The leaves of Acanthopanax henryi (Oliv.) Harms were collected in October 2012 in Xinhua (Hunan, China), 230 km west from Hunan (latitude N 27 56′ 16′, longitude E 111° 21′ 22′). The plant is not endangered or protected, and its identify was confirmed by Professor Liu Xiang-Qian (Hunan Key Laboratory of Traditional Chinese Medicine Modernization, Hunan University of Chinese Medicine, Changsha, China) and a voucher specimen (no. 20121125) was deposited within the School of Pharmacy, Hunan University of Chinese Medicine. No specific permission was required for collecting the plant.

The dried leaves of Acanthopanax henryi (Oliv.) Harms (10 kg) were cut into small pieces and extracted three times with methanol (3×100 l) by soaking at room temperature, and then concentrated to give a dark-green residue (0.8 kg), which was suspended in H₂O and separated with petroleum ether. The water fraction was fractionated by column chromatography (CC) on macroporous resin eluted with a gradient ethanol/H₂O (0, 30, 50, 75 and 95%) into five fractions, 1–5. Fraction 3 (75% ethanol, 14.0 g) was subjected to silica gel CC eluted with CHCl₃/methanol/H₂O (25:1:0/1:1:0.2) to give 15 fractions, A-O.

Fraction M (75 mg) was subjected to silica gel CC eluted with CHCl₃/methanol/H₂O (4:1:0.1/1:1:0.2) to give four sub-fractions, M1-M4 and M2-M4 were further separated using Sephadex LH-20 (methanol) to produce AS II (51 mg) (15).

The structures of the compounds were identified by mass spectrometry (MS), one-dimensional (1D)-nuclear magnetic resonance spectroscopy (NMR) and 2D-NMR, with a comparison of the spectral data with those reported previously in the literature (16).

The purity and content of AS II in the dried leaves of Acanthopanax henryi (Oliv.) Harms were determined by HPLC as previously described (17). Briefly, 4.88 mg AS II was dissolved in 50 ml 100% methanol to a final concentration of 0.0976 mg/ml for HPLC analysis. The dried leaves of Acanthopanax henryi (Oliv.) Harms (100 g) were cut into small pieces and extraction was performed three times using methanol (3X 1 l) and reflux extraction at 65°C for 2 h each time. The extract was then concentrated to generate a dark-green residue, which was suspended in 100 ml H₂O and separated with petroleum ether. The water fraction was fractionated by column chromatography (80×100 mm) on D101 macroporous resin (600 g; Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China) and eluted with a gradient of ethanol/H₂O (0, 30 and 75%) into three fractions: Fractions 1 to 3. Fraction 3 was concentrated, transferred into volumetric flasks and diluted with 100% menthol to 50 ml to produce the sample for HPLC content analysis with a Kinetex XB-C18 analytical column (100×4.6×2.6 -mm; Phenomenex, Inc., Torrance, CA, USA) at 30°C. Elution was conducted using mobile phase A (water) and mobile phase B (acetonitrile) with a gradient as follows: 0–2 min, 29–31% B; 2–13 min, 31–35% B; 13–15 min, 35–40% B; 15–23 min, 40–44% B; 23–25 min, 44–46% B; 25–31 min, 46–49% B; 31–38 min, 49–55% B. The flow rate was kept constantly at 1.0 ml/min, and the effluents were monitored at 210 nm using an Agilent 1200 HPLC system with a variable wavelength detector (Agilent Technologies, Inc., Santa Clara, CA, USA). The purity value of AS II, as evaluated by HPLC, was observed to be >98% by the peak area normalization method. The value of purity was obtained by calculating the percentage of its peak area to that of the total peaks in the HPLC chromatogram. The content of AS II in the leaves of Acanthopanax henryi (Oliv.) Harms was 3.28 mg/100 g, which was determined using the external standard method with the isolated AS II as standard.

Melting points (uncorrected) were measured using a Boetius micromelting point apparatus. Hydrogen-1-NMR (600 MHz), Carbon-13-NMR (150 MHz) and 2D-NMR were recorded at room temperature in methanol or pyridine-d5 using a Bruker ACF-500 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) and chemical shifts were presented in δ (ppm) values relative to tetramethylsilane as an internal standard. Mass spectra were obtained on an MS Agilent 1200 Series LC/MSD Trap Mass spectrometer (ESI-MS; Agilent Technologies, Inc.). Column chromatography was carried out on silica gel (200–300 mesh and 100–200 mesh; Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Merck KGaA, Darmstadt, Germany) and D101 macroporous resin (Tianjin Guangfu Chemical Co., Ltd., Tianjin, China). Reversed-phase thin-layer chromatography was performed on a precoated RP-18F254s plates (Merck KGaA). Thin-layer chromatography was conducted on self-made silica gel G (Qingdao Marine Chemical, Inc.) plates and spots were visualized by spraying with 10% H₂SO₄ in ethanol (v/v) followed by heating at 105°C.

RPMI-1640, penicillin and streptomycin were obtained from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Bovine serum albumin, LPS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Merck KGaA). Inducible NOS (iNOS; cat. no. sc-651), COX-2 (cat. no. sc-1745), TLR-4 (cat. no. sc-16240), NF-κB (cat. no. sc-8008), β-actin (cat. no. sc-47778) and peroxidase-conjugated secondary antibodies (anti-mouse, cat. no. sc-2005; anti-rabbit, cat. no. sc-2004; anti-goat, cat. no. sc-2020) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse IL-6 ELISA kit (cat. no. 555240) and mouse TNF-α (mono/mono) ELISA kit (cat. no. 555268) were purchased from BD Biosciences (San Jose, CA, USA). PRO-PREP™ Protein Extraction Solution was purchased from Intron Biotechnology, Inc. (Seongnam, Korea). An RNeasy Mini kit and a QuantiTect Reverse Transcription kit were purchased from Qiagen GmbH (Hilden, Germany). Finally, fluorochrome-conjugated secondary antibodies (anti-mouse, cat. no. A-11029 and anti-goat, cat. no. A-21432) and fluorochrome-conjugated LPS (cat. no. L-23351) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

The RAW 264.7 cells were obtained from the Korea Research Institute of Bioscience and Biotechnology (Seoul, Korea) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin sulfate. The cells were cultured in a humidified incubator with 5% CO₂ atmosphere at 37°C. To stimulate the cells, the medium was replaced with fresh RPMI 1640 medium followed by addition of LPS in the presence or absence of AS II for the indicated periods.

An MTS assay was used to determine the viability of RAW 264.7 cells. Cells (5×10⁴ cells per well) were plated in 96-well plates (SPL Life Sciences, Pocheon, Korea). Cells were treated without or with AS II (10, 20 and 40 µM) and the plates were incubated for 24 h at 37°C followed by a further 2 h with MTS solution (5 mg/ml). Optical density was measured at 490 nm. Cell viability was calculated using the formula (mean absorbance value of treated cells/mean absorbance value of untreated cells)x100.

The cells were seeded at 5×10⁴ per well in 96-well culture plates. The RAW 264.7 cells were stimulated with LPS (200 ng/ml) without or with AS II (10, 20 and 40 µM) for 24 h. Nitrite in the cultured RAW 264.7 cell supernatant was determined using Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid). An equal volume of Griess reagent was mixed with the supernatant and incubated at room temperature for 5 min. Nitrite concentrations were measured at 570 nm using an Epoch microplate spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA). A sodium nitrite (NaNO₂) standard curve was used to calculate nitrite concentration.

The amount of PGE₂ was determined using a PGE₂ Enzyme Immuno-Assay kit (GE Healthcare Life Sciences, Chalfont, UK) according to the manufacturer's protocols. Briefly, 2.5×10⁵ RAW 264.7 cells per well were cultured in 24-well culture plates. AS II (10, 20 and 40 µM) and LPS (200 ng/ml) was added to each well and incubated at 37°C for 24 h. The supernatant was collected and used to measure PGE₂ production.

RAW 264.7 macrophages (2.5×10⁵ per well) were cultured in 24-well plates and treated with LPS (200 ng/ml) in the presence or absence of AS II (10, 20 and 40 µM) for 24 h. Levels of TNF-α and IL-6 in the culture media were quantified using ELISA kits, according to the manufacturer's protocols (BD Biosciences). Briefly, ELISA plates (Nalge Nunc International; Thermo Fisher Scientific, Inc.) were coated overnight with coating buffer including anti-mouse IL-6 and TNF-α antibodies. The wells were washed and the samples and standards added. Following a 2 h incubation, biotinylated anti-mouse IL-6 monoclonal antibody and biotinylated anti-mouse TNF-α with streptavidin-horseradish peroxidase reagent were added each well and incubated for 1 h. The wells were washed and the tetramethylbenzidine substrate solution added to the wells and incubated for 30 min in the dark. A stop solution (2N H₃PO₄) was added and absorbance was read at 450 nm.

RAW 264.7 cells were plated at 6×10⁶ cells per well in 6-well culture plates and treated with LPS (200 ng/ml) in the presence or absence of AS II (10, 20 and 40 µM) for 24 h at 37°C. Following incubation, the cell pellets were lysed in PRO-PREP™ Protein Extraction Solution on ice for 20 min. Protein levels in collected supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocols. Sodium dodecyl sulfate-polyacrylamide gel (10% for iNOS and COX-2; 12% for TLR-4) electrophoresis were performed with equal amounts of protein (240 ng/lane) and transferred onto polyvinylidene membranes (EMD Millipore, Billerica, MA, USA). Following blocking with TBS-T [20 mM/l Tris-HCl (pH 7.6), 137 mM/l NaCl, 0.05% Tween 20] containing 5% skimmed milk, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000). The membranes were washed with TBS-T and incubated for 1 h at room temperature with anti-mouse, anti-goat or anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (1:2,000). The bands were evaluated by using ECL Prime Western Blotting Detection Reagent and an ImageQuant LAS 4000 Mini BioMolecular Imager (GE Healthcare).

RAW 264.7 cells were plated at 6×10⁶ cells per well in 6-well culture plates and treated with LPS (200 ng/ml) in the presence or absence of AS II (10, 20 and 40 µM) for 24 h at 37°C. Following incubation, an RNeasy Mini kit was used to isolate total cell RNA. Then, 1 µg total RNA was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription kit (Qiagen GmbH) according to the manufacturer's protocols. RT-qPCR was performed using power SYBR^® Green PCR master mix according to the manufacturer's protocols. PCR thermocycling conditions were as follows: Holding stage, 1 cycle of 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min; 1 cycle of 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec). The PCR products were measured with a StepOnePlus Real-Time RT-PCR System. Relative gene expression was calculated based on the 2^−ΔΔCq method (18) using StepOne software version 2.3 (Applied Biosystems, Foster City, CA, USA). β-actin mRNA expression was used as an endogenous control. Primer sets for RT-qPCR are presented in Table I.

RAW 264.7 cells were cultured in chambered cover glasses (Nalge Nunc International; Thermo Fisher Scientific, Inc.) for 24 h and stimulated with LPS in the presence or absence of AS II. The cells were fixed in 4% formaldehyde in PBS for 15 min at room temperature and permeabilized with 100% methanol for 10 min at −20°C. Specimens were blocked with blocking buffer (PBS with 5% serum and 0.3% Triton X-100) for 1 h and incubated overnight with polyclonal antibodies (1:200) at 4°C. Fluorochrome-conjugated secondary antibodies (1:500) were applied for 1 h at room temperature in the dark. Following washing with PBS, the nuclei were counterstained with DAPI and fluorescence was visualized using a fluorescence microscope (Ziess AG, Oberkochen, Germany). AlexaFluor 488-conjugated LPS was used to evaluate the effects of AS II on LPS and TLR-4 binding.

The statistical analysis was performed using one-way analysis of variance followed by Scheffe's test for multiple comparisons. Data are presented as mean ± standard deviation (n=5). All calculations were performed using SPSS statistics version 22 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

MTS assays were performed to determine the effects of AS II on murine macrophage viability (Fig. 1A). The following experiments were accomplished using 40 µM AS II to evaluate its anti-inflammatory effects. As demonstrated in Fig. 1B and C, LPS increased production of NO and PGE₂ compared with the untreated group. However, groups pretreated with AS II had significantly decreased levels of NO and PGE₂ production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner up to 40 µM. The effects of AS II on iNOS and COX-2 protein and mRNA expression were examined. iNOS and COX-2 mRNA and protein expression levels were undetectable in the unstimulated group; however, iNOS and COX-2 expression increased significantly in response to LPS. AS II reduced COX-2 protein and mRNA expression by 37 and 45%, respectively, at the highest concentration compared with LPS-treated cells. AS II (40 µM) decreased iNOS protein and mRNA expression by 50 and 59%, respectively, relative to the LPS=treated group (Fig. 1D and E).

The effects of AS II on synthesis of the pro-inflammatory cytokines IL-6 and TNF-α in LPS-stimulated murine macrophages were investigated. Significantly increased levels of IL-6 and TNF-α following treatment with LPS were reduced by pretreatment with AS II (Fig. 2A). The RT-qPCR data demonstrated that AS II reduced the mRNA of these cytokines. IL-6 and TNF-α mRNA expression levels were decreased by up to 49 and 56.8% respectively, compared with the LPS only treated group (Fig. 2B).

Previous studies have demonstrated that NF-κB is a key transcriptional factor involved in regulating inflammatory mediators, including iNOS, COX-2 and cytokines. As demonstrated in Fig 3, NF-κB nuclear translocation was increased in the LPS-stimulated group compared with the untreated group. NF-κB nuclear translocation decreased significantly when AS II was added with LPS (Fig. 3).

The LPS-activated TLR-4 signaling pathway was analyzed by western blot analysis and immunofluorescence staining. Receptor expression was studied 24 h following LPS treatment. TLR-4 expression in LPS-stimulated murine macrophages increased from undetectable levels compared with untreated macrophages. TLR-4 expression was decreased in the experimental groups treated with LPS and AS II. In addition, high fluorescence intensity was observed outside the cell membrane when cells were stimulated with fluorescent-dye conjugated LPS; however, fluorescence intensity weakened in the presence of AS II (Fig. 4).