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第 6 期 常柄权，等: 变叶海棠叶总黄酮的分离纯化及体外抗炎活性 ·1151·

for 4 h. After the absorption equilibrium, the resins wet-packed into a glass column (35 mm × 450 mm),
were washed twice with deionized water. Then, 50 mL with a bed volume (BV) of 50 mL. The adsorption
of 70% ethanol (volume fraction) was added to the process was performed by pumping the sample
flasks for desorption. The flasks were shaken by using solution through the pretreated glass column at a rate
a shaking incubator with a speed of 150 r/min at 25 ℃ of 2 BV/h. After the adsorption equilibration, the
for 4 h. After the adsorption and desorption equilibrium, adsorbed column was washed with distilled water and
the solutions were filtered and the corresponding subsequently desorbed with gradient ethanol-water
adsorption and desorption ratios of each resin were solutions at a rate of 2 BV/h. The FMTs concentrations
calculated using the equations below [18] : in each eluent were analysed using HPLC. Then, the
ρ  ρ eluent was concentrated to dryness under vacuum to
Adsorption ratio: /%  A 0 e  100 calculate the product purity.
ρ 0
ρ V Several variables were studied, including the initial
Desorption ratio: D /%  d d  100 concentration of the sample solution, feeding volume
( 0  e )ρ  ρ V i and different proportions of ethanol-aqueous solutions
Where,  0 : the initial concentration of total for the desorption.
flavonoids in solution (g/L);  e : the equilibrium 2.8 FTIR analysis and surface morphology
concentration of total flavonoids in solution (g/L);  d : characterization of FMTs
the equilibrium concentration of total flavonoids in the The infrared spectrum of FMTs was recorded on a
desorption solution (g/L); V d : the volume of the Fourier Transform Infrared (FTIR) spectrometer
desorption solution (mL); and V i : the volume of the (VERTEX 70, Bruker, Germany) using KBr pellets in
–1
solution in the flasks after adsorption (mL). the infrared region of 4000~400 cm at a resolution of
–1
2.6 Static adsorption isotherms 4 cm .
To determine the absorption isotherms of AB-8, five 2.9 Anti-inflammatory activity
groups of flasks containing 1 g of resin were prepared 2.9.1 Cell Culture and Viability Assay (MTT)
as described above. Then, 50 mL of the aqueous RAW 264.7 macrophages were cultured at 37 ℃ in
extract solutions at different initial FMTs (0.085, a humidified atmosphere containing 5% CO 2 in RPMI-
0.256, 0.507, 1.247, and 2.483 g/L) was added to the 1640 supplemented with 10% heat-inactivated FBS,
flasks. Thermostatic oscillation was then conducted at penicillin G (100 units/mL), and streptomycin (100 g/L).
three different temperatures, 25℃, 35℃, and 45℃, for The cytotoxicity of FMTs was evaluated with a
4 h. After the adsorption, FMTs were measured and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
the adsorption capacity of each resin was calculated bromide (MTT) assay. RAW 264.7 macrophages were
according to the following formula: seeded into 96-well plates, treated with various
(  )ρ  ρ V concentrations (50, 100, 200, and 400 mg/L) of FMTs,
Adsorption capacity:Q  0 e 0 and incubated for 24 h. Afterward, 2.5 g/L MTT was
e
W
ρ V added to each well, and the cells were further
Desorption capacity:Q  d d incubated for 4 h. The supernatant was removed, and
d
W
Where, V 0 : the initial volume of the solution added DMSO (150 µL) was added to dissolve the formazan
to the flask (ml); W: the weight of the dried resin in crystals formed in the viable cells. The absorbance of
each flask (g). each well was determined at 490 nm by using a
microplate reader.
The Langmuir and Freundlich models were used to
evaluate the adsorption behaviour between the adsorbate 2.9.2 Cytokine measurements (NO, IL-6, IL-1β, and
TNF-α)
and the adsorbent [19] . The anti-inflammatory effects of FMTs were assessed,
Langmuir isotherms:
Q  K ρ by measuring the levels of nitric oxide (NO),
Q e  1 m L  K ρ e interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumour

Freundlich isotherms: L e necrosis factor-α (TNF-α) were measured. The nitrite
concentration in the medium was measured as an
1 indicator of NO production by the Griess reaction.
 Q  K ρ n
e F e Briefly, 100 μL of each supernatant was mixed with
In addition, the adsorption properties of the selected the same volume of the Griess reagent, and the
resin were evaluated at different initial pH values absorbance of the mixture at 525 nm was determined
(2.0~6.0) of the sample solutions. with an ELISA plate reader. RAW 264.7 macrophages
2.7 Dynamic adsorption and desorption tests were cultured in 24-well plates, preincubated for 12 h
The separation properties of the selected resin were with various concentrations of FMTs (50~400 mg/L),
evaluated using dynamic adsorption and desorption and then treated for 18 h with LPS (1 mg/L). The
tests. First, 35 g (wet weight) of AB-8 resin was culture medium was collected, and the concentrations