Echinocystic Acid Isolated from Eclipta prostrata Suppresses Lipopolysaccharide-Induced iNOS, TNF-α, and IL-6 Expressions via NF-κB Inactivation in RAW 264.7 Macrophages

• Introduction
• Results and Discussion
• Materials and Methods
• Plant material
• Extraction and isolation
• LC-MS
• Chemicals and antibodies
• Cell culture and sample treatment
• Nitrite, TNF-α, and IL-6 assays
• Western blot analysis
• Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR)
• Transient transfection and luciferase assay
• Nuclear extraction and EMSA
• Statistical analysis
• Supporting information
• Acknowledgements
• References

Abstract

In this study, we aimed to identify the compounds in Eclipta prostrata responsible for its anti-inflammatory effects using an in vitro bioassay. Three triterpenoids, eclalbasaponin I, eclalbasaponin II, and echinocystic acid, were isolated from an EtOAc fraction of the 70 % EtOH extract of E. prostrata by activity-guided fractionation based on the inhibition of nitric oxide release from lipopolysaccharide-induced RAW 264.7 macrophages. Of these three triterpenoids, echinocystic acid inhibited lipopolysaccharide-induced production of nitric oxide and cytokines such as tumor necrosis factor-α and interleukin-6. Consistent with these observations, echinocystic acid concentration-dependently inhibited lipopolysaccharide-induced inducible nitric oxide synthase expression at the protein level and inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-6 expression at the mRNA level, and inhibited lipopolysaccharide-induced iNOS promoter binding activity. In addition, echinocystic acid suppressed the lipopolysaccharide-induced transcriptional activity of nuclear factor-κB by blocking the nuclear translocation of p65.

Introduction

During recent years, a great deal of research has been devoted to the identification of new natural compounds and to their recovery and purification. In this regard, unexplored natural sources of bioactive ingredients are attracting much research attention because such investigations can lead to the isolation of new biologically active compounds [1]. Eclipta prostrata L. (syn. E. alba Hassk) is a perennial herb and a member of the Asteraceae family. E. prostrata grows widely in tropical areas, especially in Asia [2], and has been reported to exhibit lipid lowering [3], antiangiogenic [4], antitumor [5], myotoxic, and hemorrhage inhibitory [6] activities. In addition, E. prostrata exhibits diverse immune-related regulatory effects. For example, it has been reported to have an immunosuppressive effect in a mouse model [7], an immunomodulatory effect on T-lymphocytes [8], antimicrobial activity [9], and anti-inflammatory activity [10]. Furthermore, in Thai traditional medicine, this plant continues to be used to treat a wide variety of inflammatory conditions, for example, its leaves are used to treat skin diseases, its stems to treat abscesses, itching, tuberculosis, amoebiasis, and asthma, and its roots are used as an antibacterial agent [11], [12]. In the present study, we focused on the anti-inflammatory effects of E. prostrata and sought to isolate its active components by activity-guided separation using an in vitro bioassay.

Inflammation is a hallmark of many human diseases, including arteriosclerosis, inflammatory bowel disease, arthritis, infectious diseases, and cancer [13]. The pathogenesis of inflammation is a complex process, which is regulated by proinflammatory mediators, such as, iNOS, TNF-α, and IL-6 [14], [15], and thus, the inhibition of proinflammatory mediator production offers a means of screening for anti-inflammatory agents [16]. Furthermore, the transcriptions of these genes are known to be regulated by MAPKs and NF-κB [17], [18].

Inflammation is triggered by various pathogens and/or tissue damage via receptor signals that stimulate NF-κB, MAPKs, and interferon response factors. In particular, NF-κB transcription factors are known to play important roles in the regulation of immune and inflammatory responses.

Previous reports have demonstrated that the CH₂Cl₂ and MeOH extracts of E. prostrata markedly inhibit NO production. In particular, orobol, a compound isolated from E. prostrata, has been reported to potently inhibit LPS-induced NO production in RAW 264.7 macrophages [2]. In addition, wedelolactone, a well-known IKK inhibitor found in many plants including E. prostrata, has been reported to inhibit the productions of NO, PGE₂, and TNF-α in LPS-induced RAW 264.7 macrophages [19]. We considered that an improved means of activity-guided separation using different solvents was needed to isolate constituent compounds with anti-inflammatory activity. Thus, the objective of the present study was to isolate the active compounds present in E. prostrata and to evaluate their anti-inflammatory potentials and their modes of action in LPS-induced RAW 264.7 macrophages. Accordingly, a solvent fraction of the 70 % EtOH extract of the whole plant with a marked NO inhibitory effect was further fractionated, and three triterpenoids were isolated.

Results and Discussion

The pharmacologically induced downregulation of LPS-inducible inflammatory mediators is considered an essential requirement for the treatment of disorders attributed to macrophage activation [20]. Leukocyte infiltration is considered a key step during the inflammatory process and is primarily controlled by chemokines, whose production is regulated positively or negatively by iNOS-derived NO. NO levels in tissues and duration of NO exposure appear to determine proinflammatory cytokine expression during the induction and resolution of inflammation [21], and thus, NO production and iNOS expression are considered pharmacologic targets for anti-inflammatory drugs.

In this study, aimed to investigate the anti-inflammatory properties of E. prostrata, we prepared n-hexane- (ECPR1H), EtOAc- (ECPR1E), BuOH- (ECPR1B), and H₂O-soluble fractions (ECPR1W) from its 70 % EtOH extract (ECPR1). We compared the effects of the extract and these fractions on LPS-induced NO production in RAW 264.7 macrophages and found that ECPR1E (IC₅₀: 41.4 ± 2.1 µg/mL) most strongly and concentration-dependently inhibited LPS-induced NO production (Table 1S, Fig. 1S A in Supporting Information). On the other hand, ECPR1, ECPR1H, ECPR1B, and ECPR1W were found to have no inhibitory effect on LPS-induced NO production up to a concentration of 100 µg/mL. To determine whether the inhibitory effect of ECPR1E on NO production was related to the modulation of iNOS expression, we examined the protein and mRNA expressions of iNOS. Both were found to be markedly upregulated in response to LPS, and ECPR1E was found to concentration-dependently inhibit these responses ([Fig. 2 A, C]). Furthermore, these inhibitory effects of ECPR1 and of the fractions were not caused by nonspecific cytotoxicity because these tested materials had no effect on cell viability at concentrations up to 100 µg/mL, as determined by the MTT assay (data not shown). Three triterpenoids, that is, eclalbasaponin I, eclalbasaponin II, and echinocystic acid, were isolated from ECPR1E by activity-guided separation ([Fig. 1]) and compared with respect to their abilities to inhibit LPS-induced NO production at nontoxic concentrations using IC₉₀ cell viability values. However, only echinocystic acid (IC₅₀: 35.5 ± 1.9 µM) suppressed LPS-induced NO production (Table 1S in Supporting Information). Interestingly, the other two isolates are glycosides of echinocystic acid, and thus, the above findings support previous reports [22], [23], [24] that aglycones are more active than the corresponding glycosides, although there are exceptions [25]. This effect has been suggested to be due to different affinities for cellular membranes, that is, the lower activities of glycosides may be due to their inability to penetrate biomembranes [26]. LC-MS showed that the concentrations of the three triterpenoids were highest in the ECPR1E fraction ([Table 1]). As indicated by the IC₅₀ values of ECPR1E and of echinocystic acid, higher levels of triterpenoids and of echinocystic acid in particular, dose-dependently inhibited LPS-induced NO production. Furthermore, echinocystic acid also suppressed LPS-induced iNOS protein and mRNA levels ([Fig. 2 B, D]), and the subsequent production of NO in LPS-induced macrophages. We then examined the transcriptional regulation of iNOS by echinocystic acid using a promoter activity assay. As shown in [Fig. 2 E], echinocystic acid significantly and concentration-dependently inhibited LPS-induced iNOS promoter activity. Accordingly, echinocystic acid was found to suppress the induction of iNOS at the protein and mRNA levels by downregulating the activity of its promoter, and thus, to reduce NO production in LPS-stimulated macrophages.

Since echinocystic acid was found to reduce NO production and iNOS expression, we also investigated its effects on LPS-induced proinflammatory cytokine release using enzyme immunoassays. We found that echinocystic acid reduced the productions of TNF-α and IL-6 ([Fig. 3 A, B]), which play key roles during the inflammatory response and are induced by a wide range of pathogenic stimuli [27], [28], and qRT-PCR showed that it also reduced their mRNA levels ([Fig. 3 C, D]). Saponins and their prosapogenins or sapogenins are known for their anti-inflammatory activities. Furthermore, in a previous study, we found that the type of linkage between sugars and saponins critically determines their anti-inflammatory activities and cytotoxicities [1]. In addition, many triterpenoids have been studied as potential anti-inflammatory agents based on their abilities to inhibit the transcription factor NF-κB [29], which is essential for the LPS or cytokine-induced expressions of proinflammatory mediators [17], [18]. For example, celastrol (from Celastrus orbiculatus) [28], glycyrrhetinic acid (from Glycyrrhizae glabra) [30], avicin (from Acacia victoriae) [31], and taraxasterol (from Taraxacum officinale) [32] were all found to block NF-κB activation and the productions of downstream inflammatory mediators induced by various stimuli in different cell types. Based on these findings, a luciferase assay was performed to determine whether the echinocystic acid-mediated inhibitions of the expressions of iNOS, TNF-α, and IL-6 were related to the suppression of NF-κB activation. Accordingly, RAW 264.7 macrophages were transiently transfected with pNF-κB-luc plasmid and then stimulated with LPS in the presence or absence of echinocystic acid. It was found that echinocystic acid prevented LPS-induced increases in NF-κB-dependent luciferase activity ([Fig. 4 A]).

Furthermore, treatment with LPS increased the binding of NF-κB to its consensus DNA sequence, and pretreatment with echinocystic acid reduced this LPS-induced NF-κB-DNA binding ([Fig. 4 B]). The nuclear translocation of the active component (p65) of NF-κB is required to activate inflammatory gene transcription [33], and thus, we investigated whether the nuclear translocation of p65 is prevented by echinocystic acid. We found that LPS markedly induced the nuclear translocation of p65, and that pretreatment with echinocystic acid suppressed this process ([Fig. 4 C]), showing that echinocystic acid inhibited the LPS-induced activity of NF-κB by suppressing the nuclear translocation of p65. Based on these results, we suggest that the inhibition of the expression of proinflammatory mediators by echinocystic acid is caused by the suppression of NF-κB activity.

Recently, it was reported that echinocystic acid (a metabolite of lancemaside A) suppressed LPS-induced acute lung inflammation in mice and the expressions of proinflammatory cytokines such as IL-1β and TNF-α, as well as that of their transcription factor, NF-κB, by inhibiting LPS binding to TLR4 receptor [34]. However, echinocystic acid did not affect LPS binding to this receptor in our system (Fig. 2S in Supporting Information). Accordingly, our findings suggested that the inhibitory effect of echinocystic acid on LPS-TLR4 binding might be cell or strain specific.

In summary, we isolated three triterpenoids from whole E. prostrata by activity-guided fractionation for anti-inflammatory activity and found that echinocystic acid effectively inhibited the LPS-induced expressions of iNOS, TNF-α, and IL-6 by inhibiting NF-κB activation in RAW 264.7 macrophages. Accordingly, our findings suggest that the anti-inflammatory activities of E. prostrata are largely due to echinocystic acid. Further studies are needed to examine the anti-inflammatory effects of echinocystic acid in an animal model.

Materials and Methods

Plant material

The whole plants of Eclipta prostrata L. were obtained from the Department of Pharmacy, Kyung Hee University Medical Center (Seoul, Republic of Korea) in October 2011 and identified by one of the authors (Prof. Dae Sik Jang). A voucher specimen (no. 2011-ECPR01) was deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Republic of Korea.

Extraction and isolation

The dried and milled plant material (400 g) was extracted with 2000 mL of 70 % aqueous EtOH three times by maceration. Extracts were combined and concentrated in vacuo at 40 °C to give a 70 % EtOH extract (45.2 g). A portion of this extract (44 g) was then suspended in distilled water (500 mL) and successively extracted with n-hexane (3 × 500 mL), EtOAc (3 × 500 mL), and BuOH (3 × 500 mL) to give n-hexane- (2.86 g), EtOAc- (4.14 g), BuOH- (6.07 g), and water-soluble fractions (30.9 g), respectively. Among the extract and solvent fractions, the EtOAc-soluble fraction showed the most potent activity against LPS-induced NO production. Thus, 4.0 g of this extract was subjected to silica gel column chromatography (4.6 × 40 cm, 70–230 mesh) using CH₂Cl₂-MeOH [(1 : 0, 19 : 1, 9 : 1, 4 : 1, 1 : 1, 0 : 1, 2000 mL of each)] as the eluent to afford 13 fractions (Fr.1–13). Fraction Fr.12 [obtained by eluting with CH₂Cl₂/MeOH (4 : 1 v/v); 570 mg] was chromatographed over Sephadex LH-20 (3.3 × 41 cm) using MeOH as the eluent to give eclalbasaponin I (280–350 mL, 150 mg) and eclalbasaponin II (380–410 mL, 121 mg). Fraction Fr.7 [obtained by eluting with CH₂Cl₂/MeOH (9 : 1 v/v; 280 mg)] was subjected to silica gel column chromatography (3.6 × 36 cm, 230–400 mesh) using n-hexane-EtOAc (9 : 1, 4 : 1, 3 : 2, MeOH, 1000 mL each eluent) to give echinocystic acid (2400–2700 mL, 14.3 mg). The purities of these compounds (> 95 %) were determined by HPLC and NMR, and their structures were determined using physical and spectroscopic data and by comparison with published values [35].

LC-MS

Triterpenoid contents were determined by liquid chromatography-mass spectrometry (LC-MS) using an ion trap mass spectrometer equipped with an electrospray source (API4000; AB Sciex) and coupled to reversed-phase HPLC (Agilent 1200; Agilent). A C-18 column (50 mm × 2.0 mm, 3 µm particle size, 110 Å pore size, Capsell pak; Shiseido and 4.0 × 2.0 mm precolumn; Phenomenex) was employed for the analysis of all extracts, using a flow rate of 0.2 mL/min and an injection volume of 3 µL. The samples were analyzed using an isocratic mobile phase consisting of 1 mM ammonium acetate buffer and methanol (10 : 90, v/v). Mass spectrometric detection was conducted in negative ion mode with MRM mode. The source temperature was maintained at 450 °C, and nebulizer gas (GS 1) and heater gas (GS 2) pressures were set to 50 psi and 55 psi, respectively. Nitrogen was used as a nebulizer and collision gas. The capillary voltage and the entrance potential were set at 4500 V and at 10 V, respectively. Echinocystic acid, eclalbasaponin I, and eclalbasaponin II standard stock solutions were prepared at a concentration of 1 mg/mL in methanol. Calibration curves over the concentration range of 5 to 5000 ng/mL were plotted as peak area of the relevant selected ion trace versus concentration. Extracts were diluted as necessary in 1 mM ammonium acetate buffer and methanol (10 : 90, v/v) to provide triterpenoid concentrations within the linear range of the calibration curve. Triterpenoids were identified in extracts by matching retention times and fragmentation patterns with standards.

Chemicals and antibodies

DMEM, FBS, penicillin, and streptomycin were obtained from Life Technologies, Inc. iNOS, p65, PARP, β-actin monoclonal antibody, and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. EIA kits for TNF-α and IL-6 were obtained from R&D Systems, and the luciferase assay kit was purchased from Promega. RNA extraction kits were obtained from Intron Biotechnology. Random oligonucleotide primers and M–MLV reverse-transcriptase were purchased from Promega. SYBR green ex Taq was obtained from TaKaRa. iNOS, TNF-α, IL-6, and β-actin oligonucleotide primers were purchased from Bioneer. PMSF, DTT, L-NIL (purity > 97 %), LPS (Escherichia coli, serotype 0111:B4), and all other chemicals were obtained from Sigma-Aldrich.

Cell culture and sample treatment

RAW 264.7 murine macrophages were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown at 37 °C in DMEM medium supplemented with 10 % FBS, penicillin (100 units/mL), and streptomycin sulfate (100 µg/mL) in a humidified 5 % CO₂ atmosphere. Cells were incubated with test samples for the indicated time and stimulated with LPS (1 µg/mL) for 24 h.

Nitrite, TNF-α, and IL-6 assays

Nitrite accumulation, an indicator of NO synthesis, was measured in culture media using the Griess reaction [22]. TNF-α and IL-6 levels in macrophages culture media were quantified using EIA kits according to the manufacturerʼs instructions (R&D Systems).

Western blot analysis

Cellular proteins were extracted from RAW 264.7 macrophages, as described previously [22], and protein concentrations were determined using Bio-Rad protein assay reagent according to the manufactureʼs instruction. Proteins were electroblotted onto PVDF membranes after being separated by 10 % SDS-polyacrylamide gel electrophoresis. Membranes were incubated for 1 h in blocking solution (5 % skimmed milk), with the primary antibody overnight at 4 °C, washed three times with Tween 20/Tris-buffer, incubated with horseradish peroxidase-conjugated secondary antibody (1 : 2000) for 2 h at room temperature and washed three times with Tween 20/Tris-buffer. Blots were then developed using an enhanced chemiluminescence (Amersham Life Science).

Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR)

Total cellular RNA was isolated using Easy Blue kits (Intron Biotechnology). For each sample, 1 µg of RNA was reverse-transcribed using MuLV reverse transcriptase, 1 mM dNTP, and 0.5 µg/µL oligo (dT_12–18).

Real-time PCR was performed using thermal cycler Dice real-time PCR system. The primers used for SYBR Green real-time reverse transcription–PCR were as follows: for iNOS, sense primer, 5′-CATGCTACTGGAGGTGGGTG-3′, anti-sense primer, 5′-CATTGATCTCCGTGACAGCCC-3′; for TNF-α, sense primer, 5′-AGCACAGA-AAGCATGATCCG-3′, anti-sense primer, 5′-CTGATGAGAGGGAG-GCCATT-3′; for IL-6, sense primer, 5′-GAGGATACCACTCCCAACAGACC-3′, anti-sense primer, 5′-AAGTGCATCATCGTTGTTCATACA-3′; and for β-actin, sense primer, 5′-ATCACTATT-GGCAACGAGCG-3′, anti-sense primer, 5′-TCAGCAAT-GCCTGGGTACAT-3′.

Transient transfection and luciferase assay

The mouse iNOS promoter plasmid (pGL3-iNOS; − 1592/+185) was prepared as described previously [36]. RAW 264.7 macrophages were cotransfected with pGL3-iNOS or NF-κB-Luc reporter plasmid vector plus phRL-TK plasmid (Promega) using Lipofectamine LTX™ (Invitrogen) according to the manufacturerʼs instructions. After transfection for 4 h, cells were pretreated with echinocystic acid for 1 h and then stimulated with LPS (1 µg/mL) for 18 h. Each well was washed with cold PBS, cells were lysed, and luciferase activity was determined using the Promega luciferase assay system.

Nuclear extraction and EMSA

RAW 264.7 macrophages were plated in 100-mm dishes and treated with echinocystic acid (10, 20, or 30 µg/mL), stimulated with LPS for 1 h, washed once with PBS, scraped into 1 mL of cold PBS and pelleted by centrifugation. Nuclear extracts were prepared as described previously [22]. They (5 µg) were mixed with double-stranded NF-κB oligonucleotide. 5′-AGTTGAGGGGACTTTCCCAGGC-3′ was end-labeled by [α-³²P] dCTP (the underline indicates a κB consensus sequence or a binding site for NF-κB/cRel homodimeric or heterodimeric complex) using a DNA labeling system (Amersham Life Science). Binding reactions were performed at 37 °C for 30 min in 30 µL of reaction buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 4 % glycerol, 1 µg of poly (dI-dC), and 1 mM DTT. Specificity of binding was examined by competition with 80-fold of unlabeled oligonucleotide. DNA-protein complexes were separated from an unbound DNA probe on native 5 % polyacrylamide gels at 100 V in 0.5 × Tris boric acid EDTA (TBE) buffer. Gels were vacuum-dried for 1 h at 60 °C and exposed to X-ray film at − 70 °C for 24 h.

Statistical analysis

The data are expressed as the mean ± SD of at least three experiments performed using different cell preparations. Statistically significant values were compared using one-way ANOVA followed by Dunnettʼs post test, and p values of less than 0.05 were considered statistically significant.

Supporting information

Data on the inhibitory effect of the 70 % EtOH extract, fractions, and isolated compounds on cell viability and LPS-induced NO production in macrophages as well as on inhibitory effects of the ECPRIE fraction and of echinocystic acid on LPS-induced NO production and LPS-binding are available as Supporting Information.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology (No. 2011–0023407). The NMR experiments were performed by the Korea Basic Science Institute (KBSI).

Conflict of Interest

The authors have no conflict of interest to declare.

^* These authors contributed equally to this work.

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Correspondence

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