Plants, compounds and chemical reagents
The SBG was purchased from Beijing Tongrentang (Beijing, China) and the ground powder was extracted twice with 80% (v/v) ethanol using an ultra-sonicator (Branson, USA.) and evaporated at 60°C and then freeze-dried. The final yield was 48.75 g (24.3%). The chromatogram of baicalein and baicalin were recorded at 315 nm and 272 nm respectively. HPLC (Shimadzu, Japan) analysis content of baicalin and baicalein was 4.1522% and 3.3075%, respectively in SBG. Baicalin and baicalein which were used for experiments were purchased from Waco (Osaka, Japan). D-Galactose, NaNO2, and LPS (Sigma-Aldrich, USA), Commercial kits for malondialdehyde, superoxide dismutase and catalase (Cayman, USA) were purchased. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco Life Technologies (MD, USA). COX-2, iNOS, bcl2, bax, procaspase-3, cleaved caspase-3, 8 and 9, and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (CA, USA).
The enzyme immunoassay (EIA) kits used for the determination of nitric oxide, prostaglandin E2 were obtained from Assay Designs Inc. (MI, USA).
Animals and administraion
ICR female mice (4 weeks old, 20-22 g) were purchased from Orient Bio Experimental Animal Center (Seongnam, Korea). Mice were housed (8 mice per cage) in a regulated environment at 25 ± 1°C with a 12 h/12 h light/dark cycle and with free access to standard rodent pellets (Purina, Korea). Animal care and experimental procedures followed requirements put forth in the Guide for the Care and Use of Laboratory Animals (Department of Health and Education, and Welfare, National Institute of Health, 1996). After 7 days of adaptation, the mice of 36 were randomly divided into six groups. The normal control group (NC, saline 0.3 ml, n = 6), aging control group (AC, D-galactose 120 mg/kg, NaNO2 90 mg/kg, n = 6), baicalin and baicalein treated group (200 mg/kg, n = 6 respectively), SBG treated group (50 mg/kg and 100 mg/kg, n = 6 respectively). Normal group mice were intraperitoneally injected saline 0.3 ml once daily for 60 days, and orally administered with saline 0.3 ml/mouse for 14 days from day 47 of the experiment. Mice in aging control and each treatment groups were intraperitoneally injected with D-galactose (120 mg/kg) and NaNO2 (90 mg/kg) once daily for 60 days. From day 47 to 60 for 2 weeks, aging control mice were orally administered with saline 0.3 ml/mouse, and the treatment groups of mice were treated with baicalin 200 mg/kg, baicalein 200 mg/kg, SBG 50 mg/kg and SBG 100 mg/kg for 14 days (orally, once daily).
The murine macrophage Raw 264.7 cells and mouse microglia BV2 cells were obtained from the Korea Cell Line Bank (Seoul, Korea) and cultured in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum. The cells were subcultured twice weekly and grown on 6-well plates at 1 ×106 cells/well at 37 °C in fully humidified 5% CO2 air.
The cell viability was assessed based on the content of metabolized blue formazan from 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) converted by mitochondrial dehydrogenases in live cells.
For Y-maze test, the experimental mice were retained and subjected to training and test on day time to assess short-term and spatial memory performance in Y-maze. The maze was placed in a separate room with enough light. The Y-maze test was of 10 minutes duration and allowed the mice to explore only two arms (start arm and the other arm) of the maze, with the third arm (novel arm) blocked. Data was expressed as percentage of alternation calculated as (successive triplet sets/total number of arm entries-2) × 100.
Brain tissue preparation
After examination of the memory behavior, all mice were deeply anesthetized and decapitated, and their brains were removed rapidly and homogenized in 50 mM (PH 7.4) cold phosphate buffer saline solution (PBS) containing a protease inhibitor cocktail (Sigma-Aldrich, USA) with 10 strokes at 1200 rpm in a homogenizer. Three brains of mice chosen randomly from each group were post-fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C, and then placed in a solution of 30% sucrose, 4% paraformaldehyde in PBS (pH 7.4).
Crystal violet staining and immunohistochemisty
The sections were incubated with antibody of bcl2 (Santa Cruz, USA). The sections were General ABC Procedure. Fixed brains were cut into 30 um sections on a sliding microtome and the sections stained with Crystal violet. Slides were immersed for 5 min in each of the following: xylene, 100% alcohol, 95% alcohol, and 70% alcohol. They were dipped in distilled water and stained in 0.5% crystal violet for 15~30 min. They were differentiated in water for 3~5 min and then dehydrated through 70% alcohol, 95% alcohol, and 100% alcohol. They were then put in xylene and cover-slipped.
Assay of SOD and CAT activities
The assay for total superoxide dismutases (SOD) is based on the ability to inhibit the oxidation of oxymine by the xanthine-xanthine oxidase system . Brain homogenates was directly centrifuged at 8000 g for 10 minutes to obtain supernatants to assay brain catalase (CAT) level and the supernatant collected for determination of superoxide dismutase (SOD) activities. The hydroxylamine nitrite produced by the oxidation of oxymine had an absorbance peak at 550 nm. One unit (U) of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%, and data were expressed as units per microgram of brain protein. Catalase activity was assayed by the previous method . In brief, to a quartz cuvette, 0.65 ml of the phosphate buffer (50 mmol l-1; pH7.0) and 50 ul sample were added, and the reaction was started by addition of 0.3 ml of 30 mM hydrogen peroxide (H2O2). The decomposition of H2O2 was monitored at 240 nm at 25°C. CAT activity was calculated as nM H2O2 consumed in 1 min per milligram of brain protein.
Measurement of MDA level
The level of lipid peroxidation in brain homogenate was indicated by the content of malondialdehyde (MDA) in brain tissue. The brain homogenates was sonicated four times for 30 seconds with 20 seconds intervals using a ultrasonicator (Branson, USA), centrifuged at 5000 g for 10 minutes at 4 °C. Thiobarbituric acid reaction (TBAR) method was used to determine the MDA which can be measured at the wave length of 532 nm by reacting with thiobarbituricacid (TBA) to form a stable chromophoricproduction. MDA content was expressed as nmol per milligram of brain protein. Protein concentration was measured using the method of Bradford . Bovine serum albumin was used as standard.
The nitrite which accumulated in culture medium was measured as an indicator of NO production according to the Griess reagent. The culture supernatant (100 ul) was mixed with 100 ul of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthyl ethylenediamine-HCl] for 10 minutes, and then the absorbance at 540 nm was measured in a microplate reader. Fresh culture medium was used as the blank in all experiments. The amount of nitrite in the samples was determined with reference to a sodium nitrite standard curve.
The cells were incubated with the SBGs or LPS or both for 18 h. After incubating the cells for 18 h, the culture medium was collected and the concentration of PGE2 secreted into the culture media was measured using a specific enzyme immunoassay according to the manufacturer's instructions (Assay Design Inc., USA).
RAW 264.7 cells and mouse microglia BV2 cells were collected by centrifugation and washed once with phosphate-buffered saline (PBS). The washed cell pellets were resuspended in extraction lysis buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM Na fluoride, and 0.5 mM Na orthovanadate) containing 5 ug/ml each of leupeptin and aprotinin and incubated with 20 min at 4 C. Cell debris was removed by microcentrifugation, followed by quick freezing of the supernatants. The protein concentration was determined using the Bio-Rad protein assay reagent according to the manufacturer's instructions. Forty micrograms of cellular protein from treated and untreated cell extracts was electroblotted onto a PVDF membrane following separation on a 10% SDS-polyacrylamide gel electrophoresis. The immunoblot was incubated overnight with blocking solution (5% skim milk) at 4°C, followed by incubation for 4 h with a primary antibody (Santa Cruz Biotechnology, USA). Blots were washed four times with Tween 20/Tris-buffered saline (TTBS) and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz, USA.) for 1 h at room temperature. Blots were again washed three times with TTBS, and then developed by enhanced chemiluminescence (Amersham Life Science, USA).
All data in the text are expressed as mean ± standard deviation (mean ± S.D.), and analyzed by one-way ANOVA and multiple comparisons were performed by Tukey HSD test. A criterion of P < 0.1 (* or #), 0.05 (** or ##) and 0.01 (*** or ###) were accepted as statistically significant and marked with * or #.