USA) or a Tissuemizer (Qiagen, Germantown, MD, USA) in lysis buffer for 30 min with rotation at 4 °C. The lysates were clarified by centrifugation at 16,000× g for 10 min at 4 °C and stored at −20 °C until needed. Nuclear lysates were prepared in a three-step procedure, as reported previously (Lee et al., 2014). Soluble cell lysates or nuclear fractions (30 µg/lane) were analysed by immunoblot method. Total or phosphorylated protein levels (p65, p50, c-Jun, and c-Fos), IκBα, IKKα/β, Syk, Src, ERK, p38, JNK, MKK4/7, MKK3/6, IRAK1, IRAK4, TAK1, TRAF6, and β-actin (as a control) were visualized, according to a previously published method (Ko et al., 2013). Immunoprecipitation analysis was performed by a previously reported method (Yu et al., 2012b). Briefly, cell lysates containing equal amounts of protein (500 µg) prepared from LPS (1 µg/ml)-treated RAW264.7 cells (1 × 107 cells/ml) were pre-cleared and incubated with 5 µl antibody to Syk, Src, or TRAF6 overnight at 4 °C. Immune complexes were mixed with 10 µl protein A-coupled Sepharose beads (50%, v/v) and rotated for 3 h at 4 °C. The immunoprecipitates were then visualized by immunoblot analysis.

2.12.

Syk and Src kinase assays

The inhibitory activity of Bd-EE on the kinase activities of Syk and Src was evaluated by kinase profiler service from Millipore (Billerica, MA, USA), as reported previously (Yu et al., 2011).

2.13.

EtOH/HCl-induced gastritis

Acute inflammation of the stomach was triggered by injection of EtOH/HCl, as reported previously (Kim et al., 2013b; Yang et al., 2013b). Bd-EE (20 and 200 mg/kg) or standard control drug [ranitidine (40 mg/kg)] was administered two times in mice.

2.14.

HPLC analysis

The phytochemical features of Bd-EE and standard compounds (quercetin, luteolin, and kaempferol) were analysed by high-performance liquid chromatography (HPLC) (Yang et al., 2012b).

2.15.

Statistical analysis

All of the data presented in this paper are expressed as the means ± SD of experiments performed with six or three samples for in vitro experiments and seven mice for in vivo tests. The rest of the data are representative of three different experiments showing similar results. For statistical comparison, the Mann–Whitney test followed by Dunn’s test for multiple comparisons was employed to analyse non-parametric data. A P < 0.05 was considered statistically significant. All statistical tests were carried out using the computer program SPSS (SPSS Inc., Chicago, IL, USA). Similar data were also obtained by additional independent experiments performed under the same conditions.

479

3.

Results

3.1.

Effect of Bd-EE on the production of NO and TNF-α

We found that Bd-EE was able to modulate the inflammatory response of macrophages after LPS treatment. Bd-EE decreased the production of NO from both LPS-treated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner (Fig. 1A). TNF-α production was also clearly suppressed by Bd-EE in LPS-treated RAW264.7 cells (Fig. 1B). In addition, two standard compounds (L-NAME and prednisolone) reduced the production of NO and TNF-α, respectively (Fig. 1C), in similar levels with previous papers (Cho, Yoo, Baik, Park, & Han, 2001; Kim et al., 2015). The effect of Bd-EE on cell viability was measured in RAW264.7 cells, peritoneal macrophages, and HEK cells using the MTT assay. There was no significant suppression of their viability at 100 and 200 µg/ml (Fig. 1D).

3.2. Effect of Bd-EE iNOS and TNF-α on mRNA upregulation Similar to previous results, NO- and TNF-α-producing inflammatory genes such as iNOS and TNF-α were identified at mRNA level in LPS-stimulated RAW264.7 cells. Bd-EE suppressed the mRNA levels of iNOS and TNF-α in a dose-dependent manner (Fig. 2). Compared with the consistent expression level of GAPDH, Bd-EE completely diminished the expression levels of iNOS and TNF-α at 200 µg/ml.

3.3. Effect of Bd-EE on the transcriptional activation and nuclear translocation of NF-κB and AP-1 We next employed a luciferase assay using HEK293 cells transfected with NF-κB-Luc or AP-1-Luc constructs to examine whether the functional activation of NF-κB or AP-1 are suppressed by Bd-EE. To test this, we established the conditions for luciferase activity by co-transfection with two adaptor molecules, MyD88 or TRIF, as reported previously (Yang et al., 2014b). Bd-EE suppressed MyD88/NF-κB-mediated luciferase activity at 100 µg/ml and 200 µg/ml (Fig. 3A left panel), but it did not reduce TRIF/NF-κB-mediated luciferase activity (Fig. 3A right panel). Under co-transfection conditions of AP-1-Luc with MyD88 or TRIF, Bd-EE only inhibited AP-1/TRIF-mediated luciferase activity (Fig. 3B). In addition, we also confirmed these activities in RAW264.7 cells overproducing TRIF or MyD88 during LPS exposure. As shown in Fig. 3C, LPS-induced luciferase activity mediated by both NF-κB and AP-1 was also diminished by Bd-EE. To reconfirm the above results, we also prepared nuclear fractions to measure NF-κB (p65 and p50) and AP-1 (c-Fos and c-Jun) component translocation levels under LPSstimulated conditions in RAW264.7 cells by immunoblot analysis. In a time-dependent manner, Bd-EE blocked the activation and translocation of NF-κB (p65 and p50) and AP-1 (c-Fos and c-Jun) from 15 to 60 min (Fig. 3D). These results indicate that Bd-EE inhibits the activation and translocation pathway of NF-κB and AP-1 regulated by MyD88 and TRIF.

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TNF-α production (% of control)

(B) RAW264.7 cells Peritoneal macrophages

120 100 80 60

**

**

**

40

**

20 0

LPS (1 μg/ml) -

+

+

+

Bd-EE (μg/ml) -

0

100

200

(C)

100 80 60

20 0

+

+

Bd-EE (μg/ml) -

0

100

200

140

*

80

**

**

60

**

**

40 20

**

+

TNF-α

100

**

40

(D) NO

120 NO and TNF-α production (% of control)

120

LPS (1 μg/ml) -

Cell viability (% of control)

NO production (% of control)

(A)

RAW264.7 cells Peritoneal macrophages HEK293 cells

120 100 80 60 40 20 0

0 0

125 250 500

L-NAME (μM)

0

20

40

80

Prednisolone (μM)

0

100

200

Bd-EE (μg/ml)

Fig. 1 – The effect of Bd-EE on the production of inflammatory mediators. (A–C) The NO and TNF-α levels were analysed using the Griess assay and ELISA, respectively, in culture supernatants of RAW264.7 cells or peritoneal macrophages treated with LPS (1 µg/ml) in the presence of Bd-EE, L-NAME, or prednisolone for 6 or 24 h. (D) Cell viability of RAW264.7 cells, peritoneal macrophages, and HEK293 cells was evaluated using the MTT assay. All of the data (A–D) are expressed as the means ± SD of experiments carried out with six samples. Similar results were obtained in two repeat experiments. L-NAME, Nω-Nitro-L-arginine methyl ester hydrochloride. *P < 0.05 and **P < 0.01 compared to the normal or control groups.

3.4. Effect of Bd-EE on the activation of a NF-κB upstream pathway To determine which proteins were involved in NF-κB activation targeted by Bd-EE, we measured the levels of phosphoproteins after treatment with Bd-EE during LPS exposure. We first detected the levels of phospho- and total forms of IκBα, IKKα/β, Syk, and Src, which are upstream for the activation, and the translocation of p65 and p50 using immunoblot analyses. After preparing whole lysates of RAW264.7 cells stimulated for 5–60 min, we investigated the level of phosphor-IκBα, which

is a critical event for the translocation of NF-κB into the nucleus (Lee, Chain, & Cho, 2009). Bd-EE showed an inhibitory effect on IκBα after 5 min of treatment (Fig. 4A). Similarly, the levels of upstream kinase phospho-forms, responsible for IκBα phosphorylation, were also clearly reduced. Thus, the phosphoprotein levels of IKKα/β and p85/PI3K were diminished after treatment with Bd-EE between 2 and 5 min (Fig. 4B). Because the phosphorylation of p85/PI3K occurs via the Syk and Src tyrosine kinases (Hung et al., 2007), we examined whether Bd-EE is able to modulate the activity of these tyrosine kinases. Interestingly, the Bd-EE extract strongly attenuated the phosphorylation of Syk and Src at early treatment times (2–5 min) (Fig. 4C).

Journal of Functional Foods 17 (2015) 476–490

RAW 264.7 cells (6 h) LPS (1 μg/ml) Bd-EE (μg/ml)

-

+ -

+ 100

+ 200

iNOS TNF-α GAPDH Fig. 2 – The effect of Bd-EE on the expression of inflammatory genes in RAW264.7 cells. RAW264.7 cells were incubated with LPS (1 µg/ml) in the presence of Bd-EE for 6 h. iNOS and TNF-α mRNA levels were determined using semi-quantitative RT-PCR.

3.5.

Effect of Bd-EE on the activation of Src and Syk

Since present data indicated that the inhibition of NF-κB activation by Bd-EE might result from the suppression of Syk and Src, we examined whether Bd-EE blocked these enzymes directly by a direct kinase assay with purified Syk and Src. BdEE almost completely blocked the enzyme activities of Syk and Src by 98 and 97%, respectively, indicating that this extract directly inhibits Syk and Src activity (Fig. 5A). To verify this possibility, we tried several validation experiments. In agreement with Fig. 5A, a reporter gene assay with the NF-κB-Luc fusion under co-transfection with Syk showed that 200 µg/ml Bd-EE was able to block Syk-induced NF-κB activity by up to 50% (Fig. 5B). In addition, under transfected conditions with Src plasmid DNA in the presence of Bd-EE in HEK293 cells, it was found that autophosphorylated Src was reduced by BdEE (Fig. 5C). Finally, Bd-EE clearly altered the binding pattern between phospho-p85/PI3K and Syk or Src, according to immunoprecipitation and immunoblot analyses (Fig. 5D). The significance of Src and Syk in LPS-stimulated inflammatory responses of RAW264.7 cells was also confirmed by treatment with their specific inhibitors. Namely, the Src inhibitor PP2 and piceatannol (Picea) strongly reduced the release of NO and TNF-α by up to 70%–90% under the same conditions (Fig. 5E).

3.6. Effect of Bd-EE on the activation of the AP-1 upstream pathway To determine which proteins were involved in AP-1 activation targeted by Bd-EE, we also measured the levels of ERK, p38, and JNK phospho-proteins. The phosphorylation levels of JNK and p38, but not ERK, were clearly suppressed by Bd-EE in a

481

time dependent manner (Fig. 6A), indicating that these two enzymes could be targeted by Bd-EE. To identify the upstream target participating in the activation of these enzymes, the phosphorylation levels of MKK3/6, MKK4/7, and TAK1 were also examined. As expected, Bd-EE strongly suppressed the phosphorylation of these enzymes at 2–5 min (Fig. 6B), while the enzyme levels of IRAK1 and IRAK4, upstream kinases of TAK1 (Cui et al., 2012), were not reduced (Fig. 6C). The level of adaptor molecule TRAF6, known to be critical downstream of IRAK4 and IRAK1 (Lu, Yeh, & Ohashi, 2008), was therefore examined to exactly verify the inhibitory target. Interestingly, TRAF6 was diminished after treatment with Bd-EE between 2 and 3 min (Fig. 6D). Then, the binding level of phospho-TAK1 to beads with immunoprecipitated TRAF6 was measured by immunoblot analysis. TRAF6 binding to phospho-TAK1 was greatly blocked in the Bd-EE treated group (Fig. 6E). Meanwhile, to determine the functional role of MAPK in LPSinduced NO and TNF-α production, the U0126 (U0), SB203580 (SB), and SP600125 (SP) inhibitors of ERK, p38, and JNK, respectively, were employed. As Fig. 6F shows, SP600125 significantly suppressed NO production and SB203580 significantly reduced TNF-α production, while U0126 strongly suppressed TNF-α production.

3.7. Effect of Bd-EE on EtOH/HCl-induced gastritis symptoms To investigate whether Bd-EE can cure inflammatory symptoms in vivo, we used a mouse gastritis ulcer model generated by EtOH/HCl injection. The EtOH/HCl treatment induced inflammatory lesions in the stomach and Bd-EE suppressed these symptoms in a dose-dependent manner at 20 and 200 mg/ kg, similar to the standard drug ranitidine (RT, 40 mg/kg) (Fig. 7A). Consistent with our in vitro results, this extract also suppressed phospho-protein levels of IκBα, Src, and Syk induced by EtOH/HCl (Fig. 7B).

3.8. HPLC analysis of Bd-EE and effects of its active compound luteolin on NO production To identify the active ingredients of Bd-EE, HPLC analysis using several representative anti-inflammatory flavonoid standards such as luteolin, quercetin, and kaempferol was carried out. Interestingly, Bd-EE displayed peaks with the same retention times as two standard compounds: luteolin (42.0 min) and kaempferol (47.4 min) (Fig. 8A). Indeed, spike experiments performed by co-injection of these standards with Bd-EE enhanced the peak areas of these flavonoids, indicating that these

Fig. 3 – The effect of Bd-EE on transcriptional activation of transcription factors and their nuclear translocation. (A–C) HEK293 cells were co-transfected with plasmid constructs (NF-κB-Luc or AP-1-Luc) and MyD88 or TRIF as well as β-gal (as a transfection control) in the presence or absence of Bd-EE. RAW264.7 cells were transfected with NF-κB-Luc or AP-1-Luc as well as β-gal, and further treated with LPS (1 µg/ml) for 12 h in the presence of Bd-EE. Luciferase activity was measured using a luminometer. (D) RAW264.7 cells (5 × 106 cells/ml) were incubated with LPS (1 µg/ml) in the presence or absence of Bd-EE for the indicated time. After preparing the nuclear fractions, the translocated levels of transcription factors (p65, p50, c-Fos, and c-Jun) were identified using immunoblot analysis. Lamin A/C was used as the loading control. All of the data (A–C) are expressed as the means ± SD of experiments carried out with six samples. Similar results were obtained in two repeat experiments. The rest (D) is representative of three different experiments showing similar results. *P < 0.05 and **P < 0.01 compared to the control groups.

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Journal of Functional Foods 17 (2015) 476–490

(A) Left panel

(A) Right panel

NF-κB -mediated luciferase activity (Fold increases)

NF-κB mediated luciferase activity (Fold increases)

300 250 200

*

150 **

100 50

1000

**

*

800 600 400 200

0

0

Tag2

+

-

-

-

pcDNA

+

-

-

MyD88

-

+

+

+

TRIF

-

+

+

+

Bd-EE (μg/ml)

-

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100

200

Bd-EE (μg/ml)

-

-

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(B) Left panel

(B) Right panel AP-1-mediated luciferase activity (Fold increases)

AP-1-mediated luciferase activity (Fold increases)

60 50 40 30 20 10

12 10 8

4 2 0

+

-

-

-

MyD88

-

+

+

+

Bd-EE (μg/ml)

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+

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Bd-EE (μg/ml) -

-

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pcDNA TRIF

(C) Left panel

AP-1-mediated luciferase activity (fold increase)

NF-κB-mediated luciferase activity (fold increase)

3.0

2.5 2.0

*

1.5 1.0

**

0.5

-

0

100

+

-

+

200 +

(D) 15 min LPS (1 μg/ml) Bd-EE (μg/ml) -

p50 c-Fos c-Jun Lamin A/C

2.5 2.0 1.5

*

1.0 0.5 0.0

0.0

p65

-

(C) Right panel

3.0

LPS (1 μg/ml)

*

6

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Tag2

Bd-EE (μg/ml)

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Bd-EE (μg/ml)

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LPS (1 μg/ml)

-

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Journal of Functional Foods 17 (2015) 476–490

(A)

5 min

LPS (1 μg/ml) Bd-EE (μg/ml) -

+ -

15 min

+ 200

+ 200

+ -

30 min

60 min

+ -

+ -

+ 200

+ 200

p-IκBα IκBα β-Actin (B) LPS (1 μg/ml) Bd-EE (μg/ml) -

2 min

3 min

+ -

+ -

+ 200

+ 200

5 min + -

+ 200

p-p85 p85 p-IKKα/β β-Actin (C) LPS (1 μg/ml) Bd-EE (μg/ml) -

2 min

3 min

+ -

+ -

+ 200

+ 200

5 min + -

+ 200

p-Src Src p-Syk Syk β-Actin

Fig. 4 – The effect of Bd-EE on the activation of upstream signalling enzymes for NF-κB translocation. (A–C) The phospho-protein or total protein levels of Syk, Src, IKKα/β, p85/PI3K, IκBα, and β-actin from LPS-treated RAW264.7 cells in the presence of Bd-EE were determined using antibodies recognizing phospho-specific or total protein. All of the data (A–C) are representative of three different experiments showing similar results.

compounds are likely present in the extract (Fig. 8A). Using the area-based standard curves (Fig. 8B right panel) of these two flavonoids, the luteolin and kaempferol content in Bd-EE was calculated. As shown in Fig. 8B (left panel), the content of luteolin and kaempferol in Bd-EE was revealed as 0.0175 and 0.003%, respectively. We next tested the inhibitory effect of the major flavonoid luteolin in Bd-EE on LPS-stimulated NO production in RAW264.7 cells. As Fig. 8C depicts, luteolin (0 to 10 µM) significantly suppressed NO production in LPS-treated RAW264.7 cells.

4.

Discussion

Considering that the levels of NO and TNF-α were increased in most chronic inflammatory diseases such as ulcerative colitis and Crohn’s disease (Murch, Braegger, Walker-Smith, & MacDonald, 1993), our present data strongly imply that Bd-

483

EE could be effective in acute and chronic inflammatory diseases through inhibition of NO and TNF-α production, leading to its traditional use. It is known that B. diffusa L. has been used ethnopharmacologically as a single or combination treatment with other plants for curing scorpion and snake bites, hepatitis, and nephritis, which are representative inflammatory symptoms induced by various inflammatory mediators (Mishra et al., 2014). Through additional systematic studies, the anti-inflammatory and antinociceptive activities of this plant in abdominal writhing, swelling, renal injury, and liver damage conditions have been demonstrated (Mishra et al., 2014; Mudgal, 1975; Olaleye, Akinmoladun, Ogunboye, & Akindahunsi, 2010; Pareta, Patra, Mazumder, & Sasmal, 2011). Considering these reports, the inhibitory activities (Fig. 1) of NO and TNF-α release by B. diffusa L. in inflammatory conditions could contribute to its ethnopharmacological and immunopharmacological activities. Compared with anti-inflammatory extracts from other plants such as Cerbera manghas (48% inhibition of NO production in LPS-treated RAW264.7 cells at 100 µg/ml), Panax ginseng (24% inhibition at 100 µg/ml), Dipterocarpus tuberculatus (29% inhibition at 100 µg/ml), Rhodomyrtus tomentosa (29% inhibition at 100 µg/ml), and Osbeckia stellata (61% inhibition at 100 µg/ ml) (Jeong et al., 2013; Yang et al., 2012a, 2013a, 2014c), Bd-EE (53% inhibition at 100 µg/ml) showed stronger anti-inflammatory activity than these extracts or similar inhibitory levels to them, implying that Bd-EE can be developed as an anti-inflammatory remedy based on its ethnopharmacological use. To understand the molecular inhibitory mechanism of BdEE, reporter gene assays and nuclear fractionation assays were employed. Indeed, this extract clearly inhibited the activation of NF-κB and AP-1, which are critical inflammationregulatory players (Delerive et al., 1999). Thus, NF-κB- and AP1-mediated luciferase activity in HEK293 cells (Fig. 3A), and translocation of NF-κB (p65/p50) and AP-1 (c-Fos/c-Jun) (Fig. 3C and 3D), which play a major role in NF-κB and AP-1 activation (Magnani, Crinelli, Bianchi, & Antonelli, 2000), were clearly inhibited by Bd-EE, suggesting that these transcription factors could be directly or indirectly targeted by Bd-EE. In a previous report, a similar ethanol fraction was shown to suppress the increased expression of NF-κB in angiotensin II-induced hypertrophy in H9c2 cardiac myoblast cells (Prathapan et al., 2013). Therefore, we explored the possibility of Bd-EE to inhibit the individual upstream signalling kinases in each pathway. Interestingly, the phosphorylation of IκBα was reduced by this extract at an early treatment time (5 min, Fig. 4A), and its upstream proteins including IKK, PI3K, Src, and Syk (Lee et al., 2008) were also diminished by Bd-EE (Fig. 4C). Moreover, direct kinase assays, immunoprecipitation analysis, and overexpression strategies (Fig. 5) revealed that the Bd-EE extract was able to directly suppress Syk and Src, which are important protein tyrosine kinases in the NF-κB pathway (Johnson, Li, & Pearlman, 2008). According to several studies, Src and Syk play varied roles in macrophage-mediated innate immunity, including immune cell cycle, phagocytosis, the production of inflammatory cytokines/mediators, and gene expression (Byeon et al., 2012; Frommhold et al., 2007; Yi et al., 2014). Alternatively, collaboration of the Syk and TLR4/MyD88 pathways results in the sustained degradation of IκBα, enhancing NF-κB nuclear translocation, resulting in the control of infectious materials

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Journal of Functional Foods 17 (2015) 476–490

(B)

(C) HEK293 cells

40

120

80 60 40 20 0

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30

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**

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+

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-

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200

p-p85 -

-

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+

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+

+

+

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-

100

200

β-Actin

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p-p85 IB: Src HC

+ -

NO

120

5 min LPS (1 μg/ml) Bd-EE (μg/ml)

p85

(E) IP : Src

5 min + 200

-

HA 0

(D) Right panel

+ -

-

Src

IP : Syk

LPS (1 μg/ml) Bd-EE (μg/ml) -

+

p-Src

10

Bd-EE (μg/ml) (D) Left panel

pcDNA

20

+ 200

NO and TNF-α production (% of control)

100

NF-κB-mediated luciferase activity (Fold increase)

Kinase activity (% of control)

(A)

TNF-α

100 80 60 **

40

**

**

20

**

0 -

PP2 Picea

-

PP2 Picea

Fig. 5 – The effect of Bd-EE on the activation of Src and Syk. (A) The Syk and Src kinase activities were determined by a direct kinase assay using purified enzymes. The control was set at 100% activity for each enzyme (Syk or Src) with vehicle treatment. (B) Syk-induced luciferase activity was measured by co-transfection of Syk and NF-κB-Luc into HEK293 cells in the presence of Bd-EE (100 and 200 µg/ml) for 12 h. Luciferase activity was measured using a luminometer. (C) HEK293 cells transfected with HA-Src (1 µg/ml) for 24 h were treated with Bd-EE for 12 h. After preparing whole lysates, the levels of phosphorylated or total of p85/PI3K, Src, HA, and β-actin were identified by immunoblot analysis. (D) RAW264.7 cells (5 × 106 cells/ml) were incubated with Bd-EE in the presence or absence of LPS (1 µg/ml) for 5 min. After preparing total lysates, the binding levels of phospho-p85/PI3K to Syk or Src were identified using immunoprecipitation with antibodies for Syk or Src and immunoblot analysis. (E) The NO and TNF-α levels were analysed by Griess assay and ELISA, respectively, in culture supernatants of RAW264.7 cells treated with LPS (1 µg/ml) in the presence of PP2 and Picea for 6 and 24 h. All of the data are expressed as the means ± SD of experiments carried out with three (A and B) or six (E) samples. Similar results were obtained in two repeat experiments. The rest of the results (C and D) are representative of three different experiments showing similar results. IP, immunoprecipitation; IB, immunoblotting; HC, heavy chain; Picea, piceatannol. *P < 0.05 and **P < 0.01 compared to the control groups.

(Lee et al., 2009; Yi et al., 2014). Furthermore, cytokine production levels were reduced in deficient Syk −/− macrophages (Dennehy et al., 2008). Bd-EE clearly ameliorated acute inflammatory symptoms in the HCl/EtOH-treated gastritis mouse model in a dose-dependent manner at 20 and 200 mg/kg (Fig. 7A), and also suppressed the phosphorylation of IκBα, Src, and Syk increased by HCl/EtOH treatment (Fig. 7B). Finally, this study and other groups (Leu, Charoenfuprasert, Yen, Fan, & Maa, 2006; Son, Chung, & Pae, 2014) found that specific inhibitors of these enzymes exhibited strong anti-inflammatory activities (Fig. 5E). Taken together, our results strongly suggest that the inhibition of Syk or Src could be responsible for the antiinflammatory effects of Bd-EE through the inhibition of NFκB, as summarized in Fig. 9. Next, we attempted to investigate the AP-1 signalling mechanism underlying the anti-inflammatory activity of Bd-EE. There was inhibition of Bd-EE in the phosphorylation of JNK and p38, while phosphorylation of ERK was not reduced (Fig. 6A). In

agreement with these results, the activation of MKK3/6, MKK4/7 and TAK1 upstream signalling enzymes was decreased between 2 and 5 min by Bd-EE treatment (Fig. 6B and 6C). In contrast, the decreased levels of upstream enzymes (IRAK1 and IRAK4) responsible for TAK1 phosphorylation indicated that these enzymes are not inhibited by Bd-EE (Fig. 6C), while other IRAK1/4 inhibitors such as kalopanaxsaponin A are known to enhance their protein levels (Joh & Kim, 2011). These data strongly suggest that the target of Bd-EE for the AP-1 pathway inhibition could be a protein simultaneously regulating IRAK1/4 and TAK1 activity, as summarized in Fig. 9. Since the literature has reported that TRAF6 plays a functional role in controlling the activity of IRAK1/4 and TAK1 (Gorjestani, Darnay, & Lin, 2012), we next examined whether Bd-EE modulates the level of TRAF6 and binding capacity with TAK1 by immunoblot and immunoprecipitation analyses. Interestingly, Bd-EE stimulated the degradation level of TRAF6 at 2 and 3 min (Fig. 6D) and also attenuated the complex formation of this protein with TAK1,

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Journal of Functional Foods 17 (2015) 476–490

(A)

(B) 5 min

LPS (1 μg/ml) Bd-EE (μg/ml) -

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+ 200

15 min + -

30 min

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(C)

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p-JNK JNK p-p38 p38

2 min LPS (1 μg/ml) Bd-EE (μg/ml)

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2 min

+ 200

LPS (1 μg/ml) Bd-EE (μg/ml) -

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IRAK4

p-MKK4/7

p-TAK1

+ -

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5 min + -

+ 200

TAK1

MKK4

β-Actin

p-ERK ERK β-Actin (D)

(E)

(F)

LPS (1 μg/ml) Bd-EE (μg/ml) -

+ -

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+ -

NO and TNF-α production (% of control)

3 min

2 min

5 min

+ 200

LPS (1 μg/ml) Bd-EE (μg/ml)

-

+ -

+ 200

p-TAK1

TRAF6

IB: TRAF6

β-Actin

NO

140

IP : TRAF6

HC

TNF-α

120 100 80 *

60 40 **

20 0 -

U0 SB SP

-

U0 SB SP

Fig. 6 – The effect of Bd-EE on AP-1 activation signalling. (A–D) RAW264.7 cells (5 × 106 cells/ml) were incubated with LPS (1 µg/ml) in the presence or absence of Bd-EE for the indicated times. After preparing whole lysates, the levels of total or phosphorylated JNK, p38, ERK, MKK3/6, MKK4/7, MKK3, MKK4, IRAK1, IRAK4, TAK1, TRAF6, and β-actin were identified using immunoblot analysis. (E) The effect of Bd-EE on the formation of the signalling complex between TRAF6 and its downstream substrate TAK1 was analysed by immunoprecipitation and immunoblot methods. (F) The NO and TNF-α levels were analysed by Griess assay and ELISA, respectively, in culture supernatants of RAW264.7 cells treated with LPS (1 µg/ml) in the presence of U0, SB or SP for 6 and 24 h. The data (F) are expressed as the means ± SD of experiments carried out with six samples. Similar results were obtained in two repeat experiments. The rest of the results (A–E) are representative of three different experiments showing similar results. IP, immunoprecipitation; IB, immunoblotting; HC, heavy chain; U0, U0126; SB, SB203580; SP. SP600125. *P < 0.05 and **P < 0.01 compared to the control groups.

indicating that TRAF6 could be a target of this extract. The inhibitory mode of action was also reported for the inhibition of hepatitis B virus replication by ginsenosides mediated by an increase in TRAF6 degradation and subsequent suppres-

(A) Left panel

sion of JNK/AP-1 (Kang, Choi, & Lee, 2013; Yoo et al., 2013). To more precisely clarify the inhibitory action of Bd-EE on TRAF6 degradation, further tests in terms of ubiquitination and proteosomal degradation will be performed in future studies.

(B)

(A) Right panel

Bd-EE (mg/kg) Normal

Vehicle

20

200

RT (40 mg/kg)

Inflamed lesion (% of control)

EtOH/HCl

Stomach lysate

120

HCl/EtOH (150 mM) Bd-EE (mg/kg) -

100 80

+ -

+ 20

+ 200

p-IκBα IκBα

60 **

40

**

**

p-Src Src

20

p-Syk

0 Normal

-

20

200

40

β-Actin

Bd-EE (mg/kg) RT (mg/kg) EtOH/HCl

Fig. 7 – The effect of Bd-EE on EtOH/HCl-induced gastritis. (A) Mice were orally treated with Bd-EE (20 or 200 mg/kg) or ranitidine (40 mg/kg) for 3 days before oral administration of HCl/EtOH. After 1 h, a technician blinded to the treatment conditions took photos (left panel) and measured the area (mm2) of gastric mucosal erosive lesions using a pixel counter (right panel). The appearance of inflamed lesions after treatment with inducer alone was considered as 100%. (B) Levels of phospho- or total forms of IκBα, Syk, and Src in stomach lysates were determined by immunoblot analysis using respective antibodies. The data (A, right panel) are expressed as the means ± SD of experiments carried out with seven mice. Similar results were obtained in two repeat experiments. The rest of the results (A, left panel, and B) are representative of three different experiments showing similar results. RT, ranitidine. **P < 0.01 compared to the control groups.

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Journal of Functional Foods 17 (2015) 476–490

(A) Standard (10 μg/ml each)

Abs (370 nm)

Luteolin Kaempferol Quercetin 42.3 min [9.2] 47.6 min [14.8] 42.0 min [16.3]

Bd-EE (50 mg/ml) 42.0 min [9.6] 47.4 min [2.4]

Quercetin 41.7 min [18.2] Luteolin 41.9 min [20.3] Kaempferol 47.3 min [17.6]

Standard + Bd-EE

Retention time (min) [Area] 20

Area (mAU x min)

(B)

0.015

R² = 0.9994

10 5 0 0 40

0.010

Area (mAU x min)

Content (%)

0.020

Luteolin

15

0.005

0.000

Kaempferol

10

15

20

15

20

Kaempferol

30

R² = 0.9997

20 10 0 0

Luteolin

5

5

10

Standard (μg/ml)

NO production (% of control)

(C) 120 100 *

80

**

60

**

40 20 0 0

2.5

5

10

Luteolin (μM) Fig. 8 – Phytochemical analysis of Bd-EE and the effects of quercetin and kaempferol on NO production in LPS-treated RAW264.7 cells. (A and B) Phytochemical characteristics of Bd-EE compared with standard compounds (quercetin, luteolin, and kaempferol). Compounds were analysed using a high-performance liquid chromatography (HPLC) system equipped with KNAUER components. The contents of luteolin and kaempferol were calculated using standard curves of flavonoids. (C) Inhibitory effects of the major ingredient of luteolin on NO production in LPS-treated RAW264.7 cells. The data (C) are expressed as the means ± SD of experiments carried out with six samples. Similar results were obtained in two repeat experiments. The rest of the results (A and B) are representative of three different experiments showing similar results. *P < 0.05 and **P < 0.01 compared to the control groups.

Journal of Functional Foods 17 (2015) 476–490

487

LPS TLR4 MyD88

TRIF

TRAF6

Bd-EE Src/Syk

Bd-EE

p85/PI3K

TAK1 MKK4/7

MEK3/6

AKT

p38

JNK

IKKα/β

c-fos

c-Jun

IκBα NF-κ κB AP-1

iNOS

Pro-TNF-α

TNF-α

NO

Fig. 9 – Putative pathway for Bd-EE-mediated inhibition of the anti-inflammatory response. Bd-EE suppresses Src and Syk activities required for NF-κB activation and TRAF6 for the AP-1 pathway. Inhibition of Src/Syk kinases and TRAF6 by Bd-EE results in downregulation of the inflammatory gene (e.g., iNOS and TNF-α) expression linked to a decrease in NO and TNF-α production. Bd-EE, Boerhavia diffusa L. ethanol extract; NO, nitric oxide; TNF-α, tumour necrosis factor-α; iNOS, inducible NO synthase; TLR, Toll-like receptor (TLR); NF-κB, nuclear factor-κB; AP-1, Activator protein-1; IKK, IκBα kinase; MyD88, myeloid differentiation primary response protein-88; TRIF, TIR domain containing adapter inducing interferon-β; Syk, spleen tyrosine kinase; LPS, lipopolysaccharide; MKK3/6, MAP kinase kinase 3/6; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositide 3-kinases; TAK1, TGF-beta activated kinase 1; TRAF, TNF receptor-associated factor; IκBα, inhibitor of κBα; IKKα/β, IκB kinase α/β.

Numerous phytochemical studies have increased our understanding of B. diffusa L. pharmacological action. Thus, punarnavine, an alkaloid from ethanol extract, has been found to display antidepressant-like activity in stressed mice by controlling plasma corticosterone levels (Dhingra & Valecha, 2014). It was reported that rotenoids including boeravinone B from root methanol extracts could act as a COX-2 inhibitor (Bairwa et al., 2013) as well as a potent anti-oxidant (Aviello et al., 2011). Eupalitin-3-O-beta-D-galactopyranoside and eupalitin from hexane, chloroform, and ethanol extracts were found to suppress NO and TNF-α production (Pandey, Maurya, Singh, Sathiamoorthy, & Naik, 2005). A lignan compound, liriodendrin, isolated from root methanol extract, was revealed to have a significant calcium (Ca2+) channel antagonistic effect (Lami, Kadota, Kikuchi, & Momose, 1991). Based on these papers, we attempted to identify the active ingredients in Bd-EE using HPLC analysis. Although the above components can be considered as anti-inflammatory ingredients, we preferred to test the peak derived from flavonoids, which are a major representative of the anti-inflammatory compounds in most plants (Georgiev, Ananga, & Tsolova, 2014). Fortunately, we identified corresponding peaks generated by standard flavonoids such as luteolin and kaempferol, and spike experiments with the standard revealed the same areas in Bd-EE (Fig. 8A). Moreover, this

compound exhibited the same NO inhibitory activity under the same conditions (Fig. 8C), suggesting that luteolin is the potential active component in the Bd-EE. However, because large numbers of different chemicals are included in this extract, further isolation of the compounds with anti-inflammatory properties will need to be undertaken by activity-guided fractionation.

5.

Conclusions

We have shown that Bd-EE and its ingredient luteolin strongly inhibit LPS-mediated inflammatory responses, such as the production of NO and TNF-α, and expression of iNOS and TNF-α mRNA during LPS/TLR4 stimulation. Furthermore, this extract markedly suppressed the activation of NF-κB by blocking a series of signalling cascades ranging from Syk/Src to IκBα. Bd-EE also clearly diminished the AP-1 signalling pathway, which is controlled by TRAF6/TAK1. In the in vivo gastritis model, inflammatory lesions in the stomach were also markedly improved with this extract. Therefore, our data strongly suggest that the anti-inflammatory property of Bd-EE linked to the suppression of Syk, Src, and TRAF6 could contribute to the ethnopharmacological role of B. diffusa L.

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Conflict of interest The authors declare no conflicts of interest.

Acknowledgements We are grateful to Drs. Deok Hyo Yoon, Khin Myo Htwe, YoungDong Kim, and Woo-Shin Lee for providing excellent technical assistance. A National Institute of Biological Resources (NIBR) grant funded by the Korean government (ME) and supported by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI12C0050), supported this work.

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