Chemicals and animals
The chemicals (+) taxifolin (CAS number: 480–18-2, 98%), dihydromyricetin (CAS number: 27,200–12-0, 98%), and 4’-O-methyltaxifolin (CAS number: 70,411–27-7, 98%) were obtained from Chengdu Biopurify Phytochemicals Ltd. Catechol (CAS number: 120–80-9, 99.5%) and DNA sodium salt (fish sperm) were purchased from Aladdin Chemistry Co. The 1, 1-diphenyl-2-picryl-hydrazyl radical (DPPH•), (±)-6-hydroxyl-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid (trolox), 2, 9-dimethyl-1, 10-phenanthroline (neocuproine), 3-(2-pyridyl)-5, 6-bis (4-phenylsulfonicacid)-1, 2, 4-triazine (ferrozine), 2, 4, 6-tripyridyltriazine (TPTZ), 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO•), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich Shanghai Trading Co. The CCK-8 (BB-4221-2) kits were from BestBio Inc. (NH4)2ABTS [2, 2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt)] was obtained from Amresco Chemical Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco. All other reagents were of analytical grade.
Sprague-Dawley (SD) rats (4 weeks old) were obtained from the Animal Center of Guangzhou University of Chinese Medicine. The protocol was performed under the supervision of the Institutional Animal Ethics Committee at the Guangzhou University of Chinese Medicine.
MTT assay to assess the protective effect against •OH-induced damage
The bmMSCs were cultured according to our previous report [15] with slight modifications. In brief, bone marrow was obtained from the femur and tibia of the rats. Marrow samples were diluted with low-glucose DMEM containing 10% FBS. MSCs were prepared by gradient centrifugation at 900×g for 30 min on 1.073 g/ml Percoll. The prepared cells were detached by treatment with 0.25% trypsin and passaged in culture flasks at 1 × 104/cm2. At passage 3, bmMSCs were evaluated for cell homogeneity using CD44 detection via flow cytometry. These cells were used for the subsequent experiments.
The protective effect of (+) taxifolin against •OH-induced bmMSC damage was investigated based on the method described in [16, 17] with slight modifications. Briefly, bmMSCs were seeded at 5000 cells per well into 96-well plates. After adherence for 24 h, bmMSCs were divided into control, model and sample [(+) taxifolin] groups.
In the control group, bmMSCs were incubated for 24 h in DMEM. In the model and sample groups, bmMSCs were incubated in the presence of FeCl2 (100 μM) followed by H2O2 (50 μM). After incubation for 20 min, the mixture of FeCl2 and H2O2 was removed. The bmMSCs in the model group were incubated for 24 h in DMEM, while bmMSCs in the sample group were incubated for 24 h in DMEM with the indicated (+) taxifolin concentrations.
After incubation, 20 μl MTT (5 mg/ml) was added, and the culture was incubated for an additional 3 h. The culture medium was discarded and replaced with 150 μl DMSO. Absorbance was measured at 490 nm on a Bio-Kinetics reader (PE-1420; Bio-Kinetics Corporation). Culture medium containing serum was used for the control group and each sample test was repeated in five independent wells.
Hydroxyl-scavenging assay based on DNA
The hydroxyl-scavenging effect of (+) taxifolin was estimated using a method developed by our laboratory [18]. Briefly, methanol sample solutions (1.2 mg/ml, 20–100 μl) were separately aliquoted into mini tubes. After completely evaporating the methanol solvent in each tube to dryness, the sample residue was treated with 300 μl of phosphate buffer (0.2 M, pH 7.4), followed by 50 μl of DNA sodium (10 mg/ml), 75 μl of H2O2 (33.6 mM), 50 μl of FeCl3 (3.2 mM), 100 μl of Na2EDTA (0.5 mM) and 75 μl of ascorbic acid (12 mM). After incubation at 50 °C for 20 min, 250 μl of trichloroacetic acid (10%, w/v) was added to the tube. After heating the mixture at 105 °C for 15 min with 150 μl of 2-thiobarbituric acid (TBA, 5% in 1.25% NaOH aqueous solution), the absorbance was measured using a Unico Spectrophotometer UV 2100 against the buffer (blank). The protective effect is expressed as follows:
$$ Protective effect\%=\frac{A_0\hbox{-} A}{A_0}\times 100\%, $$
where A0 indicates the absorbance of the blank and A indicates the absorbance of the sample (+) taxifolin.
PTIO•-scavenging assay
The PTIO•-scavenging assay was conducted based on our method [19]. In brief, 80 μl of an aqueous PTIO• solution (0.1 mM) was mixed with 20 μl of phosphate buffer at pH 5.0, 6.0, 7.0, 7.4, 8.0 and 9.0 containing 1 mg/ml of sample at the indicated concentrations. The mixture was maintained at 37 °C for 30 min, and the absorbance was measured at 560 nm on a microplate reader (Multiskan FC, Thermo Scientific). The PTIO• inhibition percentage was calculated as follows:
$$ Scavenging\%=\frac{A_0\hbox{-} A}{A_0}\times 100\%, $$
where A0 indicates the absorbance of the blank and A indicates the absorbance of the sample, (+) taxifolin.
DPPH•-scavenging assay and ABTS+•-scavenging assay
DPPH• radical-scavenging activity was determined as previously described [20]. Briefly, 1 ml of DPPH• solution (0.1 M) was mixed with the indicated concentrations of sample (0.15 mg/ml, 14–70 μl) dissolved in methanol. The mixture was maintained at room temperature for 30 min, and the absorbance was measured at 519 nm on a Unico Spectrophotometer 2100.
ABTS+•-scavenging activity was evaluated according to a previously described method [21]. ABTS+• was produced by mixing 0.2 ml of ABTS diammonium salt (7.4 mM) with 0.35 ml of potassium persulfate (2.6 mM). The mixture was maintained in the dark at room temperature for 12 h to allow completion of radical generation and then diluted with 95% ethanol. To determine the scavenging activity, the test sample (x = 15–75 μl, 0.03 mg/ml) was added to (200- x) μl of 95% ethanol followed by 800 μl of ABTS+• reagent, and the absorbance was measured at 734 nm on a Unico Spectrophotometer 2100 6 min after the initial mixing using 95% ethanol as the blank.
The percentage of DPPH•-scavenging (or ABTS+•-scavenging) activity was calculated based on the formula given in the PTIO•-scavenging assay section.
Cu2+-reducing assay
The reducing power capacity of cupric ions (Cu2+) was measured according to a previously described method [22] with a slight modification. Briefly, 125 μl of CuSO4 aqueous solution (10 mM), 125 μl of neocuproine ethanolic solution (7.5 mM) and 750 μl of CH3COONH4 buffer solution (0.1 M, pH 7.5) were added to test tubes with different volumes of sample (0.15 mg/ml, 15–75 μl). The total volume was adjusted to 1 ml with buffer and mixed vigorously. The absorbance against a buffer blank was measured at 450 nm after 30 min. An increase in the absorbance of the reaction mixture indicates an increase in reduction capability. The relative reducing power of the sample relative to the maximum absorbance was calculated using the following formula:
$$ Relative reducing effect\%=\frac{A\hbox{-} {A}_{min}}{A_{max}\hbox{-} {A}_{min}}\times 100\%, $$
where Amin is the absorbance of the control without sample, A is the absorbance of the reaction mixture with sample, and Amax is the maximum absorbance of the reaction mixture with sample.
Ferric-reducing antioxidant power (FRAP) assay
The FRAP assay was adapted from Benzie and Strain [23]. Briefly, FRAP reagent was freshly prepared by mixing 10 mM TPTZ, 20 mM FeCl3 and 0.25 M acetate buffer at 1:1:10 (pH 3.6). The test sample (x = 20–100 μl, 0.5 mg/ml) was added to (100- x) μl of 95% ethanol followed by 400 μl of FRAP reagent. The absorbance was measured at 593 nm after a 30-min incubation at ambient temperature using distilled water as the blank. The relative reducing power was calculated using the formula given in the Cu
2+
-reducing assay section.
UV-vis spectra and color reaction of Fe2+-binding
The (+) taxifolin–Fe2+ complex was evaluated using UV-Vis spectroscopy. For these experiments, 300 μl of a methanolic solution of (+) taxifolin and 100 μl of an aqueous solution of FeCl2•4H2O were added to 600 μl of an aqueous mixture of distilled water and methanol (1:1). The solution was then mixed vigorously and continuously scanned using a UV-Vis spectrophotometer (Unico 2600A) from 200 to 900 nm after 0, 10, 20, 30, and 60 min.
The above experiment was repeated using 4’-O-methyltaxifolin.
Statistical analysis
Each experiment was performed in triplicate and data were recorded as the means ± SD (standard deviation). Dose response curves were plotted using Origin 6.0 software (OriginLab). IC50 was defined as the final concentration of 50% radical inhibition (relative reducing power or binding effect). Statistical comparisons were made using one-way ANOVA to detect significant differences using SPSS 13.0 (SPSS Inc.) for Windows. p < 0.05 was considered statistically significant.