Plant and animals
Suoyang (Xinjiang) (LOT. YPA6E0003) was purchased from Caizhilin Pharmaceuticals Co., Ltd. (Guangzhou, China). Sprague–Dawley (SD) rats of 4 weeks of age were obtained from the Animal Center of Guangzhou University of Chinese Medicine.
Chemicals
Luteolin-7-O-β-D-glucoside (CAS 68321-11-9, 98%), protocatechuic acid (CAS 99-50-3, 98%), catechin (CAS 154-23-4, 98%), epicatechin (CAS 18829-70-4, 98%), naringenin (CAS 480-41-1, 98%), isoquercitrin (CAS 482-35-9, 98%), and phlorizin (CAS 60-81-1, 98%) were purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Gallic acid (CAS 149-91-7, 98%) was purchased from Shanghai Aladdin Chemistry Co., Ltd. (Shanghai, China); Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Inc. (Grand Island, NY, USA). CD44 was purchased from Wuhan Boster Co., Ltd. (Wuhan, China). PTIO• (2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) was purchased from TCI (Shanghai) Development Co., Ltd. DPPH• (1,1-diphenyl-2-picryl-hydrazl), neocuproine (2,9-dimethyl-1,10-phenanthroline), TPTZ (2,4,6-tris(2-pyridyl-s-triazine)), Trolox [(±)-6-hydroxyl-2,5,7,8-tetramethlychroman-2-carboxylic acid], and the Percoll system were obtained from Sigma-Aldrich Trading Co. (Shanghai, China); (NH4)2ABTS [2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt)] was purchased from Amresco Chemical Co. (Solon, OH, USA). Methanol and water were HPLC grade. All other reagents were analytical grade.
Preparation of the lyophilized aqueous extract of Suoyang (LAS)
The cut Suoyang was extracted with distilled water at 100 °C then freeze-dried to prepare the lyophilized aqueous extract of Suoyang (LAS). LAS with brownish red in appearance (Additional file 1) was stored at 4 °C for further analysis. The flow chart of preparation is shown in Fig. 2.
HPLC characterization of LAS
HPLC analysis was performed using a Shimadzu LC-20A (Tokyo, Japan) equipped with an Agilent 5 TC-C18 250*4.6 mm column (Beijing, China). The mobile phase consisted of methanol (A) −0.3% and formic acid in water (C) (0.01 min, remain 5% A; 0–10 min, 5% A–10% A; 10–30 min, 10% A–40% A; 30–50 min, 40% A–50% A; 50–55 min, 50% A–5% A). The flow rate was 1.0 mL/min, the injection volume was 10 μL (400 mg/mL for LAS; 0.1 mg/mL for the standards), and absorption was measured at 280 nm [10]. In the study, phenolic components were identified by comparing their retention times, and the peak areas were employed to characterize the relative content of gallic acid, protocatechuic acid, catechin, epicatechin, luteolin-7-O-β-D-glucoside, isoquercitrin, phlorizin, and naringenin.
Protective effect towards •OH-damaged MSCs (MTT assay)
MSCs were cultured according to the method described in our previous report [11]. In brief, bone marrow samples were accessed from the femurs and tibias of rats and diluted using low glucose DMEM containing 10% FBS. After gradient centrifugation at 900g/min for 30 min, the MSCs were prepared using a 1.073 g/mL Percoll system. The cells were then detached by treatment with 0.25% trypsin. The detached cells were passaged into culture flasks at a density of 1 × 10−4 cells/cm2. The homogeneity of the MSCs was evaluated at passage 3 based on their CD44 expression by flow cytometry. These cells were then used for the following experiments.
These MSCs were seeded into 96-well plates at a density of 4 × 103 cells/well. After adherence for 24 h, the cells were classified into three groups, i.e., control group, model group, and samples group. The MSCs in the control group were incubated for 24 h in DMEM. The MSCs in the model group were injured for 25 min using FeCl2 (100 μM), followed by H2O2 (50 μM). The mixture of FeCl2 and H2O2 was removed, and the MSCs were incubated for 24 h in DMEM. The MSCs in the samples group were injured and incubated for 24 h in DMEM in the presence of various concentrations of samples. After incubation, 20 μL of MTT (5 mg/mL in PBS) was added to the cells, which were then incubated for 4 h. The culture medium was subsequently discarded and replaced with 150 μL of DMSO. The absorbance of each well was then measured at 490 nm using a Bio-Kinetics plate reader (PE-1420; Bio-Kinetics Corporation, Sioux Center, IA, USA). The serum medium was used for the control group, and each sample test was repeated in five independent wells.
PTIO•-scavenging assay
The PTIO•-scavenging assay was conducted based on our method [12]. In brief, the test sample (x = 0–10 μL, 1 mg/mL) was added to (10 − x) μL of 95% ethanol, followed by 90 μL of an aqueous PTIO• solution (0.1 mM). The mixture was maintained at 37 °C for 2 h, and the absorbance was then measured at 560 nm using a microplate reader (Multiskan FC, Thermo Scientific, Shanghai, China). The PTIO• inhibition percentage was calculated as:
$${Inhibition\, \% =\, }\frac{{\text{A}_{\text{0}} - A}}{{\text{A}_{\text{0}} }}\text{ } \times { 100\% }$$
, where A0 is the absorbance of the control without the sample, and A is the absorbance of the reaction mixture with the sample.
DPPH•-scavenging and ABTS+•-scavenging assays
The DPPH•-scavenging and ABTS+•-scavenging assays were based on previous reports [13]. In the DPPH•-scavenging assay, 90 μL of an ethanolic solution of DPPH• (0.1 mM) was mixed with (10 − x) μL of an ethanolic or (x = 0–10 μL, 0.2 mg/mL) aqueous solution of the sample. The mixture was maintained at room temperature for 30 min, and the absorbance was then measured at 519 nm. In the ABTS+•-scavenging assay, the ABTS+• was produced by mixing 200 μL of (NH4)2ABTS (7.4 mM) with 200 μL of K2S2O8 (2.6 mM). After incubation in the dark for 12 h, the mixture was diluted with methanol (approximately 1:50) so that the absorbance at 734 nm was 0.30 ± 0.01. Then, the diluted ABTS+• solution (90 μL) was added to (10 − x) μL of an ethanolic or (x = 0–10 μL, 0.1 mg/mL) aqueous solution of the sample and then mixed thoroughly. After the reaction mixture stood for 6 min, the absorbance was measured at 734 nm using a spectrophotometer. The percentage of inhibition of DPPH•-scavenging or ABTS+•-scavenging was calculated using the formula described in “PTIO•-scavenging assay” section.
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was adapted from Benzie and Strain [14]. Briefly, the FRAP reagent was prepared fresh by mixing 10 mM TPTZ, 20 mM FeCl3 and 0.3 M acetate buffer at 1:1:10 at pH 3.6. The test sample (x = 0–20 μL, 0.5 mg/mL) was added to (20 − x) μL of 95% ethanol, followed by 80 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 of the sample compared with the maximum absorbance was calculated by the following formula:
$$Relative \, reducing \, effect{\text{ \% }} = \, \frac{{A - A_{{{\text{min}}} } }}{{A_{{{\text{max}}} } - A_{{{\text{min}}} } }} \, \times {\text{ 100\% ,}}$$
where Amin is the absorbance of the control without the sample, A is the absorbance of the reaction mixture with the sample, and Amax is the greatest absorbance of the reaction mixture with the sample.
Ultraviolet (UV) spectral determination of Fe2+-binding
Ultraviolet (UV) spectra of Fe2+-binding were conducted according to a previously described method [15]. Briefly, a 100-μL test sample was added to 100 μL of an aqueous solution of FeCl2·4H2O (10 mg/mL). The total volume was adjusted to 200 μL, and the solution was then mixed vigorously. The resulting mixture was incubated at room temperature for 24 h. The product mixtures were then imaged using a smartphone (Samsung, Galaxy A7, China). Subsequently, the supernatant was collected, and a spectrum was obtained using a UV/Vis spectrophotometer (Jinhua 754 PC, Shanghai, China) from 200 to 1000 nm.
Statistical analysis
The IC50 values were calculated by linear regression analysis. All linear regression analyses in this study were analyzed by the Origin 6.0 professional software. The determination of significant differences between the mean IC50 values of the sample and positive controls was performed using one-way analysis of variance (ANOVA) and a T test. The analysis was performed using SPSS software 13.0 (SPSS Inc., Chicago, IL) for windows. P < 0.05 was considered to be statistically significant.