Anti-Inflammatory Triterpenoids from the Caulophyllum robustum Maximin LPS-Stimulated RAW264.7 Cells

Caulophyllum robustum Maxim is widely distributed in China and used as a traditional herbal medicine to induce childbirth, ease the pain of labor, rectify delayed or irregular menstruation, alleviate heavy bleeding and pain during menstruation, and treat external injuries and irregular menses. According to our detailed chemical investigation, three new triterpene derivatives (1–3), together with seven known compounds, were isolated from the root and rhizome of C. robustum Maxim. Their structures were elucidated by 1D- and 2D-NMR spectroscopic analysis and physio-chemical methods. They were identified as (1) 23-hydroxy-3,19-dioxo-olean-12-en-28-oic-acid; (2) 23-hydroxy-3,11-dioxo-olean-12-en-28-oic acid; and (3) 16α,23-dihydroxy-3-oxo-olean-12-en-28-oic acid. Compounds (1–10) inhibited the LPS-activated NO production in RAW264.7 cells. Furthermore, the anti-inflammatory characteristics of these compounds were confirmed on the basis of decreases in iNOS and NF-κB protein expression in RAW264.7 cells.


Introduction
Caulophyllum robustum is a perennial herb in the family Berberidaceae, whichis well known as Hong Mao Qi in Chinese [1]. It is distributed in the northeast Tibet, Hubei provinces of China, native communities of North America, Its root and rhizome have beenused to induce childbirth, ease the pain of labor, rectify delayed or irregular menstruation, alleviate heavy bleeding and pain during menstruation [2].

Biological Activity Assays
Compounds 1-10 were tested for their ability to inhibit NO production in LPS-activated RAW264.7 macrophages as a measure of their anti-inflammatory effects, apositive control was DXMwith the value of 10 µM. The result indicated that the NO production was decreased by the presence of compound 1-5 with values of 25 µM [14] (Figure 3), since NO was made by catalytic synthesis of iNOS. NF-κB is a transcriptional factor that acts as an important mediator of the immune response and controls the expression of several proteins involved in inflammation, such as iNOS and cyclooxygenase-2 (COX-2) [15]. To further confirm the anti-inflammatory properties of 1 and 3, we investigated their effects on the protein expressions of NF-κB and iNOS in LPS-induced RAW264.7 macrophage cells using Western blotting; these proteins are involved in the pathogenesis of chronic inflammatory diseases [16]. Consistent with their inhibitory activity toward NO, compounds 1 and 3 inhibited the induction of iNOS and NF-κB protein expression in a dose-dependent manner [11] (Figures 4-7). Moreover, the housekeeping protein β-actin was not changed by the presence of compounds 1 and 3 at the same concentration.

Biological Activity Assays
Compounds 1-10 were tested for their ability to inhibit NO production in LPS-activated RAW264.7 macrophages as a measure of their anti-inflammatory effects, apositive control was DXMwith the value of 10 μM. The result indicated that the NO production was decreased by the presence of compound 1-5 with values of 25 μΜ [14] (Figure 3), since NO was made by catalytic synthesis of iNOS. NF-κB is a transcriptional factor that acts as an important mediator of the immune response and controls the expression of several proteins involved in inflammation, such as iNOS and cyclooxygenase-2 (COX-2) [15]. To further confirm the anti-inflammatory properties of 1 and 3, we investigated their effects on the protein expressions of NF-κB and iNOS in LPS-induced RAW264.7 macrophage cells using Western blotting; these proteins are involved in the pathogenesis of chronic inflammatory diseases [16]. Consistent with their inhibitory activity toward NO, compounds 1 and 3 inhibited the induction of iNOS and NF-κB protein expression in a dose-dependent manner [11] (Figures 4-7). Moreover, the housekeeping protein β-actin was not changed by the presence of compounds 1 and 3 at the same concentration.  (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) or DXM (apositive control, 10 μM) for 1 h, then stimulated with LPS (5 μg/mL) for 24 h. Nitrite levels, which reflect NO levels in culture media, were measured using Griess assays. The data shows the mean ± SD of three independent experiments. ** p < 0.01 and * p < 0.05compared to the LPS-treated values.

General
Optical rotations were recorded on an A25700-T digital polarimeter (RUDOLPH, Hackettstown, NJ, USA). IR spectra were obtained using a Thermo Tensor Nicolet-6700 spectrometer (Thermo Optics, Inc., Billerica, MA, USA) with KBr pellets, and 1D and 2D NMR spectra were recorded on a Bruker DRX-600 instrument (600 MHz for 1 H and 150 MHz for 13 C) with TMS as an internal standard, the deuterated solvent used to solubilize the samples were CD 3 OD and CDCl 3 .HR-ESI-MS was recorded on an UPLC-Q Exactive MS system (Thermo Fisher, Santa Clara, CA, USA). Silica gel (200-300 mesh (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech, Switzerland) were used for the chromatography column. Semi-preparative HPLC was performed on a DIONEX Ultimate 3000 system equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 µm, YMC Co. Ltd., Kyoto, Japan).

Plant Material
Theroot and rhizome of C. robustum Maximwere collected in May 2015 from Yi Chang City, Hubei Province, China. They were identified by Dr. Xinqiao Liu from the College of Pharmacy at South-Central University for Nationalities, China. A voucher specimen (No. CR-20150501) was deposited at the herbarium of the College of Pharmacy, South-Central University for Nationalities, China.

Cell Culture and CCK-8 Cell Viability Assay
RAW264.7 mouse macrophages were purchased from the Bio-Swamp life science lab (Bio-Swamp, MD, USA) and cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37 • C in 5% CO 2 . The subculture was carried out at 2 to 3 day intervals. When the cells were approximately 80% confluent, they were seeded in 96-well culture plates at 5 × 10 3 cells per well and incubated for 24 h for adhesion. After cells had been incubated with these treatments for 24 h, the effect of compounds 1 and 3 on cell viability was analyzed. RAW264.7 cells were treated in the absence or presence of compounds 1 and 3 (5,15, and 25 µM) for 24 h. Cell viability was determined by CCK-8 assay (data not shown).

Measurement of Nitric Oxide Production
LPS and Dexamethasone were purchased from Sigma Aldrich (Saint Louis, MO, USA). NO production was determined by the Griess reaction, which measures the accumulation of nitrite in the culture medium. When the cells were approximately 80% confluent, they were seeded in 96-well culture plates at 1 × 10 5 cells per well and incubated for 24 h for adhesion. The cells were then pretreated with phenol red-free medium containing the control (0.5% DMSO) or the indicated concentrations of compounds (1-10) for 2 h and then exposed to 5 µg/mL LPS for 24 h. The supernatant (50 µL) was collected, mixed with an equal volume (50 µL) of Griess reagent I and II. The absorbance at 540 nm was measured with a microplate reader. Sodium nitrite was used to generate a standard reference curve.

Western Blotting Analysis
Western blotting was performed as previously described with small modifications. RAW264.7 cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. The membrane was then blocked with 5% non-fat dry milk in TBST (20 mM Tris-HCl, 150 mMNaCl, and 0.05% Tween-20). After incubation with a primary antibody at 4 • C overnight, the membrane was hybridized with HRP-conjugated secondary antibody for 1 h, washed three times with TBST. The signal was detected by BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, Haimen, China). The molecular mass of bands was verified using a Multicolor Protein Marker (Beyotime, Haimen, China).

Statistical Analysis
The data were expressed as the mean ± standard deviation (SD) of at least three independent experiments. To compare three or more groups, one-way analysis of variance (ANOVA) followed by the Newman−Keuls post hoc test was used. A p-value of less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software, version 5.00 (GraphPad Software Inc., La Jolla, CA, USA).
All spectra can be found at the supplementary materials.

Conclusions
In conclusion, three new triterpene derivatives (1-3) isolated from C. robustum Maxim, a traditional medicine used for arthritis in the tujia ethnic areas in China, were demonstrated to have in vitro anti-inflammatory properties individually. The underlying mechanism may be associated with the regulation ofiNOS and NF-κB. This is the first report on the anti-inflammatory activity of the two triterpene (1 and 3) derivatives through iNOS and NF-κB. The present findings provide evidence for C. robustum Maxim as a promising treatment for inflammatory-related diseases.