Plant materials
Fresh leaves of Eucalyptus urophylla, Eucalyptus grandis and Eucalyptus camaldulensis were collected in August 2017 from the Kukeng Experimental Plantation of the Taiwan Forestry Research Institute (TFRI) in south-central Taiwan (Yunlin County, elevation 100 m, N 23° 62′ 58˝, 120° 57′ 20˝), while fresh leaves of Eucalyptus citriodora were collected in August 2019 from the Lienhwachih Research Center, TFRI in central Taiwan (Nantou County, elevation 500 m, N 23° 91′ 75˝, 120° 88′ 52˝). The samples were compared with specimen No. E-0032, E-0033, E-0034 and E-0035 from the herbarium of National Chung Hsing University (NCHU) and were positively identified by Prof. Yen-Hsueh Tseng of NCHU. The voucher specimen (CLH-068, CLH-069, CLH-070 and CLH-071) were deposited in the NCHU herbarium. The collected leaves were immediately shipped to our Taipei headquarters, and the essential oils were extracted for subsequent analyses.
Extraction of the leaf essential oils
A sample of 1 kg of fresh leaves from each species was placed in a round-bottomed flask, and 3 L of distilled water was added. After 8 h of steam distillation, the oil layers had separated from the water layers and were collected, and anhydrous sodium sulfate was added to eliminate the water. We repeated the isolation procedure for 4 times and combined the leaf essential oil for further fractionation. Yields of the essential oils were determined and the oils were stored in specimen bottles. Yields of leaf essential oils of E. urophylla, E. grandis, E. camaldulensis and E. citriodora were 3.18 ± 0.05, 2.98 ± 0.06, 3.58 ± 0.07, and 1.98 ± 0.08 ml/100 g, respectively.
Fractionation of the leaf essential oil of E. citriodora
Fifty grams of E. citriodora leaf oil was mixed with 100 g silica gel (Merck 7734, Merck Co., Germany), separated on a silica gel open column (1000 g, 85 mm i.d., 850 mm length), and then eluted with a step gradient of n-hexane and ethyl acetate that ranged from n-hexane/ethyl acetate = 100:0–0:100. The fractions were collected by thin layer chromatography (TLC) and visualized at UV 254 nm and UV 366 nm, with iodine vapor and 1% vanillin/H2SO4. The band fractions from the leaf oil that contained the same compounds were combined to produce 8 sub-fractions: fractions A (5.3%), B (7.6%), C (11.5%), D (9.8%), E (10.2%), F (35.2%), G (18.1%), and H (2.3%).
Gas chromatography-mass spectrometry (GC-MS) analysis of fractions F and G
A Hewlett-Packard HP 6890 gas chromatograph equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness, J&W Scientific) and a FID detector were used for the quantitative determination of the chemical compositions of fractions F and G. Samples were incubated in an oven at 50 °C for 2 min, and then the temperature incrementally increased to 250 °C at 5 °C/min. The injection temperature was 270 °C. The carrier gas was helium and had a 1 ml/min flow rate. The detector temperature was 250 °C, with a split ratio of 1:10. The injection volume was 1 μl. Identification of the oil components was based on their retention indices and mass spectra obtained from GC-MS analysis on a Hewlett-Packard HP 6890/HP5973 equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness, J&W Scientific). The MS reuslts were obtained in full scan mode with a 0.3 s scan time and a mass range of m/z 30–500 in the EI mode at 70 eV. All data are presented as the average of experiments performed in triplicate. Identification of the chemical compositions of fractions F and G was based on comparisons of Kovats index (KI), retention times (RT), and mass spectra with those obtained from authentic standards and/or the NIST and Wiley libraries spectra, and literature, respectively. The F and G fractions were identified using their Kováts retention indices calculated for all volatile constituents using a homologous series of n-alkanes C9-C23 on DB-5MS column, and comparing sample mass spectra with those obtained from authentic standards and/or the NIST and Wiley libraries spectra, and the literature [15].
Materials
Antibodies against PKC-α, PKC-ε and PKC-δ, COX2, p-IKK-α and anti-p-IκB-α were procured from Santa Cruz Biotechnology (Dallas, TX). Antibodies against p-ERK1/2, p-JNK1/2 and p-p38 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against iNOS and β-actin, lysis buffer and polyvinylidene fluoride (PVDF) membrane were purchased from EMD Millipore (Bedford, MA). LPS (Escherichia coli O111:B4) and protease/phosphatase inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits and enhanced chemiluminescence (ECL) reagents were purchased from Thermo Fisher Scientific (Waltham, MA). RPMI 1640 was purchased from Gibco Laboratories (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Ha-emek, Israel). L-glutamine was purchased from Life Technologies (Carlsbad, CA, USA). Alamar Blue assay kit was purchased from AbD Serotec (Oxford, UK). QUANTI-Blue medium was purchased from InvivoGen (San Diego, CA).
Cell cultures
RAW264.7 macrophage were purchased from the American Type Culture Collection (Rockville, MD). RAW-Blue macrophages were purchased from InvivoGen (San Diego, CA). All cells were propagated in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS and 2 mM L-glutamine at 37 °C in a 5% CO2 incubator [16, 17].
Cell viability assay
Cell death was quantified by the MTT reduction assay.
Cell viability was quantified by the Alamar Blue assay. Breifly, RAW264.7 macrophages were incubated with or without tested sample for 24 h. Alamar Blue reagent (10 μl) was added into the culture medium and incubated at 37 °C in a 5% CO2 incubator for 2–3 h. Fluorescence was detected with a fluorescence microplate reader (excitation/emission: 570 nm/600 nm; OPTImax tunable plate reader; Molecular Devices, Sunnyvale, CA, USA).
NO production assay
RAW264.7 macrophages were incubated for 0.5 h with tested samples, followed by incubated with or without 1 μg/ml LPS for 24 h. NO levels in the culture medium were measured by the Griess reaction as described previously [17].
Western blot analysis
Western blot was performed as described previously [16, 17]. Briefly, protein was extracted with a protease and phosphatase inhibitor cocktail containing lysis buffer. The lysates were electrophoretically fractionated through 10% SDS-polyacrylamide gel and then electrotransferred onto PVDF membranes. The membranes were incubated for 1 h at room temperature in 5% (v/v) nonfat milk in phosphate-buffered saline (PBS) with 0.1% (v/v) Tween-20). The primary antibodies were applied to the blots and incubated for 2 h at room temperature, followed by secondary antibody for 1 h at room temperature. The immunoreactive signals were detected with ECL reagents.
NF-κB reporter assay
The NF-κB reporter assay was conducted as described previously [16, 17]. Briefly, RAW-Blue cells (105 cells per 0.5 ml medium) were treated for 0.5 h with 12.5 μg/ml, 25 μg/ml, 50 μg/ml or 100 μg/ml Fraction F, followed by incubated with or without 1 μg/ml LPS for 24 h. The harvested medium (20 μl) was mixed with 200 μl QUANTI-Blue medium in 96-well plates and incubated at 37 °C for 2 h. SEAP activity was measured at 655 nm in an ELISA microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
ELISA assay
RAW264.7 macrophages (105 cells per 0.5 ml medium) were treated for 0.5 h with tested sample, followed by incubation with or without 1 μg/ml LPS for 24 h. The levels of TNF-α and IL-6 in the culture medium were measured by ELISA according to the manufacturer’s protocol. Briefly, the 96-well microplates were coated overnight with anti-TNF-α or anti-IL-6 antibody, blocked with 1% (v/v) bovine serum albumin (BSA) in PBS, and washed repeatedly. Samples or standards (100 μl) were added to the microplates, incubated at room temperature for 2 h, washed repeatedly, incubated for 2 h with biotin-conjugated detection antibody, and incubated for 30 min with 100 μl streptavidin-horseradish peroxidase (HRP) plus substrate for signal development. Then 100 μl stop solution was added to each well and the OD450 were measured in an ELISA microplate reader (Bio-Tek Instruments, Winooski, VT, USA) [16, 17].
Statistical analysis
Data were expressed as means ± standard deviation (SD) of triplicate samples. Each experiment was repeated at least three times to confirm reproducibility. Multiple groups were analyzed with one-way analysis of variance (ANOVA), followed by a post hoc Student-Newman-Keuls test. Differences among treatment means were considered statistically significant when P < 0.05.
