Ginseng and its extracts
Four-year-old NA ginseng roots collected in 2007 from five different farms in Ontario, Canada were provided by the Ontario Ginseng Growers Association. Ginseng extracts from each farm were prepared individually and combined to produce composite extracts which were used for phytochemical and pharmacological studies.
RAW 264.7 (ATCC TIB 67) murine macrophage cell lines were provided by Dr Jeff Dixon (Department of Physiology and Pharmacology, University of Western Ontario, Canada). Sephadex G75 was purchased from GE healthcare bio-sciences AB (Sweden). Cell culture medium and reagents were purchased from Gibco laboratories (USA). BD OptEIA ELISA kits tumour necrosis factor-α and interleukin-6 (BD Biosciences, USA). LPS from Escherichia coli and Griess reagent were purchased from Sigma-Aldrich (USA).
Preparation of the AQ, ALC and crude PS ginseng extracts
Dried ginseng root samples were shipped to Naturex (USA) for extraction. Samples were ground between ¼ and ½ inch and used to produce the AQ or ALC extract. Briefly, 4 kg ground ginseng roots were soaked three times during five hours in 16 L of water or ethanol/water (75/25, v/v) solution at 40°C. After extraction, the solution was filtered at room temperature. The excess solvent was then removed by a rotary evaporator under vacuum at 45°C. The three pools were combined and concentrated again until the total solids on a dry basis were around 60%. These concentrates were lyophilized with a freeze dryer (Labconco, USA) at -50°C under reduced pressure to produce AQ or ALC ginseng extract in powder form. Yield of the powder extracts from the concentrates was about 66%. The yields of the final extract (mean ± standard deviation of % extractive) from the initial ground root were 41.74 ± 4.92 and 35.30 ± 5.01 for the AQ and ALC extracts respectively.
A solution of AQ extract in distilled water (10 g/10 mL) was prepared, and the crude PS was precipitated by the addition of four volumes of 95% ethanol. The PS fraction was collected by centrifugation at 350 × g (Beckman Model TJ-6, USA) for 10 minutes and lyophilized to produce the crude PS extract.
Chromatography of ginseng extracts
High performance liquid chromatography (HPLC) analysis for ginsenoside determination
HPLC analysis on the composition of ginsenosides in AQ and ALC extracts (100 mg/ml methanol) was performed with a Waters 1525 HPLC System with a binary pump and UV detector. A reversed-phase Inspire C18 column (100 mm × 4.6 mm, i.d. 5 μm) purchased from Dikma Technologies (USA) was used for all chromatographic separations. Gradient elution consisted of [A] water and [B] acetonitrile at a flow of 1.3 mL/min as follows: 0 min, 80-20%; 0-60 min, 58-42%; 60-70 min, 10-90%; 70-80 min, 80-20%. Absorbance of the eluates was monitored at 203 nm.
Sephadex G-75 chromatography
Five hundred milligrams (500 mg) of AQ or ALC ginseng extract was dissolved in 5 mL distilled water and then fractionated by loading to a calibrated Sephadex G-75 column (47 × 2.5 cm) equilibrated and eluted with distilled water mobile phase at 4°C with a flow rate of 1 mL/min. Absorbance of the eluates was monitored at 230 nm. Fractions (5 mL) were collected and four major fractions (I-IV) were collected and lyophilized to produce four sub-fractions (I-IV) for the study of bioactivity distribution.
Size exclusion chromatography for PS analysis
Size exclusion chromatography of AQ, ALC and PS ginseng extract was carried out at 40°C with an AquaGel PAA-200 Series column (8 × 300 mm, PolyAnalytik, USA) connected to a Viscotek (Varian Instruments, USA) gel permeation chromatography system with Omnisec software (version 4.5, Viscotek, USA) for data acquisition. Solutions of AQ, ALC and PS extract (5 mg/mL) were filtered with 0.2 μm nylon filter and used for analysis. Each sample (100 μl) was injected and eluted with 0.05 M sodium nitrate (NaNO3) mobile phase at a flow rate of 1 mL/min and monitored using a multiple detectors system for light scattering, refractive index and viscosity. Pullulan polysaccharide reference standard was analyzed as a positive control.
Mouse macrophage cell line RAW 264.7 was cultured in Dulbeccos Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum, 25 mM HEPES, 2 mM Glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were seeded in 96-well tissue culture plates at a density of 1.5 × 105 cells per well and maintained at 37°C in a humidified incubator with 5% CO2 and weekly passage and used for experiments at 60-80% confluency.
Experiment to evaluate dose-related stimulation of inflammatory mediators profile in vitro was carried out by treating and incubating macrophages (1.5 × 105 cells/well) with 0, 20, 50 and 200 μg/ml of ginseng extracts or 1 μg/mL of LPS (positive control) for 24 hours. The end-points were the 24 hours-production of NO, TNF-α and IL-6 inflammatory mediators.
Immuno-suppression of LPS-induced effect
To examine the direct inhibitory effect of ginseng extracts on LPS-stimulated immune function, we pre-treated the macrophages with 0, 10, 50, 100 or 200 μg/ml of ginseng extracts two hours prior to the addition of 1 μg/mL of LPS. The 24-hour cytokine production induced by LPS was determined by measuring NO, TNF-α and IL-6 levels in the culture medium.
Suppression of AQ extract-induced macrophage NO stimulation by ALC extract
Production of NO by 1.5 × 105 macrophages/well in a 96 well-plate induced by 0, 50 and 200 μg/ml of AQ ginseng extract was determined 24 hours after the presence and absence of 200 μg/ml ALC ginseng extract.
Quantification of NO, TNF-α and IL-6
TNF-α and IL-6 concentrations in supernatants from cultured cells were analyzed with ELISA. Samples were evaluated with mouse cytokine-specific BD OptEIA ELISA kits (BD Biosciences, USA) according to the manufacturer's protocol. NO production was analyzed as accumulation of nitrite in the culture medium. Nitrite in culture supernatants was determined with Griess reagent (Sigma-Aldrich, USA). Briefly, 50 μL of culture supernatant from each sample were transferred to wells of a 96-well U-bottom microtiter plate, 50 μL Griess reagent (containing 0.5% sulfanilic acid, 0,002% N-1-naphtyl-ethylenediamine dihydrochloride and 14% glacial acetic acid) was then added. The absorbance at 550 nm wavelength was measured using Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Finland) with SkanIt software (version 2.4.2, Thermo Fisher Scientific, Finland). Sample nitrite concentrations were estimated from a sodium nitrite standard calibration curve.
Each cell culture experiment was performed at least three separate times. All statistical analyses were performed with GraphPad prism 4.0a Software (GraphPad Software Inc., USA). Data were presented as the mean ± standard deviation (SD) of triplicates from three independent experiments. Data sets with multiple comparisons were evaluated by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test. P < 0.001 was considered to be statistically significant.