Preparation and activity evaluation of chrysin-β-d-galactopyranoside

Article abstract of DOI:10.1007/s12272-016-0800-2

Chrysin-β-d-galactopyranoside was efficiently synthesized, evaluated for its inhibitory activities against H22 cell lines compared with chrysin, the scavenging of hydroxyl radical, DPPH radical and superoxide anion, inhibitory effect against bacteria and

Full text of DOI:10.1007/s12272-016-0800-2

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0800-2
RESEARCH ARTICLE
Preparation and activity evaluation of chrysin-b-D-
galactopyranoside
Zhen-Yuan Zhu1 Ling Chen1 Fei Liu1 Li-Jing Chen2 Meng Meng1
•
•
•
•
•
Hui-Qing Sun1 Yong-Min Zhang3
•
Received: 1 April 2016 / Accepted: 20 July 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract Chrysin-b-D-galactopyranoside was efficiently
synthesized, evaluated for its inhibitory activities against
H22 cell lines compared with chrysin, the scavenging of
hydroxyl radical, DPPH radical and superoxide anion,
inhibitory effect against bacteria and fungi. The structures
of all compounds were fully characterized by spectroscopic
data (NMR, MS). The anti-tumor, antioxidant and antimi-
crobial activities of chrysin-b-D-galactopyranoside were
proved to be enhanced significantly compared with chrysin.
and antimicrobial agents has turned to natural sources in
particular plants used in medicines now.
Chrysin namely 5,7-dihydroxyflavone, a form of flavo-
noids is widely found in honey and plants. The anti-in-
flammatory, anti-bacterial, anti-oxidation, anti-anxiety,
anti-strain, anti-diabetic, anti-hypertensive, anti-viral, anti-
tumor and a variety of pharmacological effects such as
vasodilation of chrysin have been reported in some studies.
These studies also proved that it have strong chemical
activity and little toxicity. Its structural can be easily
modified (Mentzer et al. 1953).
Keywords Chrysin Á Glycosylation Á Derivative Á
Antitumor activity Á Antioxidant activity Á Antimicrobial
activity
Although many common chrysin have effective physi-
ological function and little side effects, some research and
clinical application of chrysin were limited by its low water
solubility and low bio-availability. Therefore, it is of great
importance to modify the structure of dialogue salicin
chemically to enhance its water solubility and biological
activity. Thus, to improve the targeting in vivo effect of
genistein (Zheng et al. 2011; Mentzer et al. 1953).
Introduction
Despite the large number of anti-tumor drugs available for
medical use, the treatment of cancer remains a challenging
therapeutic problem (Zhu et al. 2014), and the emergence
of antimicrobial strains constitunes a substantial need
(Zheng et al. 2011). Thus, the search for novel anti-tumor
Results and discussion
Chemistry
& Zhen-Yuan Zhu
zhyuanzhu@tust.edu.cn
In the previous study, the chrysin-7-O-glucoside had been
effectively synthesized by a reaction of chrysin and Acetyl
bromgalactoside in 1999 (Alluis and Dangles 1999). In this
experiment, benzoyl group was chosen as a protecting
group of sugar ring hydroxyl group to form the compound
a under the reaction of pyridine and benzoyl chloride (Park
1
Key Laboratory of Food Nutrition and Safety, Ministry of
Education, College of Food Science and Biotechnology,
Tianjin University of Science and Technology,
Tianjin 300457, People’s Republic of China
2
Key Laboratory of Freshwater Fishery Germplasm
Resources, Ministry of Agriculture, Shanghai Ocean
University, Shanghai 200090, People’s Republic of China
¨
et al. 2005; Lonn 1985), and then hydrogen bromide was
used as a hydroxyl activating group to obtain compound b a
kind of glycosyl donor with high stereoselectivity. The
yield of benzoyl-b-D-galacopyranoside and 1-Br-benzoyl-
3
Universitq Pierre et Marie Curie-Paris 6, Institut Parisien de
Chimie Molqculaire UMR CNRS 8232, 4 place Jussieu,
75005 Paris, France
123

Z.-Y. Zhu et al.
b-D-galacopyranoside could both reach above 85 %. 1-Br-
benzoyl-b-D-galacopyranoside and chrysin were then dis-
solved in the K2CO3 and acetone solution to conduct the
following reaction. The yield of compound c obtained by
the glycosylation reaction is about 70.6 %. Followed by the
synthesis of compound d which final yield was about
52.5 % by reaction with sodium methoxide to remove the
protection group of the compound c in a solution consisted
of anhydrous dichloromethane and methanol (v:v = 1:1).
The stereochemistry of the newly introduced linkage was
determined to be b on the basis of the Gal H-1, H-2 cou-
pling constant (J1,2 = 7.6 Hz) (Bregant et al. 1999). The
specific reaction process was shown in Fig. 1. All the
synthesized compounds were characterized by IR, 1H-
NMR, 13C-NMR, and elemental analyses.
the MTT essay. As it could be clearly seen from the
Fig. 2a, the anti-tumor effect towards H22 tumor cell
decreased with the decreasing of concentration. The tumor
inhibition rate of chrysin-b-D-galactopyranosideanoside at
the concentration of 2 lmol/mL remained the highest
through out the period. To be specific, this figure reached
44.95, 69.21 and 79.34 % at the 24th, 48th and 72th h
respectively. This result was in consistence with or even
much more superior than the some past researches in
regarding to the anti-tumor activity(Tran et al. 2012).
Surprisingly, beneficial effect toward the growth of H22
cells was observed after 24 h of cultivation when the
concentration of chrysin-b-D-galactopyranosideanoside
was below 0.25 lmol/mL. The reason for this phenomenon
was yet to be determined. In comparing to chrysin-b-D-
galactopyranosideanoside, chrysin has exhibited with a
stronger anti-tumor ability under the concentration of
2 lmol/mL during throughout the period.
The ability of the new derivative such as anti-tumor,
antioxidant and antimicrobial were evaluated by the
method mentioned in MTT assay, Fenton-salicylic acid
method and bacteriostatic circle measurement method
respectively.
However, the ability is weaker than chrysin-b-D-galac-
topyranosideanoside at other concentrations. This fact was
the most obvious under the concentration of 2 lmol/mL, at
which point, its inhibition rate was almost 20 % less than
that of chrysin-b-D-galactopyranosideanoside. This phe-
nomenon might be a result of the coexisted function group
galactopyranoside depended its anti-tumor effect heavily
Anti-tumor activity
The anti-tumor activity, inhibition of H22 tumor cells of
chrysin-galactopyranoside and chrysin was evaluated by
OH
OBz
O
HO
HO
BzO
BzO
BzO
BzO
OBz
O
BzCl,Py
~Rt, 12h
HBr,DCM
Rt,6h
O
OH
OBz
0
°C
OH
Br
OBz
OBz
2'
OBz
BzO
BzO
OBz
O
3'
4'
1'
BzO
6''
O
HO
1
O
8
5''
2
4‘’
O
9
O
2''
K2CO3
6'
+
BzO
7
5'
3''
OBz1''
OBz
acetone
Br
4
3
6
10
OH
O
5
OH
O
2'
OH
3'
1'
HO
6''
5''
1
8
O
2''
NaOCH3
MeOH
2
4‘’
O
4'
9
O
6'
HO
7
5'
3''
1''
OH
4
3
6
10
5
OH
O
Fig. 1 Synthetic route of Chrysin-b-D-galactopyranoside
123

Preparation and activity evaluation of chrysin-b-D-galactopyranoside
Fig. 3 Scavenging ability of compounds on O2- redica, chrysin and
chrysin–galactopyranoside
The DPPHÁ scavenging rate of chrysin and chrysin-
galactosidase were illustrated in Fig. 4. As can be seen
from Fig. 4, the DPPHÁ scavenging rate of chrysin and
glucoside was similar without the treatment of UV irradi-
ation at a little below 30 %. This figure for chrysin was
significantly declined with prolonged UV exposure time to
only around 10 % after 7 h UV irradiation. Unlike chrysin,
the figure for glucoside remained relatively stable in the
first 5 h. It was at the fifth hour that the greatest difference
was identified in the DPPHÁ scavenging rate. At this
moment, the figure for glucose was around 23 %, only 7 %
less than without UV irradiation treatment almost twice as
that of chrysin. Surprisingly, at the 7th h, a significant
decline was observed in the figure for glucoside, to around
14 %, while the figure for glucoside stabled at 10 % still
about 5 %, less than that of glucoside.
Fig. 2 Inhibition of H22 cells incubated with for 24, 48, 72 h
a Chrysin-b-D-galactopyranosideanoside b Chrysin
on the concentration. In conclusion, the result showed that
the inhibition effect is strongly dosage dependent, and that,
this effect generally increased with the passing of time.
Since H22 cells belongs to mouse, further research is
required to confirm its effect toward human cells.
Similarly, the hydroxyl radical scavenging rate of
chrysin and chrysin-galactosidase were shown in Fig. 5. As
it could be seen from Fig. 5 the hydroxyl radical
Antioxidant activity
The O2-Á scavenge effect of chrysin and chrysin-b-D-
galactopyranosideanoside were shown in Fig. 3. From
which it could be seen that oxygen free radical scavenging
ability of chrysin-b-D-galactopyranosideanoside obtained
from glycosylation was superior to that of chrysin. Without
the radiation of the UV light, the superoxide anion scav-
enging rate of chrysin-b-D-galactopyranosideanoside was
32.79 %, about 15 % superior to that of chrysin. After 1 h
UV irradiation, growth was shown in the scavenging rate of
glucoside. The figure for glucoside reached almost as high
as 60 % almost the twice as the figure for chrysin. After
this point, decline in oxygen free radical scavenging rate
were observed both in chrysin and chrysin-b-D-galactopy-
ranosideanoside with the increasing period of exposure to
UV light.
Fig. 4 Scavenging ability of compounds on DPPHÁ redical, chrysin
and chrysin–galactopyranoside
123

Z.-Y. Zhu et al.
inhibitory effect on food spoilage bacteria like Escherichia
coli, Bacillus subtilis, Staphylococcus aureus and Sal-
monella, and the antibacterial effect of chrysin-b-D-galac-
topyranosideanoside was weaker compared with chrysin.
The diameter of antibacterial zone of chrysin was 6.18,
6.12, 6.09 and 6.11 mm, respectively. While the diameter
of antibacterial zone of chrysin-b-D-galactopyra-
nosideanoside was 6.05, 6.02, 6 and 6.03 mm, respectively.
In addition, the diameter of control group was 6 mm.
Similarly, the drugs with minimum inhibitory concen-
tration(MIC) which is more than 64 mg/mL was used in
the testing of their inhibitory effect against fungus, basi-
cally no inhibitory effects on Rhizopus, Penicillium and
Aspergillus in the two samples, but chrysin-b-D-galac-
topyranosideanoside had an obvious inhibitory effect
on mucor, which chrysin did not have. As it could be seen
from Table 1, the diameters of inhibition zone of chrysin
and the complex were 9.75, 6.37 mm. However, the
diameters of inhibition zone of these two samples on
Rhizopus, Penicillium and Aspergillus were 6 mm, which
indicated they didn’t change at all.
Fig. 5 Scavenging ability of compounds on OHÁ redical, chrysin and
chrysin-b-D-galactopyranosid
scavenging ability of both chrysin and glucoside were both
strong without UV radiation, which clearance rate was
around 35 and 40 %, respectively. The figure for the two
both gradually declined with prolonged UV exposure in the
first 3 h. During this period, the figure for glucoside
remained about 5-8 percent superior than that of chrysin.
Surprisingly, at the 5th h, with the dramatic decline of this
figure for glucoside and a slightly increase of this figure for
chrysin, the figure for the latter slightly surpassed the for-
mer at around 24 %. Afterward, the figure for glucoside
retrieved its dominant position at round 20 % with the
dramatic decline of the figure for chrysin to around 13 % at
the end of this test. It was obvious that during the whole
process, the hydroxyl radical scavenging ability of glu-
coside was generally superior to that of chrysin.
In conclusion, both compounds were inactive against
tested bacterial species except mucor that showed greatest
inhibition with a inhibitory diameter of 9.75. It suggested
that the introducing galactopyranoside to the chrysin could
not increase the antimicrobial activity of most food spoi-
lage bacteria and fungus, and that the two might exert their
antimicrobial activity through multiple mechanisms or
different pathways (Fig. 6).
Conclusion
Antimicrobial activity
One derivatives of chrysin named chrysin-b-D-galactopy-
ranosideanoside was synthesized and tested for several of
its bio-activities such as antioxidant, anti-tumor and
antimicrobial activity. The antioxidant ability decreased
with the increasing of irradiation period, however. This
trend was less obvious in the modified product which
antioxidant effect was always superior to that of chrysin. It
might be a result of the introduced group with certain
ability to improve the antioxidant effect worked as a pro-
tection group against the UV treatment. In regarding to the
anti-tumor effect, they both showed excellent anti-tumor
ability against H22 cell at certain concentrations. The
modified chemical showed a stronger dosage dependent
effect in this activity. The difference in their anti-tumor
was most obviously observed at the concentration of 2 mg/
mL. Finally, although both the two chemicals showed little
inhibition effect toward most food spoilage bacteria and
fungus, chrysin-b-D-galactopyranosideanoside showed
excellent inhibition effect toward mucor which a inhibitory
diameter of 9.75. This might due to the fact that the
In-vitro anti-microbial activity was evaluated using the
minimum inhibitory concentration (MIC) with different
strains. Table 1 showed that chrysin and chrysin-b-D-
galactopyranoside with MIC value[64 mg/mL had a little
Table 1 The antimicrobial effect of the two chemicals based on the
diameter of antimicrobial zone (mm)
Tested microorganism
Chrysin-b-D-
galactopyranosideanoside
Chrysin
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Salmonella Typhimurium
Mucor
6.18
6.12
6.09
6.11
9.75
6
6.05
6.02
6
6.03
6.37
6
Rhizopus
Penicillium
6
6
Aspergillus
6
6
123

Preparation and activity evaluation of chrysin-b-D-galactopyranoside
Fig. 6 The antimicrobial effect
of chrysin-b-D-
galactopyranosideanoside.
a common spoilage bacteria
b common fungi
introducing of galactopyranoside changed its mechanisms
or different pathways relating to the antimicrobial activity.
Further research is required to gain more knowledge about
these switch of pathways and more compounds need to be
designed and synthesized for further investigation using
chrysin as the lead compound.
After the reaction was indicated completed by TLC, the
reactant was diluted by CH2Cl2. By which means, the
organic phase was extracted. Afterward, it was washed
with 1 mol/L HCl solution and saturated NaHCO3 solution
for several times before it was washed again by saturated
NaCl solution. Then, the organic phase was dried by Na2-
SO4 and was evaporated under reduced pressure. Finally,
the purified product purified by silica gel column chro-
matography (Petroleum ether: ethyl acetate = 3:1) to fur-
nish the compound a (1.656 g, 90.5 %).
Materials and methods
Chemistry
Preparation of the 1-Br-benzoyl-b-D-
galacopyranoside (b)
Mass spectra were measured on a MS-700(GERMANY).
IR spectra were recorded(in KBr) on a FTIR1730. 1H-
NMR spectra were measured on a Bruker DRX400 spec-
trometer with TMS as the internal standard. Optical rota-
tion was measured on a Perkin Elmer 241 polarimeter type
at a condition of room temperature (25 2 °C), length
10 cm and volume 10 mL. CS Chemdraw ProTM com-
puter software was used to calculate the theoretical value of
the molecular weight, mass spectrometry and molecular
structural formula diagram draw synthetic compounds.
Reagents were at analytical grade and commercially
available (individual reagents and solvents were treated
before use). Reaction courses were monitored by TLC on
silica gel-precoated F254 Merck plates.
1.656 g of benzoyl-b-D-galacopyranoside obtained from
the previous step was dissolved in 10 mL dichloromethane
dried with specific methods, then 10 mg 4A molecular
sieves was added before it was stirred until the temperature
was cooled to 0 °C. Afterward, 6 mL 47 % HBr-acetic acid
was added to the solution slowly to react for 6 h at room
temperature. The reaction was quenched by cold saturated
NaHCO3 solution when the result of TLC indicated the
reaction was completed. The product was successively
extracted by adding a certain amount of dried CH2Cl2.
Then, the organic phase was dried by anhydrous sodium
sulfate. Eventually, it was concentrated and purified by
silica gel column chromatography (Cyclohexane: ethyl
acetate = 3:1) to furnish the compound b (1.43 g, 86.7 %).
1H-NMR (400 MHz, CDCl3):d 8.05-7.14 (m, 25H, H
arom); 6.90 (d, 1H, J = 3.9 Hz, H–l); 6.14 (d, 1H,
J = 3.1 Hz, J \ 1 Hz, H-4); 6.08 (dd, 1H, J = 10.3 Hz,
J = 3.1 Hz, H-3); 5.69 (dd, 1H, J = 10.3 Hz, J = 4.0 Hz,
H-2); 4.94 (m, 1H, H-5); 4.66 (dd, 1H, J, = 11.5 Hz,
Preparation of the benzoyl-b-D-galacopyranoside (a)
1.8 g b-D-Galactose was dissolved in pyridine 10 mL, then
cooled at 0 °C for 20 min before 6.25 mL benzoyl chloride
was added in slowly. Then, it was stirred in ice-water for
30 min followed by 12 h reaction at room temperature.
123

Z.-Y. Zhu et al.
J = 6.8 Hz, H-6); 4.48 (dd, 1H, J = 11.5 Hz, J = 6.0 Hz,
H-60).13C-NMR (100.6 MHz, CDCl3):d165.86, 165.50,
165.30, 165.26 (C=O); 133.76-128,30 (C arom); 88.41 (C-
1); 71.89 (C-5); 68.96 (C-3); 68.63 (C-2); 68.14 (C-4);
61.72 (C-6,60).
161.6 (C-5), 157.6 (C-9), 132.6-126.9 (C-10 *C-60), 106.0
(C-10), 105.9 (C-3), 100.9 (C-100), 95.3 (C-6, C-8), 76.2 (C-
500), 73.6 (C-400), 70.53 (C-200), 68.5 (C-300), 60.75 (C-600);
HRMS calcd for [M?H]? C21H20O9H: 419.1264, found
419.1285.
Preparation of the chrysin -b-D-2,3,4,6-4-O- benzoyl-
galactopyranoside (c)
Anti-tumor activity
Research on the inhibition toward H22 tumor cells chrysin
glucosidase was conducted in this experiment by MTT
method (Tran et al. 2012). H22 tumor cells was obtained
from Tianjin University of Science and Technology Lab-
oratory of functional foods. Firstly, chrysin-b-D-galac-
topyranosideanoside was weighed accurately, then 0.25,
0.5, 1.0, 1.5, 2 lmol/mL sample solution were diluted by
2 lmol/mL of concentrated 1640 cell culture medium after
filter the original sample through 0.2 lm membrane to
remove microorganisms.
100 lL tumor cells of 1 9 105 cell/mL configuration at
the logarithmic growth phase were added into every hole of
the counting board, followed by 100 lL of sample solution
at different concentrations, each concentration were repe-
ated three times. Then cultured for 20, 44, 72 h before
being centrifuged at 1000 r/min for 6 min, the supernatant
was decanted. 100 lL of MTT standard fluid was then
added in the remained precipitation and cultured for
another 4 h. Afterward, the culture was terminated by
centrifugation at 1000 r/min for 10 min. The suspension of
each hole was carefully cleaned before, the intracellular
crystals was dissolved by 150 lL dimethylsulfoxide
(DMSO) under oscillation. Finally, the OD value was
measured by using a Multi-Mode Microplate Reader at
570 nm (Sargent 2003). Control group was set by the cell
group without sample solution, while the blank group was
set by 150 lL of the DMSO solution. Samples inhibit
tumor cell inhibition rate calculation formula (1):
Chrysin (0.4 mmol), anhydrous potassium carbonate
(4 mmol) and acetone (4 mL) were added in a 50 mL
round bottom flask and stirred together. After it was fully
dissolved, another 0.6 mmol 1-Br-benzoyl-b-D-galacopy-
ranoside was dissolved in 2 mL acetone solution slowly.
The mixture was then stirred again for 4 h at 40 °C. The
organic layer was successively concentrated under reduced
pressure to furnish a yellow syrup, which was purified by
column chromatography (EtOAc-petroleum ether = 1:3)
to furnish compound c (Yellow powder, 70.6 %). [a]D
?75°(c = 0.1, CH2Cl2); IR: vmax cm-1 3435(O–H), 1617
(C=O), 1658(C=C), 1266 (Ar–O–C), 1141–1083 (C=C–O–
1
C=C); H-NMR (400 MHz, CDCl3, ppm):12.74 (s, 1H, –
OH), 8.15–7.28 (m, 25H, ArH); 6.69 (s, 1H, H-8); 6.67 (d,
J = 2.2 Hz); 6.62 (d, J = 2.4 Hz); 6.14 (d, J = 7.2 Hz,
1H, H-100); 5.76 (dd, J = 3.6 Hz, 1H, H-6); 5.74 (dd,
J = 3.6 Hz, 1H, H-600); 5.58(dd, J = 7.6 Hz, 1H, H-200);
4.67 (dd, J = 2.8 Hz, 1H, H-600); 4.64 (s, 1H); 4.62(t, 1H,
H-500); 13C NMR (400 MHz, CDCl3-d6, ppm):182.1 (C-4),
166.1, 165.5, 165.4, 165.1 (C=O, Bz)164.4 (C-7); 162.4
(C-2), 162.2 (C-5), 157.4 (C-9), 133.7–126.3 (CH, Ar),
107.1 (C-3); 106.0 (C-10); 100.1 (C-100Glc); 98.8 (C-6);
95.5 (C-8); 72.2 (C-500); 71.4 (C-400), 69.2 (C-200); 67.9 (C-
300); 62.4 (C-600); HRMS calcd for [M?H]? C49H36O13H:
835.2312, found 835.2328.
Preparation of the Chrysin-b-D-
Galactopyranoside(d)
Inhibition percent ð%Þ
Control group ðcpmÞ À Experimental group ðcpmÞ
¼
Compound c (0.6060 g, 0.7 mmol) was dissolved in
MeOH-CH2Cl2 (V:V = 1:1, 10 mL), followed by adding
in 21.5 mg CH3ONa. After stirring at 25 °C for 5 h, the
solution was neutralized by ion-exchange resin (H?), and
then filtered and concentrated. The yellow residue was then
purified by column chromatography (CH2Cl2–MeOH =
8:1) to furnish d (yellow solid, yield 74.4 %). [a]D25–15°
(c = 0.1, CH2Cl2). IR: vmax cm-1 3401(O–H), 1616(C=O),
1712(C=C), 1266(Ar–O–C), 1141–1083(C=C–O–C=C);
1H-NMR (400 MHz, DMSO-d6, ppm); 12.82(s, 1H, –OH),
8.11–7.58 (m, 5H, ArH), 7.05(s, 1H, H-8), 6.87 (s, 1H, H-3),
6.48(d, J = 2.4 Hz, H-6), 5.05(d, J = 7.6 Hz, 1H, H-100),
3.73–3.33 (m, 5H, H-200 *60’). 13CNMR (400 MHz,
DMSO-d6, ppm):182.6 (C-4), 164.1 (C-7), 163.8 (C-2),
Control group (cpm)
 100
ð1Þ
Antioxidant activity
Pyrogallol autoxidation was used in this experiment, the
specific steps were mentioned as follows: placed 50 mmol/
L, pH = 8.2 in Tris–HCl buffer 450 lL in 2 mL centrifuge
tube before it was bathed at 25 °C for 20 min. Then
100 lL of 1.0 mg/mL sample processed with different
period of UV irradiation were added into the solution.
50 lL 2.5 mmol/L phthalate solution was successively
added into it before it was water bathed at 25 °C for 5 min.
123

Preparation and activity evaluation of chrysin-b-D-galactopyranoside
Finally, 0.2 mL 8 mol/L HCl solution was added into the
solution to terminate the reaction. Result was carried out by
measuring the absorbance value of at 300 lL samples
which were placed in a 96-well microtiter plates. The
formula shown in Formula (2.1):
poured into dry sterilized dishes and melted after being
autoclaved. Followed by solidification (Cheng et al. 2006)
under room temperature.
Bacteria experiment: added 100 lL bacterial suspension
to 10 mL beef extract peptone medium and uniformed by
air blowing. Then poured it into a layer that has been
covered with plates of beef extract peptone medium.
Afterward, holes with depth of 2 mm was penetrated by a
sterile metal after it was solidified. 100 lL of each sample
gradient concentration solution was added in the holes, a
penicillin was added as a control group. The hole in the
middle of was added 100 lL of sterilized acetone controls,
three parallels were set for each bacteria sample. The plates
were then placed in a incubator at 37 °C. Diameter of the
inhibition zone were measured after 24 h incubation.
Finally different antimicrobial effects were compared by
the diameter.
OHꢀ radical scavenging activity
¼ 1 À ½As=AcŠ  100 %
ð2:1Þ
As: absorbance of the OHÁ solution containing samples.
Ac: absorbance of the control solution without sample but
with OHÁ. The percentages of OHÁ reduced were plotted
against the samples.
The antioxidant activity was performed using the method
described by Amarowicz et. al (2000). Added 500 lL
1.5 9 10-4 mol/L DPPH-ethanol solution and 500 lL
1.0 mg/mL of sample solution irradiated by UV at different
times in 2 mL centrifuge tube. After pipetting until uni-
formed, closed in the water bath 25 °C to react for 60 min,
300 lL of the reaction solution were successively added to
96-well microtiter plates, the measured absorbance values at
517 nm wavelength, was calculated by the formula (2.2):
Mold experiment: 10 mL PDA medium was melted in a
petri dish that has already been covered by a layer of PDA
medium and uniformed by air blowing. After it was
solidified in room temperature, 100 lL of each sample
gradient concentration solution was added in the holes, a
penicillin was added as a positive control group. The hole
in the middle of was added 100 lL of sterilized acetone
controls, three parallels were set for each fungi sample. The
plates were then placed in a incubator at 28 °C. Diameter
of the inhibition zone were measured after 24 h incubation.
Finally different antimicrobial effects were compared by
the diameter.
DPPHꢀ radical scavenging activity ð%Þ
¼ 1 À ½As=AcŠ  100 %
ð2:2Þ
As: absorbance of the DPPH solution containing sam-
ples. Ac: absorbance of the control solution without sample
but with DPPH. The percentages of DPPH reduced were
plotted against the samples.
Hydroxyl radical scavenging activity was measured
using Fentons reaction method with slight modification
(Bekhit et al. 2001). The reaction mixture generating
hydroxyl radicals contained 0.1 mL of H2O2 (60 mmol/L),
0.1 ml of FeSO4 (9 mmol/L), 0.1 ml of salicylic acid
ethanolic solution (9 mmol/L) and 2.7 ml of the polysac-
charides of varying concentrations. Distilled water was
used as the blank control. After being incubated at 37 °C
for 30 min, scavenging rate (%) was calculated using the
formula (2.3). 300 lL of the reaction solution were suc-
cessively added to 96-well microtiter plates, the measured
absorbance values at 510 nm wavelength.
An experiment of detecting the minimum inhibitory
concentration of the compounds against the susceptible
micro-organisms in the preliminary test (Gram-positive
bacteria and Gram-negative bacteria). A twofold serial
dilution technique (Bekhit et al. 2001) was followed to
determine the minimum inhibitory concentration (MIC) of
the compounds against. Test compounds dissolved in water
were added to culture media (Brain Heart Infusion for S.
¨
Mutans and Muller-Hinton agar for other bacteria) to
obtain final concentrations of 64–0.5 mg/mL. The final
bacterial amount was determined to be 105 CFU/mL. MIC
values were read after incubation at 37 °C for 20 h. The
lowest concentration of the test substance that completely
inhibited growth of the micro-organism was recorded as
the MIC (expressed in mg/mL). It was follow the similar
process that the minimum inhibitory concentration against
fungus was detected. All the experiments were repeated for
3 times.
Radical scavenging activity ð%Þ
¼ 1 À ½As=AcŠ  100 %
ð2:3Þ
As: absorbance of the sample containing solution con-
taining samples. Ac: absorbance of the control solution
without sample. The percentages of H2O2 reduced were
plotted against the samples.
Acknowledgments This work was financially supported by the
National Spark Key Program of China (2015GA610001), the Foun-
dation of Tianjin University of Science and Technology (Nos.
20120106), the International Science and Technology Cooperation
Program of China (2013DFA31160), and the Foundation of Tianjin
Educational Committee (20090604).
Antimicrobial activity
Prepared a 6 mm diameter metal hole punch by heat ster-
ilization at 120 °C for 20 min, the 10 g solid medium was
123

Z.-Y. Zhu et al.
Mentzer C, Ferrando R, Bost J, Pillon D, Garnier J (1953) Preparation
and pharmacodynamic action of some flavones. Bulletin De La
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