Chemicals and reagents
Acetonitrile and methanol of HPLC grade were purchased from Merck (Darmstadt, Germany). The standard products such as salidroside, verbascoside, oleanolic acid and ursolic acid were obtained from National Institutes for Food and Drug Control (Beijing, China). L (-)-2-Octanol (as the internal standard), linalool, linalool oxide, geraniol, α-ionone and β-ionone were bought from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Petroleum ether with the boiling range 80–120 ◦C was purchased from J&K Chemical Ltd. (Beijing, China). Water of HPLC grade was purified by reverse osmosis systems (Millipore, Ireland). All the reagents of HPLC grade should be filtered through a 0.45 µm membrane before experiment. Dimethyl sulfoxide(DMSO), 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), trypan, Concanavalin A (Con A), penicillin G, and strepto-mycin sulfate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Gibico 1640 medium and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corp. (San Diego, CA, USA). Other reagents and chemicals were of analytical grade.
Plant material
Fresh O. fragrans flowers were harvested from three different O. fragrans groups in our campus whose flowers were almost orange, silver and golden, respectively. They were identified separately as O. fragrans var. Aurantiacus, O. fragrans var. Latifolius and O. fragrans var. Thunbergii by Dr. Feng Li from Shandong University of Traditional Chinese Medicine. To narrate bellows conveniently, they were tentatively called as “Aurantiacus”, “Latifolius” and “Thunbergii”.
Drying methods
Shade drying (SHD), sun drying (SUD), quick-lime drying (QLD), oven drying (OD) and microwave drying (MD) were conducted on fresh flowers from three O. fragrans groups, respectively. The processing of SHD and SUD were separately carried out in doors or in the sun. The processing of QLD was carried out by placing the fresh flowers in the upper glass dryer whose lower layer was filled with quick lime. The processing of OD was operated separately at 40, 50, 60 and 70 ◦C in the drying oven (DHG-9070B, Shanghai Pei for Experimental Instrument Co., Shanghai, China). The processing of MD was carried out separately at medium-low fire (MLF), medium fire (MF), medium-high fire (MHF) and high fire (HF), respectively, in the microwave oven (M1-L213B, Midea Group Co., Foshan, China). All the dried O. fragrans flowers above should remain their constant weights before completions of their drying processing and be stored in the glass dryer filled with the desiccant silica gel.
Sample extraction and preparation
The dried flowers from three O. fragrans groups were extracted and prepared to determine 4 kinds of non-volatile components as HPLC samples as follows. 0.1 g of the dried O. fragrans flowers were measured precisely and placed into 50 mL conical flask with its stopper. Then 10 mL precise methanol was dumped into the above flask and extracted for 30 min under 100 w power and 40 kHz frequency at room temperature using an ultrasonic cleaner (KQ-100 DE, Kunshan Ultrasound Instrument Co., Suzhou, China). The lost weight was compensated with methanol after being cooled to room temperature and the extracts was filtrated through the double-layer filter paper. The continuous filtrates above was collected and stored at 4 ◦C for the subsequent content determination of 4 kinds of non-volatile components.
The dried flowers from three O. fragrans groups were extracted and prepared to determine 5 kinds of volatile components as GC samples as follows. 10 g of the dried O. fragrans flowers were measured precisely and put into 500 mL round-bottom flask. Then 300 mL of deionized water were added into the above flask. With the Soxhlet apparatuses utilized, they were extracted for 4 h. 2 mL of L (-)-2-Octanol petroleum ether solutions with the boiling range 80–120 ◦C were added into the distillated extracts. The above liquids were mixed homogeneously and their supernatants were separated and stored at 4 ◦C for the next determination of the volatile components. All experiments were carried out in triplicate.
High Performance Liquid Chromatography (HPLC)
4 kinds of non-volatile components of HPLC samples were analyzed by E2695 LC instrument (Waters Instruments Co., Massachusetts, USA) with an on-line degasser, a quaternary pump, a 2489 ultraviolet-visible detector (UVD) and an anto-sampler. 10 µL of the HPLC samples were subjected to HPLC analysis with a Symmetry C-18 analytical column (250 mm × 4.6 mm, 5 µm, Waters Corporation, USA), using a linear gradient with water- phosphoric acid (100:0.05, v/v) as solvent A and acetonitrile as solvent B. The gradient program was carried out as follows: 10% B in 0–8 min, 10–30% B in 8–22 min, 30–85% B in 23–36 min, 85–10% B in 37–66 min. The velocity of flow was 1.0 mL min− 1 and the column temperature was kept at 30 ◦C. Detection wavelength was performed at 220 nm in 0–25 min and 210 nm in 26–66 min.
Gas chromatography (GC)
5 kinds of volatile components of GC samples were analyzed by GC-4000A GC instrument (East &West Analytical Instruments Co., Beijing, China) with a nitrogen canister, a flame ionization detector (FID), a hydrogen generator, an air pump and a 1µL injection needle. 1µL of the GC samples were injected to GC instrument with an AE.SE-54 nonpolar analytical column (30 m × 0.32 mm, Lanzhou Atech Technologies co., Lanzhou, China) and the high purity nitrogenas as carrier gas, using a temperature gradient program. The gradient program was carried out as follows: 50℃ for 2 min, 50–160 ◦C at the rate of 2 ◦C min− 1, 160–250 ◦C at the rate of 22.5 ◦C min− 1, 250 ◦C for 12 min. The temperature of gasification chamber and flame ionization detector was all set at 250 ◦C. The pressure of nitrogen, air and hydrogen was set at 0.4, 0.2 and 0.09 MPa, respectively.
Assessment of cell viability
The cytotoxicity of relevant chemical components of O. fragrans flowers with various concentrations was evaluated by MTT test with some modification [42]. 1 × 105 of RAW 264.7 cells were seeded in triplicates in 96-well plates and cultured 3 h to allow cell attachment, then cultured with salidroside, verbascoside, linalool oxide, linalool, geraniol, α-ionone and β-ionone at 3.125, 6.25, 12.5, 25, 50, 100 µg mL− 1 for 24 h. Subsequently, 20 µL of MTT solution (5 mg mL− 1) was added to each well, and the cells were incubated for 4 h at 37 ◦C. The supernatants were discarded and 150 µL of DMSO were added to each well and homogenized. Absorbance values were estimated at 570 nm by Epoch2 microplate spectrophotometer (Bio Tek Instruments Co., USA). Cell viability was calculated by the following equation.
Where A1 is the absorbance value of blank control group, A2 is the absorbance value of treatment group.
Phagocytosis assay
The phagocytic activities of RAW 264.7 cells treated with various concentrations of relevant chemical components of O. fragrans flowers were examined by neutral red uptake assay [43]. RAW 264.7 cells were seeded at 2.7 × 106 cells per well in 96-well microplates. After incubation for 3 h, the supernatant was discarded. RAW 264.7 cells were pretreated with salidroside, verbascoside, linalool oxide, linalool and geraniol at different concentration (3.125, 6.25, 12.5, 25, 50, 100 µg/mL) in the presence or absence of LPS (1.0 µg mL− 1) for 24 h. Then, 100 µL of neutral red solutions (0.1%, w/v) were added into each well and incubated for another 30 min at room temperature. The supernatants were discarded and the cells were washed with phosphate buffered saline (PBS, 0.01 M, pH 7.4) thrice, the cells were in lysis for 30 min at room temperature by adding 150 µL of cell lysis buffer (Vethanol : Vacetic acid=1:1) and homogenized. The absorbance values were measured at a wavelength of 540 nm using Epoch2 microplate spectrophotometer.
Where A1 is the absorbance value of blank control group, A2 is the absorbance value of treatment group.
Statistical analysis
All data were reported as means ± standard deviation of three samples. Statistical analysis was performed with SPSS 19.0. Significance differences were tested by using the ANOVA procedure, using a significant level of p༜0.05.