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of absolute ethyl alcohol. A saturated solution of Mn(CH3COO)2
was added drop wise to the resulting solution (in the molar ratio
of luteolin : Mn2+ ¼ 2 : 1) under continuous stirring. The pH of
the solution was adjusted to 4 with 1 mmol Lꢂ1 of HCl. Aer
complete reaction, the solid was precipitated and obtained
through ltration. The solid was washed several times with
absolute ethyl alcohol and dried in desiccators over anhydrous
MgSO4. The luteolin–manganese(II) complex powder was nally
obtained. The proposed synthetic path of the co-ordination
complex was illustrated in Fig. 1.
2.6.2. Analysis of hydroxyl radical scavenging activity. The
hydroxyl radical scavenging activity of the luteolin and the
luteolin–manganese(II) complex was determined as described in
a previous literature.23
2.7. Evaluation of antimicrobial activity
Antimicrobial activities of the luteolin and the luteolin–man-
ganese(II) complex were assessed using four kinds of bacteria
including Escherichia coli, Staphylococcus aureus, Listeria mono-
cytogenes and Pseudomonas aeruginosa. These bacteria stored at
ꢂ80 ꢀC were inoculated to the LB agar culture-medium and then
incubated in a constant temperature incubator at 37 ꢀC for 18–
24 h. The bacterial suspensions were prepared via the incubated
bacteria and subsequently stored in a refrigerator at 0–4 ꢀC.
Aerwards, 0.2 mL of bacterial suspension for each bacterium
was transferred to a new LB agar culture-medium and the
susceptibility paper immersed in sample solution for 12 h was
posted on the medium. Three replicates were performed for
each bacterium. Penicillin and the sterile water were used as the
positive and negative controls, respectively. All the pre-prepared
mediums were nally incubated in a constant temperature
incubator at 37 ꢀC for 18–24 h and the inhibiting ring was
measured by a vernier caliper. Each sample was calculated
a minimum of three times, and the mean value was calculated
and reported.
2.4. Physical measurements
UV-vis spectra of 0.2 mg mLꢂ1 solutions of the luteolin and the
luteolin–manganese(II) complex were obtained in deionized
water by a UV-vis double beam spectrophotometer (UV-1601,
Shimadzu, Kyoto, Japan) using standard 1.00 cm quartz cells.
IR spectra were recorded with a FT-IR (Vertex 70, Bruker,
Rheinstetten, Germany) spectrometer in the range of 500–
4000 cmꢂ1. Briey, the luteolin and the luteolin-manganese(II)
complex dried completely were thoroughly mixed with KBr
powder, ground and pressed into a 1 mm pellet, then the
prepared samples were determined by FT-IR spectrometer. The
TG–DSC analysis measurement of the luteolin–manganese(II)
complex under the N2 atmosphere (ow rate: 10 mL minꢂ1) was
performed by a thermal analyzer (STA 449 F3 Jupiter®, Netzsch,
ꢀ
Bavaria, Germany), between the temperatures of 25–800 C at
ꢂ1
ꢀ
a heating rate of 10 C min
.
2.8. Measurement of the inhibition rate of luteolin and
luteolin–manganese(II) complex on the xanthine oxidase
(XOD)
2.5. Elemental analysis
A scanning electron microscope (Nano SEM430, Nova™, Hills- It has been reported that xanthine can be catalyzed by xanthine
boro, Oregon, USA) equipped with an energy dispersive spec- oxidase, resulting in the production of uric acid which has
troscopy (EDS, Oxford, UK) was used to perform the elemental a characteristic absorption peak at 290 nm.24 So the enzyme
analysis of the luteolin–manganese(II) complex.
activity in the present work was measured spectrophotometri-
cally by continuously measuring uric acid formation at wave-
length of 290 nm with xanthine as the substrate.25 Briey,
a series of assay solutions were prepared by dissolving 2 mL of
xanthine substrate (xed concentration: 0.6 mmol Lꢂ1) and
0.2 mL of various concentrations of luteolin or luteolin–man-
ganese(II) complex in 2.8 mL of sodium phosphate buffer
(0.2 mol Lꢂ1, pH 7.5). These assay solutions were shaken
vigorously and then incubated for 30 min in a 25 ꢀC water-bath.
Then the assay was initiated by adding 0.2 mL of xanthine
oxidase solution (0.5 U mLꢂ1). The absorbance at 290 nm was
measured every 30 s. The enzymatic activity was nally calcu-
lated according to the equation reported by a previous
literature.26
In the reaction system, the concentration of xanthine
substrate was xed at 0.6 mmol Lꢂ1. The effects of different
concentrations of luteolin or luteolin–manganese(II) complex
on the enzymatic activity were determined by changing the
concentrations of XOD. The linear equations were then ob-
tained by plotting the reaction velocities (y) and XOD concen-
trations (x) in the enzymatic reaction. A linear equation through
the origin indicates the reversible inhibition, while a straight
line which is parallel with the X axis accounts for the irreversible
inhibition. To describe the competitive inhibition mechanism,
2.6. Measurements of antioxidant activity
2.6.1. Analysis of DPPH radical scavenging activity. The
DPPH radical scavenging activity was measured according to
the method of Dehghan & Khoshkam (2012) with some modi-
cations.14 Briey, the luteolin and the luteolin–manganese(II)
complex were rstly dissolved in dimethyl sulfoxide (DMSO) to
obtain different concentrations (0.2–5.0 mg mLꢂ1). Then 1 mL
of sample solution was added to 2 mL of DPPH solution
(1 mmol Lꢂ1 in methanol). The reaction mixture was shaken
vigorously and then incubated in the dark at room temperature
for 30 min. Finally, the absorbance of the resulting solution was
measured at 517 nm (Ai) by the UV-vis double beam spectro-
photometer (UV-1601, Shimadzu, Kyoto, Japan). As a control,
the absorbance of blank solution of DPPH was also determined
at 517 nm (A0). The DPPH radical scavenging activity was
calculated according to the following equation:
!
A0 ꢂ Ai
DPPH radical scavenging activity ð%Þ ¼
ꢁ 100%
A0
(1)
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 53385–53395 | 53387
Dong, Hao
