IN VITRO ANTI-INFLAMMATORY ACTIVITY OF THE COMPONENTS OF AMOMUM TSAO-KO IN MURINE MACROPHAGE RAW 264 . 7 CELLS

Background: Plants still remain the prime source of drugs for the treatment of inflammation and can provide leads for the development of novel anti-inflammatory agents. Material and methods: An in vitro bioassay guide revealed that the 80% ethanol (EtOH) extract of the whole plant, Amomum tsao-ko (Zingiberaceae), displayed anti-inflammatory activity after assessing its effects on murine macrophage RAW 264.7 cells. Result: Phytochemical study of the 80% EtOH extract of Amomum tsao-ko led to the isolation of eight compounds: 4-hydroxy-3-methoxy-benzoic acid (1), meso-hannokinol (2), (+)-hannokinol (3), coumaric acid (4), 4-hydroxy-benzoic acid (5), (+)-epicatechin (6), (-)-catechin (7), and myrciaphenone A (8). The results indicated that two of the isolated components, (+)-epicatechin (6) and (-)-catechin (7), inhibited the production of nitric oxide (NO) significantly in lipopolysaccharide treated RAW 264.7 cells. Conclusion: LPS-induced interleukin tumor necrosis factor-alpha (TNFIL-1β and IL-10 production was also decreased in a dose-dependent manner. In addition, western blot analysis revealed that (+)-epicatechin (6) and (-)-catechin (7) reduced the expression of inducible nitric oxide synthase and inhibited nuclear localization of nuclear factor kappa-B (NF-κB).


Introduction
Amomum tsao-ko (A.tsao-ko) Crevost et Lemaire is a plant of the genus Amomum in the family Zingiberaceae, native to several countries Asia and Africa such as southern China, northern Viet Nam and Ethiopia.The dried fruit of A. tsao-ko used as an eliminate phlegm, warm the spleen, reduce abdominal pain, dyspepsia, and vomiting agent in Oriental traditional medicine (Lim et al., 2013;Zhao et al., 2010).Tsaokoin, a bicyclic nonane, and antioxidants, including hannokinol diarylheptanoids, have been isolated from this plant (Teresita et al., 2000).An antibacterial, spiroketal aculeatin (Moon et al., 2004), an antimalarial, diterpene peroxide (Kamchonwongpaisan et al., 1995), and eicosenones (Donga et al., 1988) have been identified from other species of the genus Amomum.Although the plant A. tsao-ko has long been used as a spice and perfume in addition to its medicinal usage, very few studies have reported its anti-inflammatory constituents (Lee et al., 2008).As a result of our ongoing search for novel bioactive natural products from medicinal plants, the 80% ethanol (EtOH) extract of the powdered fruit of A. tsao-ko was found to show significant anti-inflammatory activity in lipopolysaccharide (LPS)-treated RAW 264.7 cells.To investigate the anti-inflammatory properties of A. tsao-ko Ext. and its main components, we performed their effects on the survival and immune status of RAW 264.7 murine macrophage cells.Cell viability was determined using a MTT assay after treatment with various concentrations of the isolated constituents.Inhibition of NO production and iNOS expression were measured by reaction with Griess reagent and western blot analysis in LPS-induced RAW 264.7 cells, respectively.Constantly, we investigated the effects of compounds 6 ((+)-epicatechin) and 7 ((-)-catechin) on tumor necrosis factor (TNF)-α, IL-1β, and IL-10, which are related to inflammatory response at both reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).NF-κB is known to play an important role in gene expression on inflammation (Eliopoulos et al., 2002).The present study confirmed that the inhibitory effects of (+)-epicatechin and (-)-catechin were mediated via inhibition of nuclear localization of NF-κB, which were also determined by western blot analysis.

Plant Material
Dried A. tsao-ko was purchased from the Kyungdong Oriental Herbal Market in Korea (August 2012) and identified by one of the authors (Prof.Joa Sub Oh).A voucher specimen (G47) was deposited at the Natural Products Research Laboratory, Gyeonggido Business and Science Accelerator.
Cell culture RAW 264.7 mouse macrophage cells (TIB-71) were obtained from the American Type Culture Collection (Manassas, VA, USA).Cells were maintained in DMEM supplemented with 10% Korean Cell Line Bank were cultured in DMEM supplemented with 10% FBS and 1% penicillin (100 U/mL)-streptomycin (100 μg/mL) in a humidified incubator with 5% CO 2 at 37˚C.

Cell viability
RAW 264.7 cells were seeded on 96-well plates (5 × 10 4 cells/well), were treated with A. tsao-ko EtOH extracts or either of its main components for 1 h prior to LPS (1 μg/mL) stimulation for 24 h.MTT solution (5 mg/mL) was added to each well After 2 h of incubation at 37°C with 5% CO 2 , the supernatant was removed and dissolved in DMSO.The absorbance of each well was measured at a wavelength of 540 nm using a SpectraMax 190PC microplate reader (Molecular Devices, Sunnyvale, CA, USA).Data are presented as the mean ± standard deviation of three replicates.

Nitric oxide production assay
RAW 264.7 cells (5 × 10 4 cells/well in 96-well plates) were examined with A. tsao-ko EtOH extracts or either of its main components for 1 h prior to LPS (1 μg/mL) stimulation for 24 h.Nitrite in culture medium was measured by using Griess reagent (N-(1-naphthyl) ethylenediamine dihydrochloride and sulfanilamide).Absorbance was subsequently measured at 540 nm, using a SpectraMax 190PC microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Western blot analysis
Following treatment as indicated, cells were washed twice with PBS and lysed with RIPA buffer containing protease inhibitor cocktails.Cell lysates were clarified at 13,000 × g for 10 min at 4 ºC, and the supernatants were subjected to Western blot analysis as described previously.All western blot analyses are representative of at least three independent experiments.

Preparation of nuclear extract
RAW 264.7 cells were plated at a density of 1 × 10 6 cells/well in 6-well plates and treated with main components ((+)-epicatechin and (-)-catechin) for 1 h prior to LPS (1 μg/mL) stimulation for 30 min.cells were washed twice with ice-cold PBS prior to trypsinization and centrifugation at 90 x g and 4˚C for 5 min.Cells were then centrifuged at 20,000 x g and 4˚C for 5 min and resuspended in 200 μl buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF and 0.1 mM EDTA).After incubation on ice for 10 min, cells were lysed by the addition of 12.5 μl of 10% NP-40.Cells were then centrifuged at 20,000 x g for 2 min at 4˚C, and the supernatants were collected as cytosolic extract.Pellets were resuspended in 50 μl of extraction buffer (20 mM HEPES at pH 7.9, 0.4 M NaCl, 1 mM DTT, 1 mM PMSF, 1 mM EDTA and 1% NP-40) and incubated on ice for 10 min.Nuclear extract was collected by centrifugation at 15,000 x g for 15 min at 4˚C.

Statistical analysis
Statistical analysis was performed by a Student's t-test using Microsoft Excel 2007 software (Microsoft Corporation, Redmond, WA, USA).Results are presented as the mean ± standard deviation.P<0.05 was considered to indicate a statistically significant difference.

Results
LPS, a component of the cell wall of gram-negative bacteria, increases the levels of pro-inflammatory cytokines, including NO, TNF-α, IL-10, and IL-1β, in macrophages and monocytes.It also induces diverse disease-related inflammatory responses (Willeaume et al. 1995).As shown in Table 1, we showed that 80% EtOH extract of the powdered fruit of A. tsao-ko decreased LPS-induced NO production in RAW 264.7 cells.The experiments were repeated in triplicate; values are expressed as mean ± SD. b N-Monomethyl-L-arginine (LNMMA) was used as a positive control.
The concentration of extract required for 50% inhibition (IC 50 ) of activity was 59.5 μg/mL.Among the four fractions obtained by serial solvent partition of A. tsao-ko (CH 2 Cl 2 soluble fraction, EtOAc soluble fraction, n-BuOH soluble fraction, and remaining aqueous fraction), the CH 2 Cl 2 and the EtOAc soluble fraction displayed potent inhibitory activity (IC 50 < 25 μg/mL).The concentration of A. tsao-ko Ext. and the four fractions that is possibly cytotoxic to RAW 264.7 cells was determined by MTT assay.Cytotoxicity effect was not caused by CH 2 Cl 2 soluble fraction, EtOAc soluble fraction, n-BuOH soluble fraction, and aqueous fraction in LPS-induced RAW 264.7 cells.Therefore, bioassay guided purification of the active fraction, i.e., the EtOAc soluble fraction of A. tsao-ko, was conducted to purify the active components that display anti-inflammatory activity against LPS-treated RAW 264.7 cells.
Repeated column chromatography on silica gel and a RP-18 column of the EtOAc soluble fraction led to the isolation of eight compounds: 4-hydroxy-3-methoxy-benzoic acid ( 1  NMR and mass spectra were analyzed to determine the structures of the compounds.In addition, all physical and spectroscopic data obtained in the present study were compared with those of previously published manuscripts.Isolated components were tested for their inhibitory effect on NO production in LPS-stimulated RAW 264.7 cell culture system. Among the isolated compounds, compounds 6 ((+)-epicatechin) and 7 ((-)-catechin) showed the highest activity (IC 50 = 70.6 μM and IC 50 = 73.3μM, respectively) against NO production without cytotoxicity (Table 2, Fig. 2).Constantly, we conducted the effect of (+)-epicatechin and (-)-catechin on iNOS expression induced by LPS using western blot analysis.As shown in Fig. 3, (+)-epicatechin and (-)-catechin effectively inhibited LPS-induced expression of iNOS at protein level in RAW264.7 cells.These results indicate that treated with (+)-epicatechin and (-)-catechin (25-100 μM) significantly suppress LPS-induced NO production related to down-regulating iNOS expression in RAW264.7 macrophages.NF-κB plays a crucial role in general inflammatory reaction by controlling the activation of iNOS.The activation of NF-κB caused translocation of active NF-κB p50 and p65 from the cytoplasm to nucleus (Chen et al. 1995).In this study, we investigated whether (+)-epicatechin and (-)-catechin could suppress the translocation of NF-κB (p65 and p50) into the nucleus.We showed that (+)-epicatechin and (-)-catechin inhibited the translocation of NF-κB (p65 and p50) into the nucleus (Fig. 4).

Discussion
Macrophages play an important role in both passive and active immunity and regulate anti-inflammatory mediators including NO and pro-inflammatory cytokines (Alleva et al., 2002;Eliopoulos et al., 2002).iNOS (NOS II), one of the isoforms of the nitric oxide synthase (NOS) family, is found in macrophages and hepatocytes.iNOS is not expressed in most resting cells; however, upon exposure to endogenous and exogenous stimulators, such as LPS or pro-inflammatory cytokines like TNF-α, IL-1β and IL-10 the expression of the iNOS gene is increased and triggers several disadvantageous cellular responses, causing diseases, including inflammation, sepsis, and stroke (Nathan et al., 1992;Marletta et al., 1993;Duval et al., 1996).In order to extend the understanding of the anti-inflammatory effects of (+)-epicatechin and (-)-catechin, which showed the potent inhibitory activity, on the expression of inflammatory proteins, iNOS expression and the production of NO were evaluated in LPS-stimulated RAW 264.7 cells.Treatment with (+)-epicatechin and (-)-catechin inhibited NO production and iNOS expression in a dose-dependent manner in LPS-stimulated RAW 264.7 cells (Fig. 3).TNF-α is an element of the innate immune response against stimulus and IL-1β is an important factor in the acute inflammation response (Strandberg et al., 2005).NF-κB activation leads to release of pro-inflammatory proteins production.NF-κB transcription factors are in the cytoplasm as an inactive state and when stimulation, NF-κB transfer to nuclear localization (Wu et al., 2008).
Altogether, the results indicated that (+)-epicatechin and (-)-catechin, isolated from A. tsao-ko, effectively inhibited NO production in LPS-stimulated RAW 264.7 cells by suppression of iNOS expression and the production of inflammatory cytokines, such as TNF-α, IL-1β and IL-10.In addition, we confirmed that (+)-epicatechin and (-)-catechin have anti-inflammatory activity via down-regulating nuclear localization of NF-κB.

a
The experiments were repeated in triplicate; values are expressed as mean ± SD. b N-Monomethyl-L-arginine (LNMMA) was used as a positive control.

Figure 3 :
Figure 3: Effect of (+)-epicatechin and (-)-catechin on the expression of iNOS in LPS-stimulated RAW 264.7 cells Expression levels of iNOS protein were determined by western blot.β-actin served as internal control western blot.Values represent the mean ± SD of three independent replicates.

Figure 4 :
Figure 4: Regulatory effects of (+)-epicatechin and (-)-catechin on NF-κB nuclear localization in LPS-stimulated RAW 264.7 cells.Cells were pretreated with (+)-epicatechin and (-)-catechin for 1 h, followed by LPS (1 μg/mL) stimulation for 30 min.Cytosolic and nuclear extracts were analyzed via western blot with anti-NF-κB p65, anti-NF-κB p50, anti-β-actin or anti-Lamin B antibodies.β-actin served as a marker for the cytoplasm and Lamin B for the nucleus.Results shown are representative of at least three independent experiments.Next, we examined the effect of (+)-epicatechin and (-)-catechin on inflammatory cytokines, such as TNF-α, IL-1β, and IL-10 in LPS-stimulated RAW 264.7 cells.In this study, we measured the expression levels of these inflammatory cytokines by RT-PCR and ELISA analysis.As shown in Fig.5, (+)-epicatechin and (-)-catechin inhibited the expression of TNF-α, IL-1β, and IL-10 in a dose-dependent manner at both the mRNA and protein levels in RAW264.7 cells.These results showed that (+)-epicatechin and (-)-catechin have a potential activity on anti-inflammatory activity through the down-regulation of the NF-κB signaling pathway.

Figure 5 :
Figure 5: Effects of (+)-epicatechin and (-)-catechin on the expression and secretion of various cytokines in RAW 264.7 cells.A. The expression of TNF-α, IL-1β, and IL-10 mRNA was determined by RT-PCR analysis.B. Cell culture supernatant was subjected to TNF-α, IL-1β, and IL-10 cytokine ELISA as described in Materials and methods.Values represent the mean ± SD of three independent experiments.Statistical significance is indicated (*P < 0.05, **P < 0.01, compared with LPS-treated cells).

Table 1 :
Inhibitory effects of A. tsao-ko extract and isolated fractions on LPS-induced NO production RAW 264.7 cells

Table 2 :
Inhibitory effect of compounds 1-5 on LPS-induced NO production RAW 264.7 cells