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EXPERIMENTAL PROCEDURE
Materials and reagents
Fresh tuber of O. japonicus was collected in Mianyang known as
Sichuan O. japonicus. Butylated hydroxyanisole (BHA), DPPH
radical, concanavalin A (ConA) and lipopolysaccharide (LPS) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Roswell
Park Memorial Institute (RPMI) Medium 1640 and fetal bovine
serum (FBS) were obtained from GIBCO (Grand Island, NY, USA).
3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide
(MTT) were purchased from Amresco. All the reagents were of
analytical grade.
Extraction and isolation of saponin
Crushed tuber of O. japonicus was firstly extracted with 70%
alcohol at 60°C in ultrasonic washing machine (200 W) for 1.5 h.
The extracts were evaporated of alcohol at 45°C in vacuum on
rotary evaporation instrument (SHANG HAI HU XI ANAYSIS
INSTRUMENT FACTORY CO., LTD). The resulting solution was
extracted with diethyl ether, and the treatment was repeated twice.
The obtained water layer was again extracted with normal butyl
alcohol saturated with water, and the treatment was repeated four
times. The obtained normal butanol layer was re-extracted with 0.4
M NaOH (twice). The normal butanol layer was evaporated just to
dryness with a vacuum evaporator at 45°C to obtain saponin which
was again purified by non-polar macroporous adsorption resin D
101 (particle size of 0.3 to 1.25 mm, pore size of 6.0 to 10.0 nm,
TIANJIN HAIGUANG CHEMICAL CO., Ltd., China). A portion of it
was dissolved in deionized water and loaded onto a
chromatographic column (2.0 × 20 cm). It was firstly eluted with
0.1% of NaOH to colourless eluent, then eluted by deionized water
for removing carbohydrate. It was again eluted by 70% of ethanol at
a flow rate of 2 BV/h for collecting saponin after eluent exhibited
Mollish negative reaction. The purified saponin was evaporated just
to dryness with a vacuum evaporator at 45°C and was stored at
20°C for further evaluation of antioxidant and macrophagesmodulating
activities.
Physico-chemical properties of saponin
Saponin was preliminary identified by chemical methods including
foam experiment and Liebermann reaction, and thin layer
chromatography (Chen, 2004). Briefly, sample containing methanol
solution was spotted on silica gel G thin-layer plates, with
chloroform-ether (1:9) as developing solvent. Vanillin-sulfuric acid
reaction system was used for identification of saponin.
DPPH radical scavenging activity of saponin
The DPPH radical scavenging activity of saponin was measured
using the method reported by Song et al. (2010) with slight
modification. Briefly, 1 ml sample with different concentrations (1 to
5 mg/ml) was added to screw-capped tube containing 1.5 ml of
DPPH (40 mg/L, m/v) anhydrous alcohol solution. The mixture was
vortexed and set for 30 min, then the absorbance was measured at
517 nm against a blank (water instead of test sample solution)
using UNICO 2102 spectrophotometer (UNICO Instruments Co.,
Ltd., Shanghai, China). Ascorbic acid and BHA were used for
positive comparison. DPPH radical scavenging rate (SC) is
calculated as follows:
DPPH radical scavenging rate (%) = 100 × (A0 - A1)/A0.
where A0 is absorbance of blank control and A1 is absorbance value
Shuang-Li et al. 1965
of the sample. The IC50 value was calculated by nonlinear
regression algorithm SC via sample concentration. This is defined
as the amount of antioxidant necessary to reduce the concentration
of DPPH radical by 50%.
Hydroxyl radical scavenging activity of saponin
Hydroxyl radical (·OH) scavenging activity of saponin was assessed
by the method of Zhu et al. (2010). Briefly, 2 ml deionized water or
tested sample was added to screw-capped tubes which contained
0.35 ml of 8.8 mM H2O2 and 0.35 ml of 6 mM FeSO4. The mixture
was thoroughly shaken and set for 10 min before salicylic acid was
added, and shaken vigorously, which was set for 10 min and
absorbance at 510 nm was recorded. The ·OH scavenging activity
was calculated according to the following equation:
·OH scavenging rate (%) = 100 × (A0 - A1)/A0
where A0 is absorbance value of blank and A1 is absorbance value
of the sample. IC50 value was the same as that of DPPH radical.
Isolation and culture of peritoneal macrophage
Male Kunming strain mouse (7 to 8 weeks old, 19 ± 0.5 g body
weight) was purchased from the Experimental Animal Institute of
Sichuan Academy of Medical Science. Main members in research
teams have obtained the permission to work with experimental
animals. Mouse was intraperitoneally injected with 5% soluble
starch three days in advance before experiments. Macrophages
were prepared from mouse as described earlier with some
modifications (Li et al., 2011; Yang et al., 2008). Briefly, peritoneal
cell suspensions were harvested by peritoneal cavity lavage with 5
ml ice-cold sterile phosphate buffer solution (PBS). The recovered
peritoneal fluid was centrifuged at 2380 g for 10 min, the cell pellets
were suspended with RPMI-1640 cell culture media supplemented
with 10% (v/v) heat-inactivated FBS, and was cultivated for 3 h at
37°C in a humidified 5% CO2 incubator. Non-adherent cells were
removed by gentle washing twice with sterile PBS. 1 ml fresh RPMI-
1640 was added to the adherent macrophages and it was adjusted
to the concentration of 5 × 10 5 cells/ml.
Assay of phagocytic activity
The effects of saponin on the phagocytic activity of macrophage
were determined using neutral red assay as reported earlier with
some modifications (Yi et al., 2010; Xiong et al., 2011). In brief, 100
μl of macrophages (5 × 10 5 cells/ml) were pre-incubated for 3 h in a
96-well plate before removal of non-adherent with RPMI- 1640.
Various tested samples and RPMI-1640 supplemented with 10%
FBS were added to each well, and were incubated for 48 h. 100 μl
of 1% neutral red physiologic saline solution was then added. After
an additional incubation for 3 h, the mixture was removed of
supernatant and was washed 3 times with PBS prior to the addition
of cytolysate (glacial acetic acid:alcohol, 1:1, 100 μl). The
absorbance at 540 nm was recorded on Multiskan Spectrum
Microplate Spectrophotometer (Thermo Scientific, America) after
cytolysis. Higher absorbance indicates stronger phagocytic activity.
Assessment of macrophage viability
The effect of saponin on the macrophage viability was detected by
the MTT assay as previously described with some modifications
(Davicino et al., 2006). Briefly, 20 μl of MTT was added to
macrophage (100 μl, 5 × 10 5 cells/ml) which was pre-incubated for