Anti-inflammatory effects of 18-nor-ent-pimara-9(11),15-diene-4β-ol isolated from the roots of Aralia continentalis on LPS-induced in RAW264.7 cells

Aralia continentalis (A. continentalis) is a medicinal plant belonging to Araliaceae, it has been reported to exert anti-cancer, anti-bacterial, anti-inflammatory, anti-platelet and anti-oxidative activities. But the potential mechanism for the anti-inflammatory effect of compounds isolated from the roots of A. continentalis is still insufficient. So, we evaluated whether compounds isolated from the roots of A. continentalis exert anti-inflammatory effects and elucidated its potential mechanism in RAW264.7 cells. The concentrated residue was subsequently suspended in H2O and partitioned with n-hexane, methylene chloride (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The fractions were subjected to sequential column chromatography over silica-gel, RP-18, MPLC, recycling and preparative HPLC to isolated the novel compound. The novel compound was identified as 18-nor-ent-pimara-9(11),15-diene-4β-ol and confirmed anti-inflammatory activity. The 18-nor-ent-pimara-9(11),15-diene-4β-ol dose-dependently blocked NO production and inhibited iNOS, COX-2, TNF-α and IL-1β expression in LPS-stimulated RAW264.7 cells. The 18-nor-ent-pimara-9(11),15-diene-4β-ol inhibited LPS-stimulated degradation of IκB-α and nuclear accumulation of p65, which resulted in the suppression of NF-κB activation in RAW264.7 cells. Also, the 18-nor-ent-pimara-9(11),15-diene-4β-ol attenuated the phosphorylation of p38 and ERK1/2 in LPS-induced RAW264.7 cells. These results suggest that the nor-ent-pimara-9(11),15-diene-4β-ol isolated from the roots of A. continentalis may have grate potential for the development of anti-inflammatory drugs.


Introduction
In recent years, interest and expectations for medicinal resource plants have been increased, and many studies have been conducted to extract effective ingredients from natural resources and find functional materials because compounds present in medicinal resource plants have a variety of physiological vitality and are useful for living things [1][2][3].
Inflammatory reactions are reported to be involved in various pathological mechanisms, such as the introduction of bacteria and viruses into the body, in which the immune cells recognize and protect the body by secreting various inflammatory intermediaries, thereby promoting the growth of cancer cells, increasing insulin resistance, and worsening arteriosclerosis. The macrophages are known to be one of the important immune cells controlling the inflammatory response, responding to the early stages of the tumor necrosis factor-α (TNF-α), cytokines and lipopolysaccharide (LPS) infection and playing a Open Access *Correspondence: ppkh0230@korea.kr † Hyun-Ji Eo and Youngki Park contributed equally to this work 1 Forest Medicinal Resources Research Center, National Institute of Forest Science, 2009 Sobeck-ro, Yeongju, Gyeongsangbuk-do 36040, Republic of Korea Full list of author information is available at the end of the article pivotal role in the defense of the host and in maintaining the star. In particular, they are known to be involved in inflammatory reactions such as prostaglandin E 2 (PGE 2 ), nitric oxide (NO), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). These substances are reported to be involved in the development of inflammatory diseases. Inhibition of inflammatory factors is very effective in preventing the onset and progression of inflammatory diseases [4][5][6]. The nuclear factor kappa-B (NF-κB) activation is significantly associated with inflammatory diseases and much attention is focused on developing anti-inflammatory drugs targeting NF-κB. The mitogen-activated protein kinases (MAPK) cascade is one of the important signaling pathways in immune responses, and several recent studies reported that inhibition of MAPKs in mast cells can be an suitable target for pharmacological treatment of inflammatory diseases [7,8].

Cell culture
Mouse macrophage cell line, RAW264.7 cells were purchased American Type Culture Collection (ATCC, Virginia, USA) and grown in Dulbecco's Modified Eagle (Lonza, Walkerscille, MD, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 ug/ ml streptomycin. The cells were cultured in an incubator containing 5% CO 2 at 37 °C. Trypsin-EDTA (Sigma, St. Louis, MO, USA) was used to detach the cells from a T75 flask. The novel compound was dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v).

Cell viability
Cell viability was performed by MTT assay. Briefly, cells were plated at a density of 1 × 10 6 cells/well in 12-well plate and incubated for 24 h. The cells were treated with 18-nor-ent-pimara-9(11),15-diene-4β-ol at the indicated concentrations for 24 h. Then, the cells were incubated with 200 μl of MTT solution (1 mg/ml) for an additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm using UV/Visible (Perkin Elmer, Nowolk, CT, USA).

Nitric oxide generation assay
RAW264.7 cells were incubated 12-well plate for overnight. The cells were pretreated with 18-nor-ent-pimara-9(11),15-diene-4β-ol at the indicated concentrations for 6 h and then co-treated with LPS (1 μg/ml) for 18 h. NO levels were evaluated by Griess assay. Briefly, 50 μl of the cell culture supernatants were mixed with 50 μl of Griess reagent (Sigma Aldrich, St. Louis, MO, USA) and followed by reaction for 10 min at the room temperature. After 10 min, absorbance values were determined using a UV spectrophotometer (Perkin Elmer, Nowolk, CT, USA) at 540 nm.

Isolation of cytosol and nucleus fraction
Nucleus fractions from RAW264.7 cells after treatment of 18-nor-ent-pimara-9(11),15-diene-4β-ol and LPS were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocols. Briefly, we harvested RAW264.7 cells with cold 1Xhypotonic buffer and reacted at 4 ℃ for 15 min. After adding detergent and vortexing for 10 s, the cells were centrifuged at 15,000 rpm for 10 min at 4 °C and the supernatants (cytoplasmic fraction) were collected and stored at − 80 °C for further analysis. The cell pellets were used for nuclear fraction collection. Cell pellets were re-suspended with lysis buffer by pipetting up and down, and incubated at 4 °C for 30 min under shaking. After 30 min, nuclear suspensions were centrifuged at 15,000 rpm for 10 min at 4 °C, and the supernatants (nuclear fraction) were stored at − 80 °C for further analysis.
We separated the equal proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred them to nitrocellulose membrane. The nitrocellulose membranes were blocked with 5% non-fat dry milk in tris-buffered saline containing 0.05% tween 20 (TBS-T) by stirring at room temperature for 1 h and then incubated with specific primary antibodies in 5% skim dry milk in 0.05% TBS-T at 4 °C for overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL western blotting substrate and visualized in Chemi Doc MP Imaging system (Bio-rad, CA, USA).

Statistical analysis
All the data are shown as mean ± SD (standard deviation). Statistical analysis was determined by Student's t-test. Differences with *P or #P < 0.05 were considered statistically significant.
The effect of 18-nor-ent-pimara-9(11),15-diene-4β-ol on NF-κB signaling activation in LPS-induced RAW264.7 cells The nuclear factor kappa-B (NF-κB) serves to control the activation of inflammation. NF-κB is not a single gene but a family of closely related transcription factors that contain five genes: NF-κB1(p50/p105) NF-κB2(p52/ p100), RelA(p65), c-Rel, and RelB. The activities of NF-κB are strictly regulated by interaction with inhibitory IκB protein. As with the NF-κB transcription factors, there are several IκB proteins (IκBα, IκBβ, IκBγ and IκBε) that have different affinities for individual NF-κB dimers. The activation of NF-κB dimers is the result of IKK-mediated, phosphorylation-induced degradation of the IκB inhibitor, which enables the NF-κB dimers to enter the nucleus and activate specific target gene expression. The degradation and phosphorylation of IκB in response to LPS lead to NF-κB translocation to the nucleus. This event is related to the activation of a wide range of NF-κBresponsive pro-inflammatory genes [20][21][22][23][24][25].