Chemicals and reagents
Dendrobium officinale samples grown in the Huoshan area were provided by Tian-Xia-Ze-Yu Biological Technology Development Co., Ltd. (Huoshan County, Anhui, China). These samples were verified by Prof. Zhen Ouyang prior to being cut into small pieces no more than 3 mm in size.
Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). Standard monosaccharides (d-glucose, d-xylose, d-galactose, l-rhamnose, d-mannose, and d-arabinose), DEAE-52 cellulose, Sephadex G-100, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-fluorouracil (5-FU), propidium iodide (PI), trypsin, JC-1, and the Annexin V-FITC/PI apoptosis detection kit were obtained from Sigma Aldrich (St. Louis, MO, USA). The Bax, Bcl-2, and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The DCFH-DA reactive oxygen species (ROS) assay and LDH cytotoxicity assay kits were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). HepG-2 cells were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All other chemical reagents were of analytical grade. The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study.
Extraction and isolation
The components of D. officinale (20 g) were extracted three times with distilled water (400 mL × 3) for 2 h at 90 °C. The filtrate was combined and concentrated to 20 mL using a rotary evaporator at 60 °C. The concentrated supernatant was then evaporated under reduced pressure and precipitated by adding 95% ethanol until the total ethanol concentration reached 85%. The samples were subsequently stored at 4 °C overnight. The precipitate was collected following centrifugation (4000 rpm, 15 min), dissolved in distilled water, and lyophilized to obtain the crude polysaccharides. The crude polysaccharides were then deproteinized using the enzyme-Sevag method [16]. After the crude polysaccharides were dissolved in distilled water and fractionated on DEAE-52 cellulose, they were successively eluted with distilled water and an NaCl gradient (0.1–0.3 M) at 1.0 mL/min. The carbohydrate content of each fraction (10 mL/each) was monitored at 490 nm using the phenol–sulfuric acid method [17]. The obtained eluates were then combined, concentrated, dialyzed, lyophilized, and subjected to further purification on a Sephadex G-100 column. The fractions were subsequently combined and lyophilized to obtain a purified polysaccharide sample. The major fraction with only one main peak was lyophilized and named DOPA-1.
Homogeneity, molecular weight, and monosaccharide composition analysis
The homogeneity and molecular weight of DOPA-1 was determined with high performance liquid chromatography (HPLC) using a TSK-GEL G-4000 PWXL column (7.8 × 300 mm, column temperature 30 °C) and a Waters alliance refractive index detector (RID, detection temperature 30 °C). Prior to injection, the sample was filtered through a 0.45 μm membrane. Then, 10 μL of DOPA-1 (1.0 mg/mL) was injected into the HPLC column and eluted with distilled water at a flow rate of 0.45 mL/min. Dextrans of varying molecular weight (10, 40, 70, 500, and 2000 kDa) were used to establish a standard curve.
The monosaccharide composition of the purified DOPA-1 was measured by gas chromatography (GC). Briefly, 10 mg of the polysaccharides were hydrolyzed in a sealed glass tube with 5 mL of 2 M sulfuric acid at 100 °C for 8 h. The acid was then neutralized with barium carbonate. Next, the hydrolysate was acetylated with 10 mg of hydroxyl-amine hydrochloride in 1 mL of pyridine for 30 min at 90 °C. After cooling to 20–25 °C, 1 mL of acetic anhydride was added and further incubated for 30 min at 90 °C. Following cooling, the corresponding aldononitrile acetate derivatives were obtained and the corresponding monosaccharides (rhamnose, arabinose, xylose, mannose, glucose, and galactose) were then analyzed.
Infrared spectrum analysis
DOPA-1 (1.0 mg) was ground with KBr (100 mg) and pressed into a pellet. The Fourier transform infrared (FT-IR) spectra were recorded with an FT-IR spectrometer (Nicolet Nexus 470 FT-IR, USA) at a frequency range of 4000–500 cm−1.
Nuclear magnetic resonance (NMR) spectroscopy
DOPA-1 (30 mg) was dissolved in 99.9% deuterium oxide (0.5 mL). The sample was then freeze-dried three times. The 1H NMR spectra was recorded with a Bruker DRX-400 NMR spectrometer (Bruker, Rheinstetten, Germany) at 25 °C. Data processing was performed using standard Bruker XWIN-NMR software.
Periodate oxidation and Smith degradation
DOPA-1 (25 mg) was oxidized in 0.030 M NaIO4 (25 mL) in the dark for 120 h and the absorption was monitored every 12 h with a UV spectrophotometer at 223 nm until a constant absorption value was achieved. NaIO4 consumption was quantitatively measured using the UV spectrophotometric method [18] and the yield of formic acid was titrated with 0.01 M NaOH. Ethylene glycol (2 mL) was added to remove excess periodate. The periodate product was dialyzed and reduced with NaBH4 (40 mg) for 24 h at room temperature. The product was then neutralized to pH 6.0–7.0 with acetic acid. Following dialysis and lyophilization, the product was finally hydrolyzed with 2 M trifluoroacetic acid (3 mL) at 110 °C for 6 h and the hydrolysate was subjected to GC analysis.
Cell culture and treatment
HepG-2 cells were cultured in DMEM medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C with 5% CO2. The medium was refreshed 2–3 times/week.
MTT cytotoxicity assay
A colorimetric MTT assay was performed to assess cell viability as previously described [19]. Briefly, HepG-2 cells were seeded in a 96-well plate at a concentration of 2.0 × 104 cells/well in 100 μL of medium. Following incubation at 37 °C for 24 h to allow adherence, the cells were treated with different concentrations (50, 100, 200, and 400 μg/mL) of DOPA-1 for 24 h. 5-FU (50 μg/mL) was used as a positive control, while untreated cells were used as a negative control. Following treatment, the medium in each well was removed and 100 μL of MTT (1 mg/mL) was added and incubated at 37 °C for 4 h. Next, the supernatant was aspirated and the crystal violet generated by viable cells was dissolved with 100 μL of DMSO. The absorbance was measured at 570 nm using a microplate reader.
LDH assay
Cell injury was quantitatively assessed by the measurement of lactate dehydrogenase (LDH). Cells were seeded in 96-well plates. After treatments, a LDH cytotoxicity assay kit (Beyotime, Jiangsu, China) was used to measure the released LDH. Briefly, 100 μl of culture medium was collected from each well. The absorbance of the medium was measured at 490 nm wavelength in an automatic microplate reader. Blank absorbance was subtracted from insult LDH values.
Determination of ROS levels
A DCFH-DA probe is the most common and sensitive tool for detecting intracellular ROS levels [20]. Logarithmic growth phase HepG-2 cells were digested, counted, and seeded in a 96-well culture plate at a density of 4 × 104 cells/well in 100 μL of medium. After the cells were treated with DOPA-1 for 24 h, the cells were washed twice with phosphate buffered saline (PBS) and incubated with 10 μM of DCFH-DA diluted in serum-free medium at 37 °C for 30 min. The fluorescence intensity was detected with a fluorescence microplate reader at an excitation wavelength of 488 nm and emission wavelength of 525 nm.
Determination of mitochondrial membrane potential (MMP)
JC-1 is a well-known fluorescent dye used to detect MMP. Following cell treatment, the culture medium was removed and the JC-1 dye (2.5 μg/mL) was added. After incubation at 37 °C for 10 min in the dark, the cells were washed twice with PBS. The fluorescence intensity was detected with a fluorescence microplate reader at an excitation wavelength of 488 nm and emission wavelength of 525/590 nm. The changes in MMP were then observed by fluorescence microscopy.
Flow cytometry
The rate of apoptosis was determined by cellular staining with Annexin V-FITC and PI [21]. Briefly, the treated cells (4 × 105 cells/mL) were washed twice with ice cold PBS and resuspended in 100 μL of 1× binding buffer. Next, 5 µL of Annexin V-FITC and 5 µL of PI were added and incubated for 10 min in the dark. The cells were then analyzed with a flow cytometer (Guava easyCyte, Merck Millipore, USA).
Western blotting analysis
Following treatment with DOPA-1 (50, 100, 200, and 400 μg/mL) for 24 h, HepG-2 cells were washed twice with ice-cold PBS and centrifuged at 12,000g for 10 min at 4 °C. After adding lysis buffer, the cells were incubated on ice for 20 min. The protein concentration of the supernatant was determined with a BCA protein assay kit. Proteins were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and then transferred onto a 0.45-μm polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was blocked in blocking buffer (0.3 g bovine serum albumin [BSA], 20 mL PBS + Tween 20 [PBST], and 5% non-fat milk) for 2 h at room temperature, followed by incubation with the Bcl-2 (1:1000), Bax (1:1000), or β-actin (1:2000) primary antibodies overnight at 4 °C. The membranes were then washed three times with PBST (15 min each) and incubated with the appropriate secondary antibody (1:2000) for 2 h. Finally, the proteins were visualized with the ECL detection reagent and analyzed using a Chemi Doc XRS imaging system (Bio-Rad, CA, USA).
Statistical analysis
All experiments were performed in triplicate and the data were expressed as the mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 5. Analysis of variance (ANOVA) was used to compare the groups; p < 0.05 was considered a statistically significant difference.
