Materials
Glutathione (GSH), 1, 1-diphenyl-2-picrylhydrazyl radicals (DPPH), 2, 2′-azinobis-(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS), genistein, genistin, daizein, daizin, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteu’s phenol reagent was purchased from Merck Co. (Santa Ana, CA, USA). Anti-iNOS, anti-COX-2, and anti-β-actin antibody (Santa Cruz, USA) and a protein assay kit (Bio-Rad Laboratories Ltd., Watford, Herts, UK) were obtained as indicated. Poly-(vinylidene fluoride) membrane (Immobilon-P) was obtained from Millipore Corp. (Bedford, MA, USA). Plant materials were collected from Taichung, Nantou, and Hsinchu counties in Taiwan. They were identified and authenticated by Dr. Shyh-Shyun Huang, Department of Pharmacy, China Medical University, Taichung, Taiwan.
Preparing aqueous extracts of plant materials
Dried herb roots (100 g for each species) were boiled with 1 L distilled water for 1 h. Filtrate and collection of the extracts were done three times. The filtrate was concentrated to a powder by freeze dryer (Christ Alpha, Germany) and stored at −20 °C.
Chemical compositional analysis of flavonoid-related compounds and the three Glycine species by HPLC
HPLC was performed with a Hitachi Liquid Chromatograph (Hitachi Ltd., Tokyo, Japan). HPLC samples (10 mg/mL) were filtered through a 0.45 μm PVDF-filter and injected into the HPLC column. The injection volume was 10 μL and the separation temperature was 25 °C. The column was a Mightysil RP-18 GP (5 μm, 250 × 4.6 mm I.D.). The method involved the use of a binary gradient with mobile phases containing: (A) phosphoric acid in water (0.1%, v/v) and (B) H2O/Methanol: 20/80 (v/v). The solvent gradient elution program was as follows: 0–15 min, 100–75% A, 0–25% B; 15–50 min, 75–25% A, 25–75% B; and finally 50–60 min, 25–0% A, 75–100% B. The flow-rate was kept constant at 0.8 mL/min. A precolumn of μ-BondapakTM C18 (Millipore, Milford, MA, USA) was attached to protect the analytical column. Pure compounds, including daidzin, genistin, daidzein, genistein, and butyl p-hydroxybenzoate (internal control) were also analyzed using HPLC under the same conditions, and the retention time was used to identify the daidzin, genistin, daidzein, and genistein in the samples.
Determining antioxidant activities by ABTS+ scavenging ability
The ABTS+ scavenging ability was determined (Chiu et al. 2013). Aqueous solution of ABTS (7 mM) was oxidized with potassium peroxodisulfate (2.45 mM) for 16 h in the dark at room temperature. The ABTS+ solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm (Beckman UV–Vis spectrophotometer, Model DU640B). For each sample, an aliquot (20 μL) of sample (125 μg/mL) was mixed with 180 μL ABTS+ solution, and then the absorbance was read at 734 nm after 1 min. Trolox was used as a reference standard.
Determining antioxidant activity by DPPH radical scavenging ability
The effects of crude extracts and positive controls (GSH, genistein, and daidzein) on DPPH radicals were estimated (Huang et al. 2010). Aliquots of crude extracts (20 μL) at various concentrations were each mixed with 100 mM Tris–HCl buffer (80 μL, pH 7.4) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM. All the mixtures were shaken vigorously and left to stand at room temperature for 20 min in the dark. The absorbance of the reaction solutions were measured spectrophotometrically at 517 nm. The DPPH decolorizations of the samples were calculated in percentage according to the equation: % decolorization = [1 − (ABSsample/ABScontrol)] × 100. IC50 value was the effective concentration in which DPPH 50% of radicals were scavenged and was obtained by interpolation with linear regression analysis.
Determination of total polyphenol content
The total polyphenol content of the crude extracts were determined (Chiu et al. 2013). For each sample, 20 μL of the extract was added to 200 μL distilled water and 40 μL of Folin–Ciocalteu reagent. The mixture was allowed to stand at room temperature for 5 min, and then 40 μL of 20% sodium carbonate was added to the mixture. The resulting blue complex was measured at 680 nm. Catechin was used as a standard for the calibration curve. The polyphenol content was calibrated using the calibration curve based linear equation. The total polyphenol content was expressed as mg catechin equivalent/g dry weight. The dry weight indicated was the sample dry weight.
Determination of total flavonoid content
The total flavonoid contents of the crude extracts were determined (Chiu et al. 2013). For each sample, an aliquot of 1.5 mL extract was added to an equal volume of 2% AlCl3·6H2O solution. The mixtures were vigorously shaken, and the absorbances at 430 nm were read after 10 min of incubation. Rutin was used as the standard for the calibration curve. The total flavonoid content was calibrated using the linear equation based on the calibration curve. The total flavonoid content was expressed as mg rutin equivalent/g dry weight. The dry weight indicated was the sample dry weight.
Cell culture
A murine macrophage cell line RAW264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2 incubator (5% CO2 in air) at 37 °C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin–0.02% EDTA in Ca2+-, Mg2+- free phosphate-buffered saline (DPBS).
Cell viability
Cells (2 × 105) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with samples in the presence of 100 ng/mL LPS for 24 or 1 h. After that, the cells were cleaned twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read by using a microplate reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA).
Measurement of nitric oxide/nitrite
NO production was indirectly assessed by measuring the nitrite levels in BALF determined by a colorimetric method based on the Griess reaction (Shie et al. 2015). 100 μL supernatant was applied to a microtiter plate well, followed by 100 μL of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid). After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, Orleans Drive, Sunnyvale, CA, USA). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured by absorbance at 540 nm.
Protein lysate preparation and Western Blot analysis
The stimulated murine macrophage cell line RAW264.7 cells were washed with PBS and lysed in an ice-cold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, 20 mM Tris buffer (pH 7.9), 100 mM β-glycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)] on ice for 1 h, followed by centrifugation at 12,000×g for 30 min at 4 °C. Soft tissues were removed from individual mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 μg/mL, aprotinin, 1 μM pepstatin and 10 μM leupeptin. The homogenates were centrifuged at 12,000×g for 20 min, and 30 μg of protein from the supernatants was then separated on 10% sodium dodecylsulphate–polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After transfer, the membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). Later, the membranes were incubated with antibody in 5% skim milk in TBST for 2 h at room temperature. The membranes were washed three times with TBST at room temperature and then incubated with a 1:2000 dilution of anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Sigma, St Louis, MO, USA) in 2.5% skim milk in TBST for 1 h at room temperature. The membranes were washed three times and the immunoreactive proteins were detected by enhanced chemiluminescence (ECL) by using hyperfilm and ECL reagent (Amersham International plc., Buckinghamshire, UK). The results of Western Blot analysis were quantified by measuring the relative intensity compared to the control by using Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY, USA) and represented in the relative intensities.
Inhibition assay for α-glucosidase activity
The α-glucosidase inhibitory effects of the aqueous extracts from three Glycine species were assayed with minor modifications (Cao et al. 2009). Briefly, the enzyme reaction was performed using p-Nitrophenyl-alpha-d-glucopyranoside (PNP-glycoside) as a substrate in 0.1 M piperazine- N,N′-bis (2-ethanesulfonic acid) (PIPES) buffer, pH 6.8. The PNP-glycoside (2.0 mM) was premixed with samples at various concentrations. Each mixture was added to α-glucosidase solution (0.01 units, from bakers yeast) to make 0.5 mL of final volume. The reaction was terminated by adding 1 ml of 0.64% N-(1-naphthyl) ethylenediamine solution (pH 10.7). Enzymatic activity was quantified by measuring the p-nitrophenol released from PNP-glycoside at 400 nm wave length. All reactions were carried out at 37 °C for 30 min with three replications.
Measure aldose reductase activity in vitro
Crude aldose reductase (AR) was prepared as in the following steps: lenses were removed from Sprague–Dawley (SD) rats weighing 250–280 g, and were kept frozen until use. A homogenate of rat lens was prepared in accordance with the method described (Huang et al. 2011). A partially purified enzyme, with a specific activity of 6.5 U/mg, was routinely used in the evaluations of enzyme inhibition. The partially purified material was separated into 1.0 mL aliquots, and stored at −40 °C. The AR activity was spectrophotometrically assayed by measuring the decrease in NADPH absorption at 340 nm over a 4 min period, using DL-glyceraldehyde as a substrate. Each 1.0 mL cuvette contained equal units of enzyme, 0.10 M sodium phosphate buffer (pH 6.2) and 0.3 mM NADPH, both with and without 10 mM of the substrate and an inhibitor. Each 1.0 mL of cuvette containing equal units of enzyme, 0.1 M sodium phosphate buffer (pH 6.2), 0.3 mM NADPH with or without 10 mM substrate and inhibitor was prepared. One set of mixtures prepared with an equivalent volume of sodium phosphate buffer instead of tested samples was used as control. The concentration of the extracts required to inhibit 50% of AR activity under the assay conditions was defined as the IC50 value.
Statistical analyses
Experimental results were presented as the mean ± standard deviation (SD) of three parallel measurements. The statistical analyses were performed by one-way ANOVA, followed by Dunnett’s t test. A difference was considered to be statistically significant when p < 0.05, p < 0.01 or p < 0.001.