Inhibitory effects of phenolic compounds from Artocarpus styracifolius on respiratory burst of rat neutrophils

Abstract

Context: Searching for polymorphonuclear neutrophils (PMNs) respiratory burst inhibitors is an important topic in the treatment of human diseases associated with inflammation.

Objective: To investigate the inhibitory effects of phenolics isolated from Artocarpus styracifolius Pierre (Moraceae) on respiratory burst induced by phorbol myristate acetate (PMA).

Materials and methods: The anti-respiratory burst activities of eight phenolics (20 µM) were assessed by determining luminol-dependent chemiluminiscence in rat PMNs. Cytotoxicity of active compounds (1–1000 µM) was assayed by Trypan blue dye exclusion method. Cell-free models were employed to evaluate scavenging capacity of active compounds (20 µM) against reactive oxygen species.

Results: The PMA-induced respiratory burst was significantly inhibited (p < 0.05) by six isoprenylated phenolics (AS1–6) at the concentration of 20 µM (below the toxic concentration) with the inhibition rate ranging from 25.0 to 99.6%. The inhibitory potency estimated by IC₅₀ was in the order of AS1 (3.1 µM) >AS6 (5.9 µM) >AS2 (9.1 µM) >AS3 (10.0 µM) >AS5 (29.7 µM) >AS4 (57.7 µM). AS1–4, four isoprenylated flavones, potently quenched superoxide anion, hydroxyl radical, and hydrogen peroxide at the concentration of 20 µM with their scavenging rates in the range of 30.1–78.1%, 35.4–69.7%, and 65.5–86.3%, respectively. In contrast, AS5–6, two isoprenylated 2-arylbenzofurans, showed less effect than that exhibited by AS1–4.

Conclusion and discussion: The isoprenylated phenolics from A. styracifolius can potently inhibit PMA-induced respiratory burst in rat neutrophils without showing cytotoxicity. The inhibitory effects of these isoprenylated phenolics on the respiratory burst might depend on their different types of structure.

• Antioxidant
• isoprenylated phenolics
• oxidative burst
• PMNs
• ROS scavenging activity

Introduction

Polymorphonuclear neutrophils (PMNs) are well known to be the front-line defence cells and play a key role in inflammatory processes evoked by a variety of stimuli, such as bacterial and viral products, cytokines, and growth factors (Ciz et al., 2012). Through the activation of a membrane-associated NADPH oxidase, stimulated PMNs undergo a respiratory burst characterized by an abrupt increase in oxygen consumption and the production of large quantities of superoxide anion () as early as within the first minute of stimulation (Babior, 1999; Hampton et al., 1998). , the first reactive oxygen species (ROS), is generated from a one-electron reduction process mediated by activated NADPH oxidase. This subsequently initiates a cascade of radical chain reactions with the participation of some key enzymes such as superoxide dismutase and myeloperoxidase as the catalyst, which results in the formation of a suit of ROS including hydroxyl radical (HO^•), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl) (Robinson, 2008; Weiss, 1989). In normal physiological situations, ROS is formed under tight regulation and intended to participate in the destruction of viruses and bacteria within phagosomes, which have been considered as the most efficient microbicidal mechanism (Ciz et al., 2012; Daels-Rakotoarison et al., 2002). However, ROS overproduction can further promote the inflammatory process and injure neighboring host cells and tissues by lipoperoxidation, proteolysis or DNA degradation (Braga et al., 2012; Mata-Campuzano et al., 2012; Tsumbu et al., 2011). Growing evidence suggests that an abnormal ROS production attributed to an absent regulation of PMNs respiratory burst is involved in the pathogenesis of various inflammatory disorders such as rheumatoid arthritis (Mirshafiey & Mohsenzadegan, 2008; Shah et al., 2011), atherosclerosis (Cheng et al., 2013; Ding et al., 2013), reperfusion injury (Gutowski & Kowalczyk, 2013; Madamanchi & Runge, 2013), and even cancer (Li et al., 2012). Thus, it is interesting to find antioxidative substances with the ability to inhibit ROS production and/or directly scavenge ROS formed in the process of respiratory burst.

Artocarpus styracifolius Pierre (Moraceae) is an evergreen arbor tree and distributes in the southern part of China (Zhang & Wu, 1998). The roots of this plant are used in folk medicine for the treatment of rheumatoid arthritis, psoatic strain, hemiplegia, and fracture. In our previous experiments, we observed that the ethanol extract of the root bark of A. styracifolius displayed strong inhibitory effects on respiratory burst induced by phorbol 12-myristate 13-acetate (PMA) in rat PMNs (unpublished data). Further phytochemical investigation on this active extract led to the isolation of the major A. styracifolius phenolics (AS), including four isoprenylated flavones, two isoprenylated 2-arylbenzofurans, and two flavan-3-ol derivatives (Ren et al., 2013). Some of the AS were reported to possess antiplasmodial and antitrypanosomal activities (Bourjot et al., 2010). However, there is no information available for the effects of these compounds on PMNs respiratory burst. Thus, the focus of this paper is on such properties of AS. For this purpose, a cellular respiratory burst model reproduced by chemical stimulation of rat PMNs was used to evaluate the inhibitory ability of phenolics against ROS production. In addition, cell-free systems were also used in which ROS and the compounds were brought together in order to find a direct interaction between the phenolic compounds and ROS.

Materials and methods

Chemicals and solvents

All reagents used were of analytical grade. Luminol (3-aminophthalhydrazide), PMA, and Trypan blue (TB) were purchased from Sigma Aldrich (St. Gallen, Switzerland). Quercetin (QC) was obtained from National Institutes for Food and Drug Control. Dimethyl sulfoxide (DMSO) was purchased from Beijing Solarbio S & T Co., Ltd (Beijing, China). Ethylenediaminetetraacetic acid (EDTA) and Tris were purchased from BIO BASIC INC (Markham, ON, Canada). Pyrogallic acid (1,2,3-trihydroxybenene) and CuSO₄ were purchased from Guangdong Shantou Xilong Chemical Factory (Guangdong, China). Lymphocyte separation medium (LSM) was purchased from Shanghai Huajing Bio-tech Co., Ltd. (Shanghai, China). Hanks’ balanced salt solution (HBSS) and fetal calf serum (FCS) were purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd. (Zhejiang, China). Neocuproine (2,9-dimethyl-1,10-phenanthroline hemihydrate), ascorbic acid, hydrogen peroxide, hydrochloric acid, glucose, NaCl, KCl, CaCl₂, MgCl₂, KH₂PO₄, Na₂HPO₄, NaH₂PO₄, Na₂CO₃, NaHCO₃, H₃BO₄, and MgSO₄·7H₂O were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Isolation and structure identification of phenolic compounds

The plant collection and phytochemical study of eight phenolics were carried out according to the procedure described in our previous work (Ren et al., 2013). In brief, the air-dried root bark of A. styracifolius (1.3 kg) was macerated with 95% EtOH three times (10 L for each extraction) at room temperature. The filtrate was evaporated in vacuum to produce a residue (453.2 g). The residue was suspended in H₂O and then partitioned successively with petroleum ether (PE), CHCl₃, and ethyl acetate (EtOAc) to provide PE-soluble, CHCl₃-soluble, and EtOAc-soluble portions, respectively. These three portions were further separated using various chromatographic columns packed with silica gel, Toyopearl HW-40 C, Sephadex LH-20, octadecylsilyl-silica gel, etc. The PE-soluble portion (41.2 g) yielded AS6 (32.0 mg) and CHCl₃-soluble portion (16.6 g) yielded AS4 (120.4 mg), AS5 (10.0 mg), AS2 (17.0 mg), AS3 (10.5 mg), and AS1 (15.0 mg). AS7 (2.2 g) and AS8 (335.7 mg) were obtained from the EtOAc-soluble portion (194.4 g).

By comparing their ¹H-, ¹³C-NMR, and ESI-MS spectral data with those reported in the literature, the known compounds AS1–8 (Figure 1) were identified as styracifolin C (AS1), albanin A (AS2), artocarpone B (AS3), artonin A (AS4), artopetelin B (AS5), lakoochin A (AS6), catechin (AS7), and catechin-3-O-α-l-rhamnopyranoside (AS8). The degree of purity (>98%) was assessed by HPLC-DAD and ¹H-NMR analyses.

Figure 1. Chemical structures of AS and QC (positive control).

Animals

Sprague–Dawley (SD) rats weighing 280–320 g were obtained from the Experimental Animal Center of Jiangxi University of TCM (Jiangxi, China). They were kept in plastic cages at 22 ± 2 °C with free access to pellet food and water and on a 12 h light/dark cycle. This study complied with current ethical regulations on animal research (National Research Council of USA, 1996) and followed principles in the Declaration of Helsinki. All animals used in the experiment received humane care.

Isolation of rat PMNs

Blood from the orbit veins of healthy SD rats was collected into glass tubes containing 1 mL of 1% heparin sodium, then mixed with 2 mL of 4.5% Dextran T500 inverted ×5, and stored at 4 °C for 40 min. Polymorphonuclear neutrophils were isolated according to the method described previously (Bei et al., 1998). Briefly, 6 mL of supernatant was slowly pipetted down the side of a clean 10 mL Eppendorf tube containing 2 mL of LSM and centrifuged (500 × g) for 15 min at 0 °C. The supernatant was aseptically aspirated and discarded. The remaining cells were suspended in 2 mL of special separation liquid (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM NaH₂PO₄, 10 mM HEPES, 5 mM Tris, pH 7.2) and the suspension was centrifuged (1000 × g) for 5 min at 0 °C. The supernatant, mononuclear cell layer and lymphocytes were discarded and then a pellet composed of PMNs and erythrocytes were retained. Erythrocytes were lysed by mixing remained cells with 2 mL of distilled water and vibrating the tube for 20 s. Tonicity was restored by the addition of 2 mL of 1.8% NaCl. The tubes were centrifuged (1000 × g) for 5 min at 0 °C. The cell pellet was washed twice by resuspension in 2% FCS-HBSS and re-centrifugation for 5 min at 0 °C. Cells were enumerated using an electronic particle counter (Coulter Electronics, Inc., Hialeah, FL). Cell viability and differential cell counts were determined by Trypan blue dye exclusion and Wright staining, respectively. Purity of PMNs was >95% and viability >95%. Cells suspended in 2% FCS-HBSS were maintained on ice until used in the various assays described below.

Determination of inhibitory activities against the respiratory burst of rat PMNs

The inhibitory activities of compounds against the respiratory burst of rat PMNs was measured by luminol-dependent chemiluminiscence (CL) assay, as previously described (Liu et al., 2008). Briefly, PMNs (2 × 10⁶) suspended in 200 μL of luminol were incubated with 10 μL of either DMSO (control) or tested samples (final concentration 20 µM) in a luminous cup for 15 min. About 20 μL of PMA (final concentration 0.2 µM) was added and CL was counted per 5 s on a BPCL Model Ultra Weak Chemiluminescence Analyzer (Institute of Biophysics, Academia Sinica, Beijing, China) at 37 °C, 1000 V. The percentage of inhibition was calculated using the following formula: where CL_control is the luminosity peak value of control, CL_sample is the luminosity peak value of sample, and CL_background is the background value of autoluminescence of unactivated PMNs. The 50% inhibitory concentration (IC₅₀) was determined from a dose–response curve. QC, a flavonoid compound well known for its antioxidant property, was used as a positive control in all experiments of this study.

Cytotoxicity assay

Cell viability was determined using the standard Trypan blue dye exclusion method described previously (Koko et al., 2008). In brief, PMNs (1 × 10⁶) suspended in 1 mL of HBSS supplemented with 2% FCS was incubated with 10 μL of either tested samples (final concentrations ranging from 1 to 1000 µM) or DMSO for 30 min at 37 °C. PMNs viability was determined by adding each sample to 112 µL of 0.4% Trypan blue and scoring the first 100 cells encountered in the field of view of a light microscope as either alive or dead based on the uptake of Trypan blue. The 50% cytotoxic concentration (CC₅₀) was calculated from a dose–response curve.

Evaluation for the capacity of scavenging

was generated from a pyrogallol autoxidation system described before (Liu et al., 2005). Briefly, the reaction mixture composed of 10 μL of either tested samples (final concentration 20 µM) or DMSO (control) and 20 μL of pyrogallol (1.0 mM). About 320 µL of luminol (1.0 mM) diluted in carbonic acid-buffered saline solution (CBSS, pH 10.2) was added into the reaction system to trigger the CL reaction. The CL was counted per 1 s on a BPCL Model Ultra Weak Chemiluminescence Analyzer at 37 °C and calculated with the peak value. Scavenging activities (%) were calculated by the following equation: where CL_control is the luminosity of the control, CL₀ is the luminosity of the background, and CL_sample is the luminosity of the tested compounds.

Evaluation for the ability of scavenging HO^•

For hydroxyl radical scavenging effect assays, HO^• was generated by a Fenton-type reaction system described previously (Qin et al., 2003). In brief, reactants were added in turn and mixed as follows: 50 μL of tested samples (final concentration 20 µM) or DMSO (control), 50 μL of CuSO₄ (1.0 mM), 20 μL of ascorbic acid (1.0 mM), 50 µL neocuproine (1.0 mM), and 50 µL H₂O₂ (0.15%, v/v). Then, 780 μL of boric acid-buffered solution (BBS, 0.1 mM, pH 9.0) was added into the mixture to start the CL reaction. The CL was counted per 5 s on a BPCL Model Ultra Weak Chemiluminescence Analyzer at 37 °C. The CL was calculated with the peak value. Scavenging activity was calculated according to Equation (2).

Evaluation for the ability of scavenging H₂O₂

H₂O₂ scavenging activity was assayed according to the method described previously (Qin et al., 2003) with some modifications. In brief, the reaction system consisted of 50 μL of either tested samples (final concentration 20 µM) or DMSO (control) and 50 μL of 0.5% H₂O₂. About 600 µL of luminol (1.0 mM) diluted in CBSS (pH 9.5) was added, which immediately triggered the CL reaction. The CL was counted per 2 s on BPCL Model Ultra Weak Chemiluminescence Analyzer (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) at 37 °C. Scavenging activities (%) were calculated according to Equation (2).

Statistical analysis

SAS 8.0 (SAS Institute, Cary, NC) was used to analyze the results. Data were expressed as mean ± SD of four independent experiments. One-way ANOVA and Turkey’s tests were used to compare mean values between all experimental groups. Values of p < 0.05 were considered to be significant in all cases.

Results

Inhibitory effects of AS on rat PMNs respiratory burst activated by PMA

In this study, respiratory burst was assessed by determining luminol-dependent CL. As shown in Figure 2, the addition of 0.2 µM PMA to the rat PMNs resulted in a rapid and drastic ROS generation, as revealed by a 167-fold rise in CL (measured in arbitrary units) compared to cells during stable stage of self-luminosity. After pretreatments with 20 µM of AS for 5 min, the CL was potentially attenuated by 99.4% for AS1, 99.1% for AS2, 81.8% for AS3, 25.0% for AS4, 39.7% for AS5, and 99.6% for AS6 compared to control cells (p < 0.05). Apart from these six isoprenylated phenolic compounds (AS1–6), the two flavan-3-ol derivatives (AS7 and AS8) failure to take effects as the inhibitors of rat PMNs respiratory burst. The differences in the potency for the reduction of PMA-stimulated rat PMNs respiratory burst were observed among these six active isoprenylated phenolics. AS1, AS2, and AS6 offered the strongest inhibitory effects, with a significantly greater effect than the well-known potent antioxidant quercetin revealed, while AS3, AS4, and AS5 showed inhibitory effects to a lesser degree when compared to quercetin at a concentration of 20 µM. Moreover, it was also found that AS suppress PMA-activated oxidative in a dose-dependent manner and potentiation of inhibitory effects was observed with the increase of AS concentration. Especially, when the concentration of AS1, AS2, AS3, and AS6 was up to 20 µM, over 80% inhibition was maintained. The IC₅₀ of active AS was calculated and the order of activity was as follows: AS1 > AS6 > AS2 > AS3 > AS5 > AS4 (Table 1).

Figure 2. Inhibitory effects of AS on rat PMNs respiratory burst activated by PMA. (A) Dynamics curve of rat neutrophils-CL reaction stimulated by PMA. (B) Inhibitory effects of 20 µM of AS and QC (positive control) on PMA-stimulated respiratory burst of rat PMNs. Data are means ± SD of four independent experiments. *p < 0.05 (Turkey’s test) in comparison with that of control. NS indicates non-significant. (C) Inhibitory effects of AS1–6 on PMA-induced respiratory burst of rat PMNs at various concentrations (5–20 µM). Data are means ± SD of four independent experiments.

Table 1. The selectivity of anti-respiratory burst properties of phenolics from A. styracifolius.

Compounds	Inhibiting PMNs respiratory burst (IC50, µM)	Cytotoxicity (CC50, µM)	SI^a
QC	4.7 ± 0.3	226.2 ± 19.2	48.1
AS1	3.1 ± 0.3	198.7 ± 11.6	64.1
AS2	9.1 ± 0.8	1784.1 ± 101.2	196.1
AS3	10.0 ± 1.1	209.2 ± 14.5	20.9
AS4	57.7 ± 6.6	826.5 ± 28.2	14.3
AS5	29.7 ± 2.3	2588.2 ± 124.3	87.1
AS6	5.9 ± 0.6	1501.3 ± 104.8	254.5

^aSI = CC₅₀/IC₅₀.

The cytotoxicity of AS on rat PMNs

The potential cytotoxicity of active AS was determined using the Trypan blue assay at different concentrations. AS exhibited high toxicity to PMNs at concentrations as high as 200 µM, but when the concentrations decrease to lower than 20 µM, the viability of PMNs was not substantially modified and did not exceed 5%. In order to determine the selectivity of the inhibitory effects against rat PMNs respiratory burst evoked by PMA, the selectivity index (SI) values (ratio of the CC₅₀ versus PMNs cytotoxicity to the IC₅₀ versus PMNs respiratory burst inhibition) was employed. Among the tested compounds, AS6 showed the highest selectivity towards inhibitory activities against PMNs respiratory burst (Table 1).

The capacity of AS to scavenge , HO^•, and H₂O₂

To further understand the inhibitory efficacy of tested AS on respiratory burst of PMNs, the capacity of scavenging ROS was evaluated by cell-free models. As observed in Figure 3, all tested compounds discriminately revealed the scavenging abilities toward various ROS produced by cell-free systems at a concentration of 20 µM, respectively. In view of , 20 µM of AS1–4 displayed substantial scavenging effects with the % scavenging ranging from 30.7 to 78.1% and the scavenging capability is in the order of AS1 > AS2 > AS3 > AS4. In contrast, AS5–6 revealed mild scavenging capability toward at the same concentration, although they were detected to be the powerfully effective compounds in the experiments designed to assess inhibitory activity against PMNs respiratory burst. Inspection of HO^• and H₂O₂ provided similar results. AS1–4 potently quenched HO^• and H₂O₂ at the concentration of 20 µM, with the scavenging rates ranging from 35.4 to 69.7% and from 65.5 to 86.3%, respectively, while AS5–6 showed less effect against both ROS.

Figure 3. The scavenging capacity of 20 µM of AS1–6 and QC (positive control) towards , H₂O₂, and HO^• in acellular systems. Data are means ± SD of four independent experiments. *p < 0.05 (Turkey’s test) in comparison with that of control. NS indicates non-significant.

Discussion

A large number of phenolic compounds including flavonoids, stilbenoids, and 2-arylbenzofurons have been isolated from Artocarpus plants and possess a wide range of biological properties such as anti-inflammatory (Hsu et al., 2012; Lin et al., 2011), antiplatelet (Jagtap & Bapat, 2010; Weng et al., 2006), antimycobacterial (Loizzo et al., 2010; Siritapetawee et al., 2012), and antiprotozoal activities (Bourjot et al., 2010). Among these activities, the antioxidant actions of these polyphenols derived from Artocarpus species have been frequently reported in vivo and in vitro (Omar et al., 2011; Sidahmed et al., 2013). However, an evaluation of their effect on the respiratory burst of neutrophils has not yet been investigated. This study presented the first documentation of inhibitory effects of eight phenols from A. styracifolius on rat PMNs respiratory burst induced by PMA.

In the present work, AS1–6, the six isoprenylated phenolic constituents from A. styracifolius, was endowed with significant (p < 0.05) inhibitory effects on the respiratory burst of rat PMNs. The order of inhibitory potency was as follows: AS1 > AS6 > AS2 > AS3 > AS5 > AS4 (Figure 1 and Table 1). Interestingly, AS7 and AS8, the two catechin derivatives with the weakest lipid-solubility among tested compounds, showed no activity in suppressing PMNs respiratory burst. Previous studies suggested that a lower lipid-solubility might make the molecule difficult to penetrate the cell membrane where ROS is generated by membrane-bound NADPH oxidase (Hernandes & Britto, 2012; Maridonneau-Parini et al., 1986; Pagonis et al., 1986; Varga et al., 2001). On the basis of our results, it is supposed that the absence of inhibition of respiratory burst by AS7 and AS8 might be associated with the poor lipid-solubility of these molecules. This hypothesis is underlined by the fact that with two methoxy group in B ring, AS6 possessed higher inhibitory capability on PMNs respiratory burst than AS5 which carries two polar hydroxyl groups. Furthermore, the cytotoxicity assay indicated that the toxic concentration of all active compounds (AS1–6) were much higher than 20 µM, where most compounds (except for AS4 and AS5) exhibited over 80% inhibition against the respiratory burst of rat PMNs induced by PMA. The SI values used to assess selectivity of inhibitory efficacy of all active compounds remained high and no less than 14.3 (Table 1), which indicated that their inhibitory activities on PMNs respiratory burst was not due to general toxicity.

Apparently, the respiratory burst of neutrophils is inevitably accompanied with the generation of massive ROS metabolites which are mainly represented by , HO^•, and H₂O₂ (Blokhina et al., 2003). The excessive production of ROS leads to the imbalance of intracellular redox status which can be pharmacologically modulated by antioxidants that act by donating an electron to a free radical and converting it to a non-radical form. Therefore, many studies asserted that the effects of phenolics on the respiratory burst of phagocytes can be explained by their ROS scavenging properties (Ciz et al., 2012; Pincemail et al., 1987; Zielinska et al., 2000). Our results showed that AS1–6 have significant oxidant scavenging activity towards , HO^•, and H₂O₂ (Figure 3). It is worth noting that the differential relations of the potency for oxidant scavenging with the efficacy for the inhibition of respiratory burst were observed depending on the structural types of the tested compounds. In view of AS1–4, four isoprenylated flavones, the order of potency for ROS scavenging is well consistent with that of potency for the suppression of respiratory burst of PMA-activated PMNs, which suggested that the variant oxidant-scavenging capacity of isoprenylated flavones may account, at least in part, for their differential anti-respiratory burst activities. This result is in accordance with previous research which affords similar results when investigating the effects of Rosa canina fruit extracts abound in proanthocyanidins and flavonoids on neutrophil respiratory burst (Daels-Rakotoarison et al., 2002). Surprisingly, inspection of AS5–6 affords a converse suggest. AS5–6, two isoprenylated 2-arylbenzofurans, exhibited strong inhibitory effects on respiratory burst of PMA-induced PMNs, but appeared to be weak scavengers in the assay for capacity of direct scavenging ROS (Table 1 and Figure 3). According to published studies reviewed by Ciz et al. (2012), the anti-respiratory burst activities of phenolics may be mediated via inhibition of enzymes involved in cell signaling besides by modulation of redox status. Thus, we speculated that an inhibition of ROS formation rather than a scavenging of ROS played a dominating role in PMA-stimulated neutrophils exposed to AS5–6 in the present study. However, this is first report dealing with the effects of 2-arylbenzofuran derivatives on respiratory burst of neutrophils. Further studies are needed to explore the reasonable mechanism underlying AS5–6 anti-respiratory burst effects.

Conclusions

In summary, we demonstrated that the isoprenylated phenolic compounds from A. styracifolius inhibited PMA-induced respiratory burst in rat neutrophils without showing cytotoxicity. The explanations for the inhibition of the respiratory burst by isoprenylated phenolics in our study might be various and depend on structural types of different isoprenylated phenolics. The anti-respiratory burst effects of isoprenylated flavones might be due to their direct ROS scavenging properties. In contrast, the inhibition of PMA-induced respiratory burst by isoprenylated 2-arylbenzofurans might involve in the suppression of intracellular ROS generation rather than the direct scavenging of ROS. Based on the above results, we suggest that isoprenylated phenolics could have potential therapeutic possibilities as a class of promising candidate agents for the prevention or treatment of diseases in which the pathogenesis of the activated PMNs or an oxidative stress play a key role.

Declaration of interest

The authors report no conflict of interest.

Acknowledgements

Our sincere thanks go to Mr. Shihong Lv, Guangxi Zhuang Autonomous Region Institute of Botany, Chinese Academy of Sciences, for the collection of medicinal materials and to Dr. Jiahui Peng of the Department of Nutrition and Food Science, Texas A&M University for critical review of the manuscript. This study was supported by National Natural Science Foundation of China (Nos. 81160509, 81360475, and 81060326), Subject of Jiangxi Education Department (No. GJJ10552), and Research Project of Jiangxi Health Department (No. 2009A058).