Lonimacranthoide VI was isolated from the flowers of Lonicera macranthoides Hand.-Mazz. and the structure is shown in Fig. 1. Lonimacranthoide VI was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as a stock solution stored at −20°C and was subsequently diluted with medium prior to each experiment. The final DMSO concentration did not exceed 0.1% DMSO throughout the study (all the control groups comprised 0.1% DMSO).

RAW264.7 murine macrophages were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium containing 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO₂. The effect of lonimacranthoide VI on cell viability was assessed by the 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product. Briefly, the RAW264.7 cells were seeded at 5×10⁴ cells/well in a 96-well plate and treated with various concentrations of lonimacranthoide VI or vehicles. Each treated or control group contained six parallel wells. After incubation for 24 h at 37°C in a humidified incubator, cell viability was determined. MTT (5 mg/ml in phosphate-buffered saline) was added to each well and incubated for 4 h; subsequently, 100 μl of the solubilization solution (10% sodium dodecyl sulphate in 0.012 M HCl) was added into each well and the plate remained in the incubator overnight. Absorbance was recorded on a microplate reader (Tecan Austria GmbH, Salzburg, Austria) at a wavelength of 570 nm (reference wavelength, 690 nm). The percentage of cell proliferation was calculated as a ratio of the optical density (OD) value of the sample to the OD value of the control. All the experiments were performed under the same conditions at least three times. The cell inhibitory ratio was calculated by the following formula: Inhibitory ratio (%) = (1−average absorbance of treated group/average absorbance of control group) ×100%.

RAW264.7 cells were plated at a density of 2.5×10⁵/ml cells in a 24-well plate with 1 ml of culture medium per well and cultured overnight. The cells were pre-incubated for 2 h with various doses of lonimacranthoide VI and stimulated for 24 h with 100 ng/ml LPS. The cell culture supernatants were collected immediately following treatment and centrifuged at 1,000 × g for 15 min to remove the particulate matter. PGE₂ was determined using an enzyme immunoassay (EIA) kit (catalog no. ADI-900-001, Enzo Life Sciences, Switzerland). The medium and PGE₂ EIA conjugate was added to a 96-well plate pre-coated with goat anti-mouse IgG and left to react for 2 h, followed by a final wash to remove any unbound antibody-enzyme reagent. A substrate solution was added and the intensity of the color produced was measured at 405 nm (correction wavelength set at 570–590 nm).

The ability of lonimacranthoide VI to inhibit ovine COX-1 and COX-2 was determined using an enzyme immunoassay (EIA) kit (catalog no. 560101; Cayman Chemical Co., Ann Arbor, MI, USA). COX catalyzes the first step in the biosynthesis of AA to PGH₂. PGF_2α, produced from PGH₂ by reduction with stannous chloride, was measured by EIA (ACE™ competitive EIA, Cayman Chemical, Ann Arbor, MI, USA). Briefly, to a series of supplied reaction buffer solutions [960 μl 0.1 M Tris-HCl (pH 8.0) containing 5 mM EDTA and 2 mM phenol] with either COX-1 or COX-2 (10 μl) enzyme in the presence of heme (10 μl), 10 μl of various concentrations of test drug solutions (1, 10, or 100 μM in a final volume of 1 μl) were added. These solutions were incubated for 5 min at 37°C and subsequently 10 μl AA solution (100 μM) was added. The COX reaction was stopped by the addition of 50 μl 1 M HCl after 2 min. Then 100 μl of stannus chloride was added to produce PGF_2α, which was measured by EIA. This assay is based on the competition between PGs and a PG-acetylcholinesterase conjugate (PG tracer) for a limited amount of PG antiserum. The amount of PG tracer that is able to bind to the PG antiserum is inversely proportional to the concentration of PGs in the wells since the concentration of the PG tracer is held at a constant while the concentration of PGs varies. The specific antiserum-PG complex bound to a mouse anti-rabbit IgG that had been previously attached to the well. The plate was washed to remove any unbound reagents and 200 μl Ellman’s reagent, which contains the substrate to acetylcholine esterase, was added to the well. The product of this enzymatic reaction generates a distinct yellow color that absorbs at 406 nm. The intensity of this color, determined by spectrophotometry, is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the well during the incubation. Percent inhibition was calculated by the comparison of the compounds treated to the various control incubations.

Total cellular RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and the concentration of RNA was determined at 260 nm. cDNA was synthesized by extension of oligo (dT) primers with 10 units of avian myeloblastosis virus reverse transcriptase in a mixture containing 1 μg total RNA. The cDNA amplification was performed using the PCR kit (Takara Bio, Inc., Shiga, Japan), denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min. The primer sequences used were as follows: COX-2 sense, 5′-GGGAAGCCTTCTCCAACC-3′ and antisense, 5′-GAACCCAGGTCCTCGCTT-3′; and GADPH sense, 5′-AACGACCCCTTCATTGACC-3′ and antisense, 5′-TCAGATGCCTGCTTCACC-3′, which was used as an internal control. The PCR products (10 μl) were separated on 2% agarose gel and visualized by ethidium bromide staining. The gel image was captured and analyzed using Quantity One software (Tanon Science and Technology Co., Shanghai, China).

All the experiments were repeated at least three times. Data are reported as means ± standard deviation. The statistically significant differences of the test compounds compared to the untreated control were calculated using the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the anti-inflammatory effects of lonimacranthoide VI, a murine RAW264.7 macrophages in vitro model was used. RAW264.7 cells were treated with various concentrations of lonimacranthoide VI and cell viability was measured using the MTT assay. As shown in Fig. 2, the resulting survival curve shows that lonimacranthoide VI does not have cytotoxic effects on the proliferation of cells. As lonimacranthoide VI showed no cytotoxicity at concentrations ≤100 μM in RAW264.7 macrophages, lonimacranthoide VI was used at a concentration of 0–100 μM for the remaining experiments.

Since PGE₂ is one of the most important inflammatory mediators, the effects of lonimacranthoide VI on the LPS-induced release of PGE₂ from RAW264.7 cells were observed. The cells were pretreated with lonimacranthoide VI for 2 h followed by incubation with 100 ng/ml LPS. One day after LPS treatment, the PGE₂ contents in the culture medium were detected. The LPS-induced PGE₂ secretion level was inhibited by treatment with the compound at all the doses examined (IC₅₀=0.25 μM) and the maximum inhibition was observed at a dose of 100 μM (Fig. 3). Indomethacin (IM) was used as a positive control.

COX-1 and COX-2 catalyze the biosynthesis of PGH₂ from the AA substrate. The inhibition of COX-1 results in certain undesirable side-effects, whereas COX-2 inhibition provides therapeutic effects in pain, inflammation, cancer, glaucoma, Alzheimer’s and Parkinson disease (7). Therefore, the present study aimed to examine the COX-1 and COX-2 inhibitory activity of lonimacranthoide VI on purified enzymes as a mechanism of topical anti-inflammatory action. The compound showed inhibitory effects on COX-1 and COX-2 (Tables I and II). Furthermore, 10 μM lonimacranthoide VI inhibited COX-2, whereas the dose had no effect on COX-1.

To investigate the effects of lonimacranthoide VI on mRNA expression of COX-1 and COX-2, the RAW264.7 macrophage cells were pre-treated with the compound at various concentrations ranging 1–100 μM and were stimulated with 100 ng/ml LPS for 24 h. The COX-2 non-selective inhibitor, IM, was used as a standard drug for comparing the ability of lonimacranthoide VI in modulating the pro-inflammatory genes, COX-1 and COX-2. As shown in Fig. 4, 100 ng/ml LPS can stimulate COX-2 mRNA expression, whereas it had no significant effects on the COX-1 mRNA expression. In addition, 100 μM lonimacranthoide VI can significantly suppress the mRNA expression of COX-1 and COX-2 in the LPS-stimulated RAW264.7 macrophage cells as compared to LPS-treated cells alone. However, the presence of IM only produced a significant reduction of COX-2 mRNA expression in LPS-treated cells.