Quantitative real-time reverse transcriptase

RAW 264.7 cells were pretreated with ebractenoid F for 2 h, and then LPS (1 μg/mL) for 18 h. After incubation, the total RNAs were extracted using the Trizol reagent kit (Invitrogen, Carlsbad, CA). Both the amount and purity of RNAs were measured using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). The cDNA was synthesized from one microgram of total RNAs using the amfiRivert Platinum cDNA Synthesis Master Mix (GenDepot, Barker, TX) according to the manufacturer’s instructions. Quantitative real-time reverse

Quantitative real-time PCR was performed using forward and reverse primers and a SYBR Green working solution (AccuPower^® 2XGreenStar qPCR Master Mix, Bioneer, Daejeon, Korea), with the following conditions: 95℃ for 30 s, followed by 40 cycles of 95℃ for 15 s, 55℃ for 20 s, and 72℃ for 35 s. The following primers were used (Table 1): iNOS, 5’-TCC TAC ACC ACA CCA AAC-3’ (sense) and 5’-CTC CAA TCT CTG CCT ATC C-3’ (antisense); COX-2, 5’-CCT CTG CGA TGC TCT TCC-3’ (sense) and 5’-TCA CAC TTA TAC TGG TCA AAT CC-3’

(antisense); IL-6, 5’-AGG CTT AAT TAC ACA TGT TCT CTG G-3’ (sense) and 5’-TTA TAT CCA GTT TGG TAG CAT CCA T-3’ (antisense); IL-1β, 5’-GCC ACC TTT TGA CAG TGA TGA G-3’ (sense) and 5’-AGT GAT ACT GCC TGC CTG AAG-3’ (antisense); MCP-1, 5’-ATG CAG TTA ATG CCC CAC TC-3’ (sense) and 5’-TTC CTT ATT GGG GTC AGC AC-3’ (antisense); GAPDH, 5’-GCC ATC AAT GAC CCC TTC ATT-3’ (sense) and 5’-GCT CCT GGA AGA TGG TGA TGG-3’

(antisense); β-actin, 5’-CTG ACT ACC TCA TGA AGA TCC TC-3’ (sense) and 5’- CAT TGC CAA TGG TGA TGA CCT G-3’ (antisense).

Table 1. Primer sequences of inflammatory medicators

2.13 Preparation of the cytoplasmic and nuclear extract

RAW 264.7 cells were treated with ebractenoid F for 2 h prior to LPS (1 μg /mL) stimulation. The cells were washed and suspended in 100 μL of iced-cold lysis buffer A [10 mM HEPES [pH 7.9], 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail] for cytoplasmic extract on ice for 15 min. After ice incubation, 12.5 μL of 10% NP-40 was added. The tubes were agitated on a vortex for 20 s and then centrifuged for 5 min. The resulting supernatant represented the cytosolic extract. The remain pellets were the nuclear extract. The pellets were resuspended in 20 μL of ice-cold nuclear extraction buffer (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.4mM PMSF and a protease inhibitor cocktail) and incubated on ice for 1 h with intermittent vortexing. This nuclear extract was centrifuged for 10 min at 15,000 rpm; the resulting supernatant represented the nuclear fraction.

2.14Statistical analysis

The results represent the mean ±the standard deviation (SD) from three different experiments. A one-way analysis of variance (ANOVA) followed by a Dunnett’st- test was applied to assess the statistical significance of the differences between the study groups. P values < 0.05 were considered statistically significant [*P < 0.05;

**P< 0.01; ***P<0.001].

Ⅲ. RESULTS

1.Isolation of Ebractenoid F (EF) from E. ebracteolata by bioassay-guided fractionation

The bioassay-guided fractionation was performed to isolate the most effective bioactive compound. The subject fractions for isolation were selected by bioassays at every single step (Figure 6). Five kilograms of Euphorbia ebracteolata Hayata was grinded and extracted with 18 L of methanol for 6 h at the room temperature.

This process was repeated for five times. After filtration, the extracts were evaporated under reduced pressure and lyophilized. The methanol extract (125.9 g) was partitioned with n-hexane (n-Hex), methylene chloride (MC), ethyl acetate (EA), n-butanol (n-BuOH), and distilled water (DW) at 1:1 ratio for three times (Figure 7). All fractions were evaporated to be dried under vacuum. The n-Hex fraction (39.2 g), which potentially reduced nitric oxide (NO) production at the non-toxic dose on the RAW 264.7 cells, was selected to be sub-fractionated and thoroughly evaporated. Thirty grams of n-Hex fraction was dissolved in 80%

acetonitrile and sub-fractionated using high speed counter current chromatography (HSCCC, Tauto TBE-1000A, Shanghai Tauto Biotech Co. Ltd, Shanghai, China)

Figure 6. Separation scheme of E. ebracteolata

E. ebracteolata was extracted with MeOH, and sequentially partitioned with n-Hex, MC, EA, n-BuOH, and DW. The n-Hex layer (30 g of 39.2 g) was injected into HSCCC to obtain 12 sub fractions. After then, the selected H10 sub fraction was further isolated using preparative HPLC.

Figure 7. HPLC-UV chromatogram of (A) MeOH extract and (B) n-Hex fraction (A) The total MeOH extract of E. ebracteolata (B) The n-Hex fraction which includes less polar components in E. ebracteolata. The mobile phases were composed of 0.1%

formic acid in water and 0.1% formic acid in acetonitrile. The flow rate of 0.8 mL/min was employed with INNO C18 column (I.D. 4.6 mm x 150 mm, 5 μm particle size) in HPLC-UV analysis. The gradient system was illustrated in section 2.1.

Figure 8. (A) HSCCC chromatogram of n-Hex fraction and (B) HPLC-UV chromatogram of H10 sub-fraction

(A) The HSCCC chromatogram of n-Hex layer. The grey region, designated as H10, was collected from 290 to 320 min. H10 was selected as the most potential fraction in the n- Hex layer. (B) The HPLC-UV chromatogram of H10 sub-fractionated from the n-Hex layer. Following solvent partition at the previous experiment, n-Hex was subjected to be separated by HSCCC. The 0.1% formic acid in n-Hex was used as the stationary phase, 0.1% formic acid in acetonitrile, and 0.1% formic acid in distilled water were used as the mobile phase. The flow rate of 3 mL/min and revolution speed of 460 rpm were employed in HSCCC separation. The gradient system was illustrated in section 2.2.

Figure 9. (A) Preparative-HPLC chromatogram of H10 sub-fraction and (B) HPLC- UV chromatograms of separated compounds

(A) The prep-HPLC chromatogram of H10 sub-fraction. (B) The HPLC-UV chromatograms of pure compounds, H10-4, H10-5, and H10-6, isolated from the sub- fractionated H10 layer. Following the previous sub-fractionation using HSCCC, H10 was subjected to be purified by preparative-HPLC. The mobile phases were composed of 0.1%

formic acid in water and 0.1% formic acid in acetonitrile. The flow rate of 3 mL/min was

2.Screening fractions/compounds from E. ebracteolata

To access the anti-inflammatory potency of extracts, fractions, sub-fractions, and compounds from E. ebracteolata, anti-inflammatory effects were evaluated at the nontoxic doses. First of all, we determined the cytotoxicity of them on RAW264.7 cells using MTT assay. And then, nitric oxide (NO) and NF-κB Secretary Alkaline Phosphatase (SEAP) assay were applied at each separation step. The results were shown in Table 2.

First, the roots of E. ebracteolata were extracted with methanol, and the extract showed promising reduction of nitric oxide (NO) at the nontoxic dose (IC₅₀= 1.1± 0.2 μg/ml). Next, the methanol extract was partitioned with solvents (n-Hex, MC, EA, n-BuOH, and DW). Among the solvent fractions, the solvent fraction which exhibited the most effective NO reduction was then- Hex fraction (IC50= 2.4±0.2 μg/mL). For the further separation, the n- Hex fraction was sub-fractioned using HSCCC to offer 12 sub-fractions. All the sub-fractions had outstanding NO suppression. Hence, NF-κB Secretary Alkaline Phosphatase (SEAP) assay was used to screen samples in that NO production could be evoked by the NF-κB signaling pathway. Based on the results of NF-κB SEAP inhibitory effects, the H10 sub-fraction exhibited the most promising anti-inflammatory effect (IC₅₀ = 4.0 ± 1.7 μg/mL). Finally, the H10 sub-fraction was separated into the final fractions (H10-1~H10-6) using preparative-HPLC. Among them, the three compounds, including H10-4, H10-5, and H10-6, were elucidated as pure compounds. As shown in Figure 10, H10-6 was chosen to explore its bioactive mechanism for the further study (IC₅₀= 7.7± 0.82 μM).

Table 2. Inhibitory effects of extract, fractions, sub-fractions, and compounds on LPS-induced NO and NF-κB SEAP production in macrophage RAW 264.7 cells

including MeOH extract, solvent partitions, CCC fractions, prep-HPLC sub- fractions, and single compounds for 2 h before LPS (1 μg/mL) stimulation. Cell viability was determined by MTT assay after 24 h sample treatment. NO production was estimated by NO assay after 18h LPS stimulation, and NF-κB SEAP was measured after 16 h LPS treatment.

a: IC80by MTT assay; ^b: IC50by NO assay; ^c: IC50by SEAP assay; IC50: half maximal inhibitory concentration; -: no effect; N.D.: not determined

Figure 10. The effects of the selected H10-6 on LPS-stimulated inflammatory mediators, NO and NF-κB SEAP, in macrophages

Cells (1 Χ 10⁵cells/well) were seeded in 24-well plates and incubated for 24 h. After then, cells were pretreated with the indicated doses of the sample for 2 h before LPS (1 μg/mL) stimulation. Cell viability was determined by MTT assay after 24 h sample treatment. NO production was estimated by NO assay after 18 h LPS stimulation, and NF-κB SEAP was measured after 16 h LPS treatment. Data were obtained from three independent experiments and expressed as the mean ± standard deviation (S.D).

3.Elucidation and Determination of Ebractenoid F (EF)

The fraction H10-6 was analyzed and the purity was accessed by HPLC-UV and HPLC-MS. In addition, the chemical structure of the isolated compound was determined based on the 1D/2D NMR data.

H10-6 is obtained as yellow oil. Its molecular formula is determined to be C₁₉H₂₄O₂ based on ESI-MS at m/z 287 [M+H]⁺and NMR analysis (CDCl₃). It is detected under UV (MeOH) λ_max(logε) 220/280 nm. The ¹H and ¹³C NMR data of H10-6 indicated that it is an 18-norrosane diterpenoid with an aromatic A-ring.

According to the ¹H NMR (CDCl₃, 500 MHz), one proton (H-15) at δ5.86 (dd, J=

17.4, 10.8 Hz) and two protons (H-16 and H-16') at δ4.87 (dd, J= 10.8, 0.9 Hz) and 4.95 (dd, 17.4, 0.9 Hz) indicate the terminal methylene group. The HMBC correlations from H-1 to C-3 (δ139.9), C5 (δ127.1), and C9 (36.4), from H-6 to C- 4 (δ122.6), C-5 (δ127.1), and C-10 (δ140.4), from Me-19 to C-3 (δ139.9), C-4 (δ 122.6), and C-5 (δ127.1), and from 3-OH to C-2 (δ140.8), C-3 (δ139.9), and C-4 (δ 122.6), reveal the presence of a 3,4,5-trisubstituted catechol as ring A. The relative configuration is determined by the ROESY spectrum. The correlations of Me-20/H-12β, H-12β/H-15, and H-8/Me-17 indicate a β-orientation for Me-20 and α-orientations for H-8 and Me-17 (Figure 12-17, and Table 3). Thus, the chemical structure of H10-6 was defined as shown in figure 11 and represents ebractenoid F (EF).

Figure 11. Chemical structure of ebractenoid F

The isolated compound was determinated by 1D/2D NMR and MS spectrometry.

H10-6 was called Ebractenoid F (EF).

Figure 12. LC-ESI/MS spectra and UV spectrum of H10-6 (EF)

The H10-6 (EF) generated the protonated ions [M+H]⁺ at m/z287.1 in positive ion mode and the deprotonated ions [M-H]^-at m/z 285.1 in negative ion mode. The compound has maximum UV absorption at 220 and 280 nm.

Table 3. ¹H and ¹³C NMR assignment of H10-6 (EF) (δin ppm, Jin Hz, 500 and 125 MHz in CDCl₃)

Figure 13. ¹H NMR spectra of H10-6 (EF) (Dissolved in CDCl3, 500 MHz)

Figure 14. ¹³C NMR spectra of H10-6 (EF) (Dissolved in CDCl3, 125 MHz)

Figure 15. HSQC spectrum of H10-6 (EF) (Dissolved in CDCl3, 500 MHz)

Figure 16. HMBC spectrum of H10-6 (EF) (Dissolved in CDCl₃, 500 MHz)

Figure 17. ROESY spectrum of H10-6 (EF) (Dissolved in CDCl3, 500 MHz)

4.Effects of Ebractenoid F (EF) on pro-inflammatory mediators in LPS-stimulated RAW 264.7 cells

Besides NO and NF-κB SEAP screening (Figure 10), excessive pro-inflammatory mediators produced by macrophages, such as cytokines and chemokines, can trigger various chronic inflammatory diseases. Among them, interleukin (IL)-6, IL- 1β, and MCP-1 could be regarded as potent biomarkers to evoke inflammation. In this vein of thought, the inflammatory mediators in RAW 264.7 cells were induced by LPS. The effects of EF on the inflammatory mediators were examined by real- time PCR and western blot. As a result, EF decreased the pro-inflammatory cytokines, which are interleukin (IL)-6, IL-1β, and MCP-1. Furthermore, it reduced the pro-inflammatory enzymes like inducible Nitric Oxide Synthatase (iNOS), and Cyclooxygenase-2 (COX-2) generating the inflammatory mediators (NO and PGE₂) at both mRNA and protein levels. The data indicated that EF inhibited NO, NF-κB SEAP, IL-6, IL-1 β, and MCP-1 by affecting on enzymes like iNOS and COX-2 expression at the transcriptional level (Figure 18).

Figure 18. Ebractenoid F (EF) inhibited the downstream signaling pathways of NF- κB in LPS-stimulated RAW 264.7 cells.

Cells (1 Χ 10⁶ cells/well) were seeded in 6-well plates and incubated for 24 h. After then, cells were pretreated with the indicated doses of the sample for 2h before LPS (1 μg/ml) stimulation.

(A) The relative level of pro-inflammatory cytokines (IL-6, IL-1β, and MCP-1) mRNA expression (2^-ΔCt) was determined by real-time PCR and calculated by subtracting the C_t

value for β-actin from the C_tvalues for IL-6, IL-1 β, and MCP-1. ΔC_t = CtIL-6 or IL-1 β or MCP-1 - C_{t β-actin}. (B) At the same condition like above, the relative level of pro- inflammatory enzymes (iNOS, and COX-2) mRNA expression was examined by real- time PCR. The data were obtained from three independent experiments and expressed as the means ± S.D. (C) After 18h LPS treatment, the whole lysates were subjected to Western blot analysis immunoblotting with iNOS, COX-2, and β -actin.

5. Effects of Ebractenoid F (EF) on LPS-mediated NF-κB translocational and transcriptional activity

Since NF-κB is the key regulator of inflammation, we examined whether EF affected on the NF-κB activity. After LPS stimulated RAW 264.7 cells, there were the sequential signaling pathways. To describe the effects of EF on NF-κB, when treated LPS for 15 min followed by EF (15 μM) treatment for 2 h, the extracted whole lysates and nucleus lysates were analyzed by western blot. Using the whole lysates, both inhibition of phosphorylation and degradation of iκB-α in cytosol were observed. In addition, the nuclear translocation of NF-κB dimer was attenuated by 15 μM EF treatments after 10 to 15 min LPS stimulation. Plus, NF- κB transcriptional activity by EF treatment was measured by luciferase assay. After NF-κB transfected RAW 264.7 cells were stimulated by LPS for 4-6 h, luciferase activity from the cell lysates was weakened. According to the results, we confirmed that EF could attenuate both translocational and transcriptional activity of LPS- medicated RAW 264.7 cells (Figure 19).

Figure 19. Ebractenoid F (EF) inhibited NF-κB in LPS-stimulated RAW 264.7 cells.

Cells (1 Χ 10⁶ cells/well) were seeded in 6-well plates and incubated for 24 h. After then, cells were pretreated with the indicated doses of the sample for 2h before LPS (1 μg/ml) stimulation.

(A) After 0-15 min LPS treatment, the whole lysates were subjected to Western blot analysis with iκB-α, p-iκB-α, and β -actin. (B) After 0-15 min LPS treatment, cytoplasmic and nuclear extracts were extracted and analyzed by Western blot with p65, p50, iκB-α and β-actin. (C) The cells were transfected with NF-κB luciferase reporter plasmid for 4h, followed by LPS stimulation for 6h.

6. Effects of Ebractenoid F (EF) on the upstream of NF-κB and MAPKs pathway

As shown in previous results, EF regulated transcriptional and translocational activity of NF-κB. We investigated the upstream signaling pathway to regulate NF- κB. It is reported that LPS can evoke NF-κB activation via TLR4-MyD88-TIRAP pathway. EF decreased the phosphorylation of AKT and IKKα/β at the right upper factors of NF-κB. However, the uppermost factors, TLR4, MyD88, and TIRAP, were not affected by EF treatment. Hence, it may pass through the plasma membrane and specifically target on NF-κB.

As another important fact, the MAPKs pathway is one of the most extensively investigated signal transduction pathways related to inflammation process.

Previous studies have reported that MAPKs pathway plays an important role during the release of pro-inflammatory cytokines and inflammatory mediators in LPS-induced RAW264.7 cells. Therefore, we examined the effects of EF on the phosphorylation levels of p38 MAPK extracellular signal-regulated kinase (ERK) and jun NH2-terminal kinase (JNK) in the presence of LPS treatment by western blot analysis. As shown in Figure 20, EF down-regulated the phosphorylation of JNK, and ERK1/2 induced by LPS stimulation within 15 min.

Through these works, we settle that EF can modulate the inflammatory transcription factor, NF-κB, by regulating the inhibitory effects of NF-κB upstream pathways (AKT, and IKK α/β) and MAPKs (JNK, and ERK1/2) signaling pathways (Figure 21).

Figure

20

Cells (1 Χ 10⁶cells/well) were seeded in 6-well plates and incubated for 24 h. After then, cells were pretreated with the indicated doses of the sample for 2h before LPS (1 μg/ml) stimulation.

Within 15min LPS treatment, the whole lysates were subjected to Western blot analysis (A) with p-Akt, Akt, p-IKK α/β, IKK α/β, MyD88, TIRAP, and β -actin. (B) with p-p38, p38, p-JNK, JNK, p-ERK1/2, ERK1/2, and β -actin.

Figure x. The effects of EF on inflammatory mechanisms

Ⅳ. DISCUSSION

Understanding how the NF-κB pathway influences and is influenced by signaling pathways provides crucial insight into the regulation of inflammatory responses [29]. Though lots of treatments for inflammation-associated diseases have been launched and diagnosed, the drugs have still side-effects [4]. The studies of new agents from natural products have been conducted continuously [42]. We now know that LPS-evoked TLR4 signaling pathways are clearly related to inflammation [28], through these pathways aforementioned in figure 21.

As the efficient separation method, activity-guided fractionation and purification processes were employed to identify the anti-inflammatory compound from Euphorbia ebracteolata [1]. Powdered roots of the medicinal herb were extracted with ethanol and then partitioned with solvents, n-Hex, MC, EA, n-BuOH, and DW.

Among them, n-Hex layer showed strong activity and therefore, subjected to separation and purification using various chromatographic techniques. The extract was fractionated into 1-12 fractions by HSCCC, and the fraction H10 showing potent activity was isolated using prep-LC. Isolated fraction was identified by comparing spectral data (LC-UV, NMR, and ESI-MS) with literatures [19-25, 39- 41] to be a rosane-type diterpene, ebractenoid F [21, 22].

the most proficient compound by bioassay-guided fractionation against inflammation. Using the non-toxic doses, inflammatory mediators dissolved in supernatant excreted from the cells were investigated by NO assay [30, 31]. To concrete its anti-inflammatory activities, NF-κB SEAP assay was conducted as well [32]. Through all the processes, H10-6, ebractenoid F, was selected as the therapeutic agents against inflammation by narrowing the candidates.

Ebractenoid F is one of active constituents of Euphorbia ebracteolata [21, 22]. It has been reported that Ebractenoid F reduces nitric oxide in murine macrophages at the non-toxic doses [22]. Macrophage plays a central role in a host’s defense against bacterial infection through phagocytosis, cytotoxicity, and intracellular killing [7]. Although the research has shown that ebractenoid F induced anti- inflammatory effects in macrophages, the inherent mechanism underlying the effect of EF on LPS-induced inflammation remains unknown. The results indicated that ebractenoid F decreased nitric oxide and NF-κB SEAP in supernatant into which inflammation-induced cells secreted (Fig. 10). We demonstrated that ebractenoid F can inhibit many inflammatory mediators, such as enzymes, cytokines and chemokines, which induce inflammation at both protein and mRNA levels.

Ebractenoid F suppressed iκB-α phosphorylation and degradation, staying in inactive (Fig. 11A). In addition, the results showed that ebractenoid F inhibited nuclear translocation of NF-κB and NF-κB -DNA binding activities (Fig. 11B).

Furthermore, we investigated whether ebractenoid F may not only inhibit the binding of NF-κB to the DNA but it also inhibits the upstream signaling proteins.

When engaged by LPS, the LPS receptor, TLR4, transduces signals through MyD88 and TRAF6 [28]. It is possible that ebractenoid F suppresses inflammatory genes and transcription factors by blocking the TLR4 or other accessory proteins, such as MyD88 and TIRAP [14]. The induction of MyD88 and TIRAP leads to the activation of NF-κB followed by the production of pro-inflammatory cytokines [28]. PI3K, existing in a complex with TLR4 and MyD88 in murine macrophages, and its downstream target kinase, AKT, appear to be important components of LPS-induced NF-κB activation. NF-κB is also activated by NF-κB upstream signaling cascades such as IKK-α/β [29]. As a result, although ebractenoid F did not reduce the uppermost protein, TLR4, MyD88, and TIRAP, ebractenoid F had an effect on the protein levels of constitutively phosphorylated AKT and IKKs (Fig.

12A).

MAPKs are a highly conserved family of protein serine/threonine kinases and have been shown to play significant roles in inflammation induced by various stimuli [43]. MAPKs are the main kinases involved in activation of a number of downstream pathways such as ERK, JNK and p38, which are also associated with NF-κB. The result showed that ebractenoid F suppressed the phosphorylation of ERK, and JNK in a dose-dependent manner without affecting their total protein levels (Fig. 13).

In conclusion, this study provides the concrete evidence on anti-inflammatory effects of ebractenoid F. Anti-inflammatory effects of ebractenoid F are associated