Suffruticosol A isolated from Paeonia lactiflora seedcases attenuates airway inflammation in mice induced by cigarette smoke and LPS exposure

Journal of Functional Foods 17 (2015) 774–784

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Suffruticosol A isolated from Paeonia lactiflora seedcases attenuates airway inflammation in mice induced by cigarette smoke and LPS exposure Hyung Won Ryu a,1, Hyuk-Hwan Song b,1, In-Sik Shin c, Byoung Ok Cho d, Seong Hun Jeong e, Doo-Young Kim a, Kyung-Seop Ahn a, Sei-Ryang Oh a,* a

Natural Medicine Research Center, KRIBB, 30-Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si 363883, Republic of Korea b R&D Team, Agency for Korea National Food Cluster (AnFC), 460 Iksan-daero, Iksan, Jeonbuk 507-749, Republic of Korea c College of Veterinary Medicine, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea d Ato Q&A corporation, 303 Cheonjam-ro, Wansan-gu, Jeonju 560-759, Republic of Korea e Namhae Garlic Research Institute, Namhae 668-812, Republic of Korea

A R T I C L E

I N F O

A B S T R A C T

Article history:

Resveratrol oligomers from the extract of Peaonia lactiflora seeds were investigated for the

Received 2 February 2015

first time and their potential role as nutraceuticals to treat airway inflammation based on

Received in revised form 8 June

their anti-inflammatory activity examined. After the isolation of five major peaks (suffruticosol

2015

B, suffruticosol A, trans-ε-viniferin, trans-gnetin H, and cis-suffruticosol D) of a P. lactiflora extract

Accepted 8 June 2015

using a one-step high-speed counter current chromatograhy (HSCCC) process, the anti-

Available online

inflammation activity of each compound was evaluated in the LPS-stimulated RAW264.7 macrophages. Suffruticosol A showed potent levels of inhibition in the expression/

Keywords:

production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in the assay.

Anti-inflammation

Additionally, suffruticosol A exhibited a significant suppression of the increased inflam-

Airway inflammation

matory cell count, protein content, pro-inflammatory cytokines and the production of reactive

Counter-current chromatography

oxygen species in the bronchoalveolar lavage fluid of acute airway inflammation murine

Paeonia lactiflora

model, in which cigarette smoke and LPS exposure were both used as pro-inflammatory

Resveratrol oligomers

stimuli. These experimental results indicate that suffruticosol A has significant effects both in vitro and in vivo to be a potential candidate for airway inflammation therapeutics based on its anti-inflammatory activity. © 2015 Published by Elsevier Ltd.

* Corresponding author. Natural Medicine Research Center, KRIBB, 30-Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si 363883, Republic of Korea. Tel.: +82 43 240 6110; fax: +82 43 240 6029. E-mail address: [email protected] (S.-R. Oh). 1 These authors equally contributed this work. Chemical compounds: Resveratrol (PubChem CID: 445154); Suffruticosol B (PubChem CID: 10652146); Suffruticosol A (PubChem CID: 5321548); trans-ε-Viniferin (PubChem CID: 5315233); trans-Gnetin H (PubChem CID: 9852931); cis-Suffruticosol D (PubChem CID: 46830468). http://dx.doi.org/10.1016/j.jff.2015.06.036 1756-4646/© 2015 Published by Elsevier Ltd.

Journal of Functional Foods 17 (2015) 774–784

1.

Introduction

Chronic inflammatory respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) have continuously increased its prevalence in the recent decade (Welte & Groneberg, 2006). Although medicinal treatments that can alleviate the symptoms of these diseases exist, active research efforts are currently focusing on functional foods that possess the ability to ameliorate symptoms of these diseases. Respiratory disease is characterized by a loss of lung function and abnormal inflammatory cell infiltration and closely related with the exposure to various stimuli such as cigarette smoke, air pollutants and chemicals (O’Donnell et al., 2006). In particular, cigarette smoke is believed to be an important factor in the development of COPD. Cigarette smoke induces the recruitment of inflammatory cell such as neutrophil and macrophage via release of proinflammatory cytokines and chemoattractants (Shin et al., 2014). Inflammatory cells release reactive oxygen species (ROS), proinflammatory cytokines and tissue lysis enzyme including matrix metalloproteinase and elastase. These mediators aggravate airway inflammation and induce the destruction of normal alveolar structure (Barnes, 2014). Therefore, the inhibition of the inflammatory response is considered an important factor in controlling respiratory disease (Angelis et al., 2014). Indeed, roflumilast, a PDE4 inhibitor, is a recommended medication for treating COPD via inhibiting airway inflammatory responses (Wedzicha et al., 2013). Many researchers have also developed therapeutic materials for controlling respiratory disease with a focus on reducing inflammatory response (Bai et al., 2011; Yang et al., 2012). Peony (Peaonia lactiflora) is an herbaceous perennial flowering plant native to central and eastern Asia (Choi et al., 2011). It has been used for many years as a traditional medicine for the treatment of abdominal pain and syndromes, such as abdominal muscle stiffness (Tsai, Wu, Liu, Wu, & Tsai, 2005). Previously, monoterpene glucosides (roots), flavonoids (flowers), tannins (fruits), stilbenes (seeds), triterpenes and steroids (roots, root cortex, rhizomes, leaves, flowers, and callus tissues) have been isolated from this plant (He et al., 2010a). Among the many constituents, the stilbenes have been shown to exhibit a more potent bioactivity than the monoterpene glucosides, flavonoids, tannins, triterpenes and steroids in various tissues of P. lactiflora (Choi et al., 2011; He et al., 2010a; Tsai et al., 2005; Yuk et al., 2013) and have inspired significant interest for drug development due to their potential for usage in pharmacotherapeutics (Wan, Wang, Yang, Wang, & Kong, 2011). Resveratrol oligomers, a type of stilbene, have attracted interest because of their multi-faceted chemical and biological activities (Shen, Wang, & Lou, 2009; Snyder, Gollner, & Chiriac, 2011), including tyrosinase (Ohguchi et al., 2003), neuraminidase (Yuk et al., 2013), ecdysteroid antagonist activity (Sarker, Whiting, & Dinan, 1999), and α-glucosidase (Choi et al., 2009). In the following study, we describe a pioneering method for the successful preparative separation and identification of resveratrol oligomers from 60% EtOH extracts of P. lactiflora seedcases in a one-step process using HSCCC and examine their anti-inflammatory activities in vitro and in vivo to evaluate their pharmacological potential as candidates for airway inflammation treatment.

2.

Materials and methods

2.1.

Reagents and materials

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Analytical grade n-hexane, ethyl acetate and methanol for the HSCCC separation were purchased from Fisher Chemical (Loughborough, UK). Distilled deionized water was prepared using a Milli-Q system (18 MΩ) (Millipore, Bedford, MA, USA). The seedcase samples of P. lactiflora were collected from a farm in Uiseong-gun, South Korea, in October 2011 (Gyongsangbuk-do Agricultural Research & Extension Services). Voucher specimens (KHPark120906) were identified by Prof. Jae-Hong Park (Institute of Life Science and Biotechnology, Kyungpook National University, Korea).

2.2.

Apparatus

Ultra-performance liquid chromatography (UPLC) analysis was performed using an ACQUITY UPLC™ system (Waters Corporation, Milford, MA, USA) equipped with a binary solvent delivery manager, a photodiode array (PDA) and a sample manager coupled to a Micromass Q-TOF Premier™ mass spectrometer equipped with an electrospray interface (Waters Corporation, Milford, MA, USA). The corona-charged aerosol detector (CAD) was a Corona Ultra CAD from Thermo Fisher Scientific (Chelmsford, MA, USA). The nebulizer gas was compressed nitrogen. The gas pressure and temperature were maintained at 35 psi and 25 °C, respectively. The HSCCC instrument used in our study was a TBE-1000A HSCCC system (Tauto Biotechnique Company, Shanghai, China) equipped with a tri-rotor column (1000 mL; coil diameter, 190 mm; tube diameter, 3.0 mm; revolution radius, 126 mm). The β-value of the preparative column varied from 0.59 at the internal layer to 0.75 at the external layer. The mobile phase was delivered using a Tauto TBP5003 pump (Tauto Biotechnique Company, Shanghai, China). The temperature of the HSCCC centrifuge separation column was maintained at 25 °C using a Model SH-WB7R constanttemperature controller (Samheung instrument, Sejong, Korea). Continuous monitoring of the effluent was achieved with a Chrom Tech model-500 detector (Chrom Tech, Inc., Apple Valley, MN, USA) at a 280 nm wavelength, and an autochro-2000 data module (Young Lin instrument Co., Ltd, Anyang-si, Korea) was employed to record the autochro-WIN software (version 2.0, Young Lin instrument Co., Ltd, Anyang-si, Korea) chromatogram.

2.3.

Sample preparation

Hand-peeled seedcases (180 g) of P. lactiflora were extracted with 60% ethanol (4 L × 3) by ultrasonication for 30 min at 25 °C. The 60% ethanol extract was concentrated using a rotary evaporator (35 °C) to obtain the crude extract (23.6 g). The residue was stored at −20 °C before use.

2.4. Selection of the two-phase solvent system for highspeed counter current chromatograhy Measurements of the K values of the target compounds were performed as follows: the crude extract (10 mg) was weighed

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into a 25-ml glass tube, and 5 mL of each phase of a preequilibrated two-phase solvent system was added. The glass tube was then shaken vigorously for 5 min to equilibrate the sample. After the contents had settled, 1 mL of each phase was transferred to two separate test tubes. An aliquot of each phase (1 mL) was subsequently analysed by UPLC-CAD chromatography because although all of the compounds contained resveratrol chemotypes, their λmax values were relatively different. Compounds 3, 4 and 5, each of which contained a stilbene unit, absorbed much more strongly at 320 nm than at 280 nm. Conversely, the suffruticosol derivatives (1 and 2) absorbed almost 10 times less strongly at 320 nm than at 280 nm. The K value was analysed using the following equation:

K U L = AU AL

(1)

where AU and AL are the peak area of the compounds in the upper and lower phase of the UPLC chromatograms. If the upper phase was used as the stationary phase, the K values were equal to KU/L. For the separation factor (α), the relationship between the change in the pair peak and the following peak can be described by the following equation:

α = Ka K b

(2)

where Ka and Kb are the K values for each compound, and Ka > Kb (Zhong, Wan, Ding, Wu, & Xie, 2014).

2.5.

HSCCC separation

In each separation, the coiled column of the TBE-1000A (1000 mL) was first entirely filled with the upper phase as the stationary phase. The lower phase was then pumped into the head end of the column at a flow rate of 6.0 mL/min while the apparatus was run at a revolution speed of 600 rpm. After the mobile phase front emerged and hydrodynamic equilibrium was established in the column, the sample solution (10 mL) containing 500 mg of crude extract was introduced through the injection valve (Kang, Ha, Chun, Kang, & Kim, 2012). There were multiple attempts using trial and error to adjust a proper concentration of the enriched resveratrol chemotype extracts to observe the particularly different λmax values (Yuk et al., 2013). For the entire duration of the experiment, the separation temperature was controlled at 25 °C. The effluent was continuously monitored at 290 nm using a UV–Vis detector and automatically collected in 18-mL test tubes for 3 min using a fraction collector. The chromatogram was recorded immediately following sample injection. The peak fractions were collected according to the elution profile. Following the completion of the separation procedure, the solvents were expelled from the column. The numerical values for the retention of the stationary phase for the solvent systems with the volume ratio of 2:5:2.5:5 (v/v/v/v) were found to be close to one other (73.0%). The retention of the stationary phase (SF) was defined as the retained stationary phase volume (VS) divided by the total capacity of the multilayer coiled column (VC), as follows (Zhong et al., 2014):

SF = VS VC = (VC − VSF ) VC

(3)

2.6.

Cell culture

RAW264.7 macrophage cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA) and were maintained in a humidified incubator at 37 °C with 5% CO2.

2.7.

Cytotoxicity assay

To measure cell viability, we used an EZ-Cytox cell viability assay kit (DAEIL lab, Seoul, Korea). RAW264.7 cells were cultured in a 96-well plate at a density of 2 × 105 cells/mL for 24 h. The cells were subsequently treated with various concentrations of each compound (10, 20, 40, 60, 80, and 100 µM). After culturing for 24 h, 10 µL of the kit solution was added to each well and incubated for 4 h at 37 °C and 5% CO2. The cell viability was determined in terms of the formazan production by measuring the absorbance at 480 nm using an ELISA reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The reference wavelength was 650 nm. Cell viability was determined relative to the untreated control cells.

2.8.

Measurement of NO production

RAW264.7 cells were cultured in a 96-well plate at a density of 2 × 105 cells/well for 24 h. After incubation, the cells were pretreated with various concentrations of each compound (10, 20, and 40 µM) for 1 h and were treated with 1 µg/mL of LPS (Sigma-Aldrich) for an additional 16 h. The culture supernatant was collected at the end of the culture period for a nitrite assay. The culture supernatant (100 µL) was mixed with an equal volume of Griess reagent (Sigma-Aldrich) in a 96-well plate. After a 15-min incubation period at room temperature, the absorbance was measured at 540 nm. The concentration of nitrite was calculated using a standard curve produced from known concentrations of sodium nitrite dissolved in DMEM. The results are presented as the mean ± SD of four replicates of one representative experiment.

2.9.

Western blotting

RAW264.7 cells were cultured in a 100-mm dish at a density of 2 × 105 cells/mL for 24 h. After incubation, the cells were pretreated with various concentrations of each compound (10, 20, and 40 µM) for 1 h and were treated with 1 µg/mL of LPS for the indicated times. The cells were harvested and lysed by cell lysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP40, 0.02% NaN3, and 1 mM PMSF) containing a protease inhibitor cocktail (Sigma-Aldrich) for 30 min on ice, and the cell extracts were then centrifuged. After quantification of the protein concentration, equal amounts of protein were separated in SDS–polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond ECL Nitrocellulose; Amersham Biosciences, Bucks, UK). The membranes were then blocked with 5% nonfat dried milk in TBS-T (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and incubated with the target

Journal of Functional Foods 17 (2015) 774–784

antibodies overnight at 4 °C. After incubation, the membranes were washed, incubated with HRP-conjugated secondary antibody for 2 h at room temperature, and then washed again. The protein bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Bucks, UK).

2.10.

Measurement of TNF-α, IL-6, and IL-1β

The quantities of TNF-α, IL-6, and IL-1β in the cell supernatant were measured using an ELISA kit (R&D Systems) according to the manufacturer’s protocol. The results are presented as the mean ± SD of three replicates from one representative experiment.

2.11. LPS and cigarette smoke induced airway inflammation mouse model Specific pathogen-free male C57BL/6N mice (6 weeks old, weight 20–25 g) were purchased from Koatech Co. (Pyeongtaek, Korea) and used after 2 weeks of quarantine and acclimatization. The mice were given sterilized tap water and standard rodent chow. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-15010). The mice were divided into five groups: normal control (NC), airway inflammation (cigarette smoke with LPS intranasal instillation), Rof [cigarette smoke with LPS intranasal instillation + Roflumilast (10 mg/kg, p.o.)], and suffruticosol A 20 or 40 [cigarette smoke with LPS intranasal instillation + suffruticosol A (20 mg/kg and 40 mg/kg, p.o., respectively)]. The smoke was generated from a 3R4F research cigarette (Kentucky reference cigarette, University of Kentucky, Lexington, KY, USA) containing 11.0 mg of total particulate matter, 9.4 mg of tar, and 0.76 mg of nicotine per cigarette. Cigarette smoke exposure (1 puff/min, 35 mL puff volume over 2 seconds, every 60 seconds, eight cigarettes per day) was carried out using a cigarette smoke generator (Daehan Biolink, Incheon, Korea). The mice were subjected to 1 hour of cigarette smoke exposure in the exposure chamber (50 cm × 30 cm × 30 cm) for 7 days. Roflumilast and suffruticosol A were administered to the mice by oral gavage 1 hour before cigarette smoke exposure for 7 days. LPS was intranasally instilled 10 µg dissolved in 50 µL PBS under anaesthesia 1 hour after cigarette smoke exposure on Day 4.

2.12. Inflammatory cell count in bronchoalveolar lavage fluid (BALF) Twenty-four hours after final cigarette smoke exposure, the mice were sacrificed by intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co., Seoul, Korea), and tracheostomy procedures were performed according to a previously established protocol (Shin et al., 2014). To obtain the BALF, icecold PBS (0.5 mL) was infused into the lung and withdrawn via tracheal cannulation three times (total volume 1.5 mL). To determine differential cell counts, 100 µL of BALF was centrifuged onto slides using a Cytospin (Hanil Science Industrial, Seoul, Korea) (200 g, 4 °C, 10 min). The slides were dried, and the cells were fixed and stained using Diff-Quik® staining reagent (B41321A; IMEB Inc., Deerfield, IL) according to the manufacturer’s

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instructions.The supernatant obtained from the BALF was stored at −70 °C for biochemical analysis.

2.13.

Analysis of BALF

The induction of oxidative stress was monitored using 2′,7′dichloroflurorescein diacetate (DCF-DA, Sigma-Aldrich, Carlsbad, CA, USA) according to a previous study (Lee, Shin, Seo, Ha, & Shin, 2011). In brief, the BALF cells were washed with PBS and total cells (5 × 103) were counted. Cells were treated with 20 µM DCF-DA for 10 min at 37 °C. Intracellular ROS activity was quantified by measuring the fluorescence at 488 nm excitation and 525 nm emission on a fluorescence plate reader (Perkin– Elmer, Waltham, MA, USA). To measure neutrophil elastase activity, BALF was reacted with N-succinyl-(Ala)3-p-nitroanilide (Sigma-Aldrich) in 37 °C for 90 min in accordance with the protocol of a previous study (Sakuma et al., 1998). The absorbance was measured at 405 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Additionally, BALF contents were evaluated using a protein assay kit (Bradford assay, Bio-Rad, Hercules, CA, USA). The absorbance was measured at 595 nm using an ELISA reader (Molecular Devices). The levels of IL1β, IL-6 and TNF-α (Invitrogen, Carlsbad, CA, USA) in the BALF were quantified by ELISA according to the manufacturer’s protocol. The absorbance was measured at 450 nm using an ELISA reader (Molecular Devices).

2.14.

Statistical analysis

The data are expressed as the means ± SD. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by a multiple comparison test with the Dunnett adjustment using GraphPad InStat v. 3.0 (GraphPad Software Inc., LaJolla, CA, USA). A p value <0.05 was considered significant.

3.

Results and discussion

3.1.

Optimization of HSCCC conditions

According to the basic tenets of HSCCC (Ito, 2005), when the polarity of the target analyte is unknown, a two-phase solvent system comprising n-hexane/ethyl acetate/methanol/water is employed, and the optimal solvent conditions are subsequently identified by changing the solvent polarity. This solvent system is a basic two-phase solvent system because it provides a wide polarity range (Zhang et al., 2014). The partition coefficients of the target compounds should be in an acceptable range (0.5 ≤ K ≤ 2.0). A smaller K value results in coelution near the solvent front with poor resolution, whereas a larger K value tends to give better resolution but broader, more dilute peaks with a longer elution time. The separation factors (α) were lower than 1.5, which was difficult for the HSCCC to separate theoretically (He et al., 2012; Ito, 1992; Zhao, Han, Li, & Yue, 2013; Zhong et al., 2014). To separate the five major compounds in this experiment, the following four solvent systems were tested by UPLC-CAD, and their partition coefficient (K) values are displayed in Table 1. The K values for compounds

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Table 1 – Spectral characteristics and collected weights of isolated phytochemicals from P. lactiflora. Compounds

Measured [M-H]−

Error (ppm)

Molecular formula

UV–vis Maxima (nm)

[α]D20

Peak area (%)a

Weight (mg)b

Suffruticosol A (1) Suffruticosol B (2) trans-ε-Viniferin (3) trans-Gnetin H (4) cis-Suffruticosol D (5)

679.2007 679.1929 453.1363 679.1968 679.1981

5.7 −5.7 5.5 1.9 0.0

C42H32O9 C42H32O9 C28H22O6 C42H32O9 C42H32O9

227, 282 232, 281 228, 326 237, 325 230, 308

+95 +157 +151 +136 +296

98.9 95.3 95.1 93.7 96.4

100.1 ± 5.5 25.3 ± 1.5 30.3 ± 2.7 224.3 ± 11.2 60.0 ± 4.1

a b

(c0.5,CH3OH) (c0.2,CH3OH) (c0.2,CH3OH) (c0.2,CH3OH) (c0.2,CH3OH)

Peak area percentage of components determined by UPLC-PDA. All compounds were examined in a set of experiments repeated three times.

3–5 were evaluated successfully at 3:5:3:5 (v/v/v/v) solvent systems, but the small K values (K = 0.05 and 0.32) for compounds 1 and 2 were unsatisfactory. The 2:5:2.5:5 (v/v/v/v) systems resulted in greater resolution for compounds 1 (K = 0.63) and 2 (K = 1.25), and the separation seemed dramatically improved compared to that of the 3:5:3:5 (v/v/v/v) systems. Considering the variations of K values of the target compounds in the above solvent systems, a two-step gradient elution was designed: the first step separated suffruticosol B

(1) and suffruticosol A (2) using n-hexane/ethyl acetate/ methanol/water (2:5:2.5:5, v/v), and the second step isolated trans-ε-viniferin (3), trans-gnetin H (4) and cis-suffruticosol D (5) using n-hexane/ethyl acetate/methanol/water (3:5:3:5, v/v/ v/v) (Fig. 1). The K values of the target compounds 3–5 were between 0.5 and 2.0, and the separation factors (α) were large enough for the these compounds to exhibit resolutions relative to one another (α 3,4 = 1.66 and α 4,5 = 1.50) (Appendix: Supplementary material); furthermore, the successful HSCCC

Fig. 1 – (A) HSCCC chromatogram of 60% EtOH extracts of P. lactiflora. (B) Chemical structures of resveratrol and compounds 1–5 isolated from the seedcases of Paeonia lactiflora. Compounds: 1, suffruticosol A; 2, suffruticosol B; 3, trans-ε-viniferin; 4, trans-gnetin H; 5, cis-suffruticosol D.

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separation largely depends on the retention of the stationary phase. In general, the higher the amount of the stationary phase retained in the separation column, the better the peak resolution will be. The retention of the stationary phase for the solvent systems with the volume ratio of 2:5:2.5:5 (v/v/v/v) was much closer to 73.0%. The crude extract (500 mg) was separated and purified in one step using HSCCC with a stepwise elution with a pair of two-phase solvent systems using n-hexane/ethyl acetate/ methanol/water (2:5:2.5:5, v/v/v/v) and (3:5:3:5, v/v/v/v) as the optimum solvent system. Five compounds were well isolated in less than 650 min on the HSCCC chromatogram (Fig. 1). Following UPLC-PDA analysis, the five compounds yielded 25.3 mg of suffruticosol B (1) with a purity of 95.3%, 100.1 mg of suffruticosol A (2) with a purity of 98.9%, 30.3 mg of transε-viniferin (3) with a purity of 95.1%, 224.3 mg of trans-gnetin H (4) with a purity of 93.7%, and 60.0 mg of cis-suffruticosol D (5) with a purity of 96.4%, respectively (Table 1). Interestingly, the values for the molecular weight and retention time (tR) of compound 5 were similar to those of compound 4. The separation with HSCCC-produced compounds exhibited exceptional resolution and purity. This is the first report of the successful use of HSCCC for the concurrent isolation of these five compounds from P. lactiflora seedcases.

3.2.

Structural identification

The structures of compounds 1–5 were identified as suffruticosol B (1), suffruticosol A (2), trans-ε-viniferin (3), transgnetin H (4), and cis-suffruticosol D (5) (Fig. 2A). The isolated compounds were characterized using spectroscopic data, including 1D and 2D-NMR, in comparison with previously published data (Choi et al., 2009, 2011; He et al., 2010b; Yuk et al., 2013). The retention times, mass spectral data of molecular ions, UV–Vis absorption maxima, and optical rotation of all isolated compounds are shown in Table 1.

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using the EZ-Cytox cell viability assay kit. As shown in Figs. 2B and 3A, compounds 2–4 at concentrations of up to 40 µM did not exhibit cytotoxicity against RAW264.7 cells. Based on these results, compounds 2–4 at concentrations less than 40 µM were examined against NO production. The experiment of antiinflammatory effects will focus on Suffruticosol A (1) which emerged to be the most potent NO inhibitor (Appendix: Supplementary material). Furthermore, the most active inflammation inhibitor 1 was proven to be present in the native seedcases in high quantities by an UPLC chromatogram. Suffruticosol A was selected as a potent anti-inflammatory compound for the next step.

3.4. Effects of suffruticosol A on NO production and iNOS expression in LPS-stimulated RAW264.7 cells LPS-stimulated cells showed a significant increase in NO production compared with the non-treated cells; however, suffruticosol A treatment decreased NO production markedly in a dose-dependent manner compared with the LPSstimulated cells (Fig. 3B). In addition, suffruticosol A treatment suppressed iNOS expression induced by LPS treatment in a dose-dependent manner (Fig. 3C–D).

3.5. Effects of suffruticosol A on TNF-α, IL-6, and IL-1β production in LPS-stimulated RAW264.7 cells LPS-stimulated cell exhibited a significant increase in IL-6 production compared with non-treated cells; however, suffruticosol A treatment markedly reduced IL-6 production in a dosedependent manner compared with the LPS-stimulated cells (Fig. 4A). Similar to the results of IL-6 production, LPS-stimulated cell showed significant elevations of TNF-α and IL-1β compared with the non-treated cells. By contrast, suffruticosol A treatment significantly inhibited the elevated TNF-α and IL1β induced by LPS treatment (Fig. 4B and 4C, respectively).

3.3. Effect of isolated compounds on cell cytotoxicity in RAW264.7 cells

3.6. Effects of suffruticosol A on inflammatory responses in airway inflammation mice induced by cigarette smoke and LPS exposure

RAW264.7 cells were treated with various concentrations of isolated compounds 1–5 for 24 h, and cell viability was examined

Cigarette smoke and LPS exposed mice showed a significant increase in the number of inflammatory cells, including

Fig. 2 – Effect of tested compounds on cell viability in RAW 264.7 cells. Cell viability in LPS-stimulated RAW264.7 macrophages treated with compounds 1–4 for 24 h. The error bars represent the mean ± SD.

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Fig. 3 – Effect of suffruticosol A on cell viability (A), NO production (B) and iNOS expression levels (C) in LPS-stimulated RAW264.7 macrophages. The error bars represent the mean ± SD. #p < 0.001 vs. control, *p < 0.05 vs. LPS, **p < 0.001 vs. LPS. (C–D) The iNOS expression levels were determined by western blotting.

Fig. 4 – Effect of suffruticosol A on TNF-α (A), IL-6 (B), and IL-1β (C) production in LPS-stimulated RAW264.7 macrophages. The error bars represent the mean ± SD. #p < 0.001 vs. control, *p < 0.05 vs. LPS, **p < 0.001 vs. LPS.

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neutrophils and macrophages, in the BALF compared with the normal controls. The suffruticosol A-treated mice had a markedly decreased number of inflammatory cells, particularly neutrophils, in BALF compared with the cigarette smoke and LPS exposed mice (Fig. 5A). ROS production markedly increased in the cigarette smoke and LPS exposed mice compared with the normal controls. By contrast, suffruticosol A-treated mice had significantly decreased ROS production compared with the cigarette smoke and LPS exposed mice (Fig. 5B). As shown in Fig. 5C, the BALF contents markedly increased in the cigarette smoke and LPS exposed mice, whereas this was markedly decreased in suffruticosol A-treated mice compared with the cigarette smoke and LPS exposed mice. The result of elastase activity was consistent with those of ROS production and BALF contents. Cigarette smoke and LPS exposed mice had significantly elevated elastase activity compared with the normal control; however, suffruticosol A-treated mice meaningfully reduced elastase activity compared with the cigarette smoke and LPS exposed mice (Fig. 5D). TNF-α in BALF significantly increased in the cigarette smoke and LPS exposed mice compared with the normal control. However, suffruticosol A-treated mice had markedly reduced TNF-α in BALF in a dose-dependent manner compared with

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the cigarette smoke and LPS exposed mice. The result of TNF-α was similar to those of IL-6 and IL-1β. Cigarette smoke and LPS exposed mice had markedly elevated the production of IL-6 and IL-1β in BALF compared with the normal controls; however, suffruticosol A-treated mice significantly decreased IL-6 and IL-1β compared with the cigarette smoke and LPS exposed mice (Fig. 5E, 5F and 5G, respectively).

4.

Discussion

COPD is characterized by an increased number of neutrophils, macrophages and inflammatory mediators in the airways and lung parenchymal (Shin et al., 2014). Currently, COPD is a major global health problem that estimates to be the third leading cause of death in 2020 (Barnes, 2007; Ham et al., 2012). In this study, we investigated the protective effects of suffruticosol A isolated from Paeonia lactiflora seedcases in an airway inflammation model in mice induced by cigarette smoke and LPS exposure. In addition, we evaluated the protective effects of suffruticosol A in LPS-stimulated RAW264.7 cells. Suffruticosol A suppressed the recruitment of inflammatory

Fig. 5 – Effect of suffruticosol A on inflammatory cell count (A), ROS production (B), protein content (C), elastase activity (D), TNF-α (E), IL-6 (F), and IL-1β (G) production in BALF of CS + LPS mice. NC, normal control mice treated with PBS only; CS + LPS, cigarette smoke + LPS intranasal instillation; ROF, roflumilast (10 mg/kg) + cigarette smoke + LPS intranasal instillation; suffruticosol A −20 and 40, suffruticosol A (20 mg/kg and 40 mg/kg, respectively) + cigarette smoke + LPS intranasal instillation. Error bars represent the mean ± SD. ##p < 0.01 vs. control; *p < 0.05 vs. CS + LPS, **p < 0.01 vs. CS + LPS.

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cells with reduction in the ROS production, elastase activity and proinflammatory mediators in cigarette smoke and LPSexposed mice. Consistent with in vivo experiment, suffruticosol A inhibited the increased NO production, proinflammatory mediators, and iNOS expression in LPS-stimulated RAW264.7 cells. Inflammatory cells are closely associated with the development of COPD via the production of proinflammatory cytokines, chemokines, ROS and tissue lysis enzymes, resulting in airway inflammation and alteration (O’Donnell, Breen, Wilson, & Djukanovi, 2006). Among the inflammatory cells, neutrophils are known to produce ROS, cytokines that induce TNFα, IL-1β and IL-6, elastase and matrix metalloproteinases (Hoenderdos & Condliffe, 2013). ROS provoke airway inflammation via activation of specific inflammatory signalling such as NF-κB and MAPKs (Barnes, 2014). Cigarette smoke is believed to be a potent stimulus that recruits inflammatory cells into the airway (Stampfli & Anderson, 2009). Previous studies have demonstrated that cigarette smoke increases the recruitment of neutrophils into the airway by elevating the production of ROS (Onizawa, Aoshiba, Kajita, Miyamoto, & Nagai, 2009; Sato et al., 2008). The results of the 40 mg/kg-treated mice were similar to those of the mice treated with a positive control (roflumilast, a commercial COPD drug). This finding demonstrated that suffruticosol A effectively inhibited the recruitment of inflammatory cells into the airway that were induced by cigarette smoke and LPS exposure. In addition, tissue lysis enzymes have been found to destroy the normal airway structure, which eventually aggravates airway inflammation and reduces lung function (Cataldo et al., 2003; Lungarella, Cavarra, Lucattelli, & Martorana, 2008). Suffruticosol A suppressed the increased elastase activity induced by cigarette smoke and LPS exposure. Proinflammatory cytokines, such as TNF-α, IL-6 and IL-1β, are closely involved in inflammatory responses (Hsu, Fang, Huang, & Yen, 2015) by the activation of inflammatory signalling near stimuli-damaged lesions (Park et al., 2012); therefore, the suppression of proinflammatory cytokine production seems to be an important step in controlling the inflammatory response. In this study, cigarette smoke and LPS-exposed mice exhibited significant increases in protein content of the BALF with an elevation of proinflammatory cytokines including TNFα, IL-1β and IL-6; however, suffruticosol A significantly inhibited the elevated BALF cytokines induced by cigarette smoke and LPS exposure. These effects were considered to be closely related with the development of airway inflammation induced by cigarette smoke and LPS exposure. Consistent with in vivo experiment, the anti-inflammatory effect of suffruticosol A was supported by in vitro experiment. NO production is closely associated with inflammatory processes. NO is synthesized from L-arginine by inducible nitric oxide synthase (iNOS) (Shin et al., 2013). The expression of iNOS increased in inflammatory cells stimulated with various stimuli and overproduced NO in inflammatory lesions (Kwon et al., 2012; Zhang et al., 2013). The iNOS-derived NO is a source of reactive nitrogen species (RNS) that induces oxidative stress in damaged tissue. NO also acts as an inflammatory signalling activator, which aggravates inflammatory responses (Jeon et al., 2014). LPS-stimulated cells showed a significant increase in NO production and iNOS expression compared with the non-treated cells; however, suffruticosol A treatment decreased NO production and iNOS expression markedly in a

dose-dependent manner compared with the LPS-stimulated cells. Similar to results of in vivo experiment, suffruticosol A treatment markedly decreased the levels of proinflammatory cytokines in a dose-dependent manner in LPS-stimulated RAW264.7 cells. These results indicate that suffruticosol A suppresses inflammatory response. Our collective results suggest that suffruticosol A exhibits potent anti-inflammatory activities in LPS-stimulated RAW264.7 cells and in the cigarette smoke- and LPS-exposed airway inflammation model in vivo; therefore, these findings suggest that suffruticosol A could be a potential candidate for treating respiratory disease based on its anti-inflammatory properties. In summary, we have demonstrated that an important medicinal plant, P. lactiflora, contains resveratrol oligomers, which are exhibitors of potent anti-inflammatory effects. This is the first presentation of the HSCCC method that was specifically developed for the preparation and isolation of five resveratrol oligomers from P. lactiflora. The most potent inhibitor was presented in the seedcases at very high concentrations via UPLCPDA; furthermore, the anti-inflammatory effects of suffruticosol A were demonstrated in both in vitro and in vivo experiments. Suffruticosol A inhibited the production of NO, iNOS, and proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPSstimulated RAW264.7 macrophages and the airway inflammation mice model; therefore, this study suggests that P. lactiflora extracts may be a potent therapeutic agent for airway inflammatory diseases, including asthma and/or COPD.

Acknowledgments This work was supported by the KRIBB Research Initiative Program (KGM1221521).

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2015.06.036. REFERENCES

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