The dried plant materials were obtained from the Beijing Tong Ren Tang Chinese Herbal Medicine shop (Sydney, Australia). A voucher specimen of each sample has been deposited in the Herbal Analysis Laboratory, University of Western Sydney. The scientific names and family names of the herbs are shown in Table 1. The samples were ground to a fine powder in a grinder before extraction.
Chemicals and reagents
Gallic acid, quercetin, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), sodium carbonate, aluminium chloride, sodium nitrate, sodium hydroxide, H2O2, Folin-Ciocalteu (F-C) reagent, ascorbic acid, 95% ethanol, bovine serum albumin (BSA), lipopolysaccharide (LPS: E. coli serotype 0127:B8), EDTA, N-(1-1-napthyl) ethylenediamine dihydrochloride, penicillin G sodium benzyl, resazurin sodium 10%, streptomycin, sulfanilamide, tetramethyl benzidine (TMB) and trypan blue were purchased from Sigma (Australia) and Lomb Scientific Pty Ltd. (Australia). Antibiotics, Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) and glutamine were purchased from GIBCO. Interferon-γ (murine) and tumour necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Bioscience (San Jose, CA, USA).
Preparation of water extracts
Sample powder (3 g) was autoclaved with 30 mL deionised water at 121°C for 1 h. The extracted samples were centrifuged at 10,447 × g (Sorvall Ultra pro 80, Kendro Instruments Australia Pty, Ltd) for 20 min and the supernatants were transferred to a 50-mL volumetric flask. The residues were rinsed two more times, and the pooled extract was adjusted to 50 mL with deionised water. The samples were stored at −20°C until analysis of phenolic and flavonoid contents.
Preparation of ethanol extracts
Sample powder (3 g) was extracted with 30 mL of 95% ethanol in a water bath at 70°C for 6 h. The extracted samples were centrifuged and the supernatants were transferred to a 50-mL volumetric flask. The residues were rinsed two more times and the pooled extracts were adjusted to 50 mL with 95% ethanol. The samples were stored at −4°C until analysis of phenolic and flavonoid content. All water and ethanol extracts were filtered through a 0.45-μm nylon filter before analysis.
Determination of total phenolic and flavonoid content
The total phenolic content was determined according to the Folin-Ciocalteu (F-C) colorimetric method . Briefly, 50 μL of sample and 50 μL of F-C reagent were pipetted into an Eppendorf tube. The contents were vortexed for 10 s and then left to stand at room temperature for 2 min before the reaction was stopped by adding 500 μL of 5% (w/v) sodium carbonate solution and 400 μL of distilled water, and the volume was adjusted to 1 mL. The mixture was then vortexed and incubated at 45°C for 30 min before cooling rapidly with ice. The absorbance of the solution was measured at 760 nm. Gallic acid concentrations ranging from 25 to 300 μg/mL were prepared and a calibration curve was obtained using a linear fit . The samples were analysed in duplicate.
The total flavonoid content was determined according to the aluminium chloride method . Briefly, 0.5 mL of sample and 300 μL of NaNO2 (1:20 w/v) were pipetted into a test tube and the contents were vortexed for 10 s and left to stand at room temperature for 5 min. After standing, 300 μL of AlCl3 (1:10 w/v), 2 mL of NaOH (1 M) and 1.9 mL of distilled water were added to the reaction mixture, which was then vortexed for 10 s, and the absorbance was measured at 510 nm. Quercetin concentrations ranging from 0 to 1200 μg/mL were prepared and a standard calibration curve was obtained using a linear fit. The samples were analysed in duplicate.
Free radical DPPH scavenging assay
The DPPH assay was carried out according to procedures of Brand-William et al.
], with minor modifications. Different volumes (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μL) of ethanol and water extracts were mixed with DPPH radical in methanol (2.2 mg/L, 200 μL) in a 96-well microplate. The final volume of each well was made up to 300 μL by adding the appropriate amount of methanol. The mixture was shaken gently on a microplate reader (Bio-Rad, Hercules, CA ,USA) and the absorbance at 515 nm was measured every 2 min for 30 min or until the absorbance reached its maximum value. The DPPH concentration in the reaction medium was calculated from a calibration curve derived from serial dilution of the DPPH standard. The control (containing all reagents except the test compound) and standards were subject to the same procedure. The free radical scavenging activity was expressed as the percentage inhibition of free radical generation by the sample, and calculated using the following formula:
where Acontrol is the absorbance of the control, and Asample is the absorbance of the sample at 515 nm. The samples were analysed in triplicate.
Assay for scavenging activity using Saccharomyces cerevisiae
The antioxidant activities of the ethanol and water extracts were also measured according to a S. cerevisiae-based high throughput assay . S. cerevisiae BY4743 was cultured overnight in a 50-mL volume by inoculation of a single colony. The culture was then diluted to an optical density at 600 nm (OD600) of 0.2 in media, and 180 μL of the culture broth was added to each well in a 96-well microtitre plate to which 10 μL per well of each herbal extract had been added in duplicate. H2O2 (10 μL, 32% v/v) was added to give a final concentration of 4 mM. The initial OD600 reading was taken using a microplate reader (Multiskan EX, Thermo Electron, USA), and the plates were then incubated in an incubator at 30°C with shaking at 750 rpm. Yeast growth was monitored by reading the OD600 until 20 h. Ascorbate was used as the positive control. The samples were analysed in duplicate.
Ferrous ion-chelating effect
The ferrous ion-chelating effects of the extracts were estimated according to the method of Chua et al.
]. Briefly, 740 μL of methanol and 200 μL of sample were incubated with 20 μL of FeCl2
solution (2 mM). The reaction was initiated by adding 40 μL of ferrozine (5 mM) into the mixture, which was then allowed to stand at ambient temperature for 10 min. The absorbance of the reaction mixture was measured at 562 nm. Distilled water instead of ferrozine solution was used as a blank, to correct for any unequal colour of the sample solution. EDTA-Na2
was used as a reference standard. The ferrous ion-chelating ability was calculated as follows:
where Acontrol is the absorbance of the control, Asample is the absorbance of the sample or standard, and Ablank is the absorbance of the blank.
Ferric-reducing antioxidant power assay
The FRAP of the extracts was tested according to Oyaizu . Different concentrations of the samples (1 mL) and the reference chlorogenic acid were added to 2.5 mL of phosphate buffer (0.1 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1.0%, w/v). Each mixture was incubated at 50°C for 20 min and 2.5 mL of trichloroacetic acid (10%) was added. The mixture was shaken vigorously, and the solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%, w/v). After a 30 min incubation, absorbances were read at 700 nm. Each analysis was carried out in duplicate. Increased absorbance of the reaction mixture indicates stronger reducing power.
Maintenance, preparation and activation of J774A.1 macrophages
J774A.1 macrophages were incubated in 50-mL flasks in DMEM containing 10% FBS supplemented with antibiotics (1%) and glutamine (1%). The cells were maintained in 5% CO2 at 37°C, with the media being replaced every 3 days. Once the cells had grown to confluence in the culture flask, they were removed using a rubber policeman. The cell suspension was concentrated by centrifugation for 5 min at 180 × g, the supernatant was removed and the pellet was resuspended in a small volume of fresh DMEM (with 1% antibiotics and 10% FBS). The cell density was estimated using a Neubauer counting chamber (Neubauer Precicdor HBG, Germany). Cell concentration was adjusted using DMEM (with 1% antibiotics and 10% FBS) to obtain 75,000 cells/well, after which 180 μL of the cell suspension was dispensed into the 60 inner wells of a 96-well plate. Sterile distilled water was added to the outer row of wells. For assays of the extracts, 20 μL of the extract solution was added with or without activator (1 μg/mL LPS and 10 U/mL IFN-γ). In most cases, the maximum dose of extract used was 1 mg/mL with a minimum of five doses made by serial dilution. The cells were incubated for 24 h at 37°C and 5% CO2. Cells with media alone were used as negative controls and activated cells were used as positive controls.
NO level was determined with the Griess reagent , which quantifies nitrite, one of the stable reaction products. Briefly, the supernatant was mixed with an equal volume of Griess reagent (0.1% w/v naphthyl ethylenediamine and 1% w/v sulfanilamide in 5% v/v phosphoric acid) in the wells of a 96-well flat-bottomed microtitre plate. After standing for 5 min at room temperature, the absorbance was measured at 550 nm using a Bio-Rad microplate reader. The remaining supernatant was removed from each well for the TNF-α assay by ELISA.
Cell viability was assessed using a modified MTT assay . Briefly, cells (7×105 cells/well) were seeded in a 96-well plate and treated with 20 μl of sample extract. After incubation for 24 h, the supernatant was replaced with 200 μL of media with MTT solution (500 μg/mL). After further 4 h incubation at 37°C, 200 μL of DMSO was added to each well to solubilise the deposited formazan. The optical density of each well was measured at 490 nm by a microplate reader.
TNF-α determination by ELISA
A sandwich ELISA method was used as described previously  to determine the TNF-α concentration. The capture antibody was used with 0.1 M sodium carbonate buffer (pH 9.5). Serial dilutions of TNF-α standard from 0 to 1000 pg/mL in diluent (10% BSA in PBS) were used as the internal standard. TNF-α was detected using a biotinylated secondary antibody and an avidin peroxidase conjugate with TMB as the detection reagent. After 30 min, the reaction was stopped by 2 M sulfuric acid and the absorbance was measured at 450 nm.
Data presentation and analysis
The results were expressed as mean ± standard deviation (SD), calculated from duplicate determinations and the linear relationship was visually determined. The significance of differences among groups of data were determined with one-way analysis of variance (ANOVA) by using statistical software SPSS 18.0 for Windows (IBM, USA) and Duncan’s multiple range test . The threshold for statistical significance was P < 0.05.