Engineering, 2013, 5, 455-458
http://dx.doi.org/10.4236/eng.2013.510B093 Published Online October 2013 (http://www.scirp.org/journal/eng)
Oxidization Resistance in Vivo for Raspberry Flavone
Jinxu Sun1,2, Huixia Zhu1,2, Guangxiao Dong1*
1Key Laboratory of Industrial Microbiology, Ministry of Education,
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China
2Department of Biology, Hengshui College, Hengshui, China
Received 2013
ABSTRACT
The experiments on the effect of oxidization resistance in vivo for raspberry flavone extract manifests that the extract of
raspberry flavone could effectively increase CAT, SOD and GSH-Px enzyme activity in rats’ liver, kidney, blood and
skeletal muscle and decrease decomposition product of lipid peroxidization, with a significant antioxidant effe ct.
Keywords: Raspberry; Flavone; Rats
1. Introduction
Flavonoid compound extensively exist in plants’ fructi-
fication, stems, flowers and leaves, possessing a great
many kinds of biological activities. Domestic and over-
seas scholars have studied and proved that flavonoid
compounds are anti-neoplastic, antibacterial, anti free ra-
dical and anti-virus. In recent years, there are related re-
ports on the studies on flavone oxidization resistance in
plants, but few on raspberry flavone oxidization resis-
tance [1,2].
2. Materials and Method
The first extract of raspberry flavone
Weigh and take 100 g comminuted raspberry (first
grade, bought from the pharmacy of Heng shui City
Hospital of Chinese medicine. It was comminuted and
sieved with 60 mesh and ready for use after drying at
50˚C) to put into triangular flask, and adding into 1 L 95%
alcohol, which was conducted extract at 50˚C with 36h
of extraction. During extraction, sonic extract was done
at the 12th and 24th hour, with 300W of ultrasonic power
and 25min of ultrasonic time. When the extract was at
50˚C (Ultrasonoscope, JL-60DTH Shanghai Tianpu Ana-
lytical Instrument Co., Ltd.), jolt it constantly. After ex-
traction, vacuum filtrate and vacuum freeze-dry to get the
powder of crude extraction.
The second extract of raspberry flavone
The crude extraction liquid of rapberry flavone
achieved in last step (rotated membrane evaporimeter,
RE201D Shanghai Bocai Instrument Co., Ltd.) was va-
cuum rotated and evaporated to gain paste-like solids,
which were dissolved by ultrasound after adding into pu-
rified water. Extract for three times by petroleum ether,
and the upper aqueous layer was taken to be extracted for
three times by ethyl acetate, and yellow powder was got
after vacuum rotation and evaporation.
The purified products of raspberry flavone
The powder of raspberry flavone extraction got in
previous step was dissolved by 70% alcohol and purified
by AB-8 rasin column (chemically pure Nanjing Univer-
sity Synthetic Resin Co., Ltd.), and then yellow powders
were gained after vacuum rotation and evaporation.
The grouping and preparation of experimental rats
The rats for experiments (purchased from Chinese
Academy of Medical Sciences Experimental Animal In-
stitute): 3-week old male rats were used whose weights
were between 80 and 100 g.
The grouping and preparation of experimental rats:
randomly divide rats into 5 groups with 7 rats in each
group as high dose raspberry flavone group, low dose
group, positive control group, negative control group and
blank group. Force and feed blank group on normal sa-
line and base without cholesterol every day; force feed
negative control group on normal saline and cholesterol
base every day; force and feed positive control group on
VE and base with cholesterol every day, while the VE
feeding amount was calculated according to 0.0025 g/kg
(body weight); feed high and low dose groups on fodder
with cholesterol and high and low dose of raspberry fla-
vone extraction every day. The raspberry flavone extrac-
tion was calculated based on 400 mg/kg and 100 mg/kg
rat’s weight; the addition amount of cholesterol in base
was 0.5%. After 6 wee ks ’ fe eding, rats we re fast for 12 h,
and 7 rats were randomly taken to conduct experiments.
The fundamental ingredient of base (%): starch: 50;
*Corresponding a uthor.

J. X. SUN ET AL.
456
soybean oil: 5.6; cellulose compound: 3.2; mineral ele-
ment compound: 6.7; mixed vitamin: 1; protein: 23.2; wa-
ter: 9.8.
The collection of rats’ liver and kidney samples
After 12 h’ fasting, the experimental rats which were
fed for 6 weeks were anaesthetized by 4% pentobarbital
sodium, and the dose was based on 40 mg/kg rats’ weight.
Take about 100 mg rats’ skeletal muscle, renal cortex
and liver, and 10 ml blood. Add 0.9% normal saline for
10 ml/g. Grind tissues to get tissue homogenate, which
was centrifuged at 4˚C, 4000 rpm for 15 min, and take
the supernatant to detect CAT, SOD and GSH-Px en-
zyme activity, an d d e tect MDA [3].
The determination of CAT activity
CAT can resolve H2O2, and this reaction can be rapid-
ly terminated by adding ammonium molybdate. The ex-
tra H2O2, together with ammonium, can form a kind of
faint yellow complex compound, and its quantity of gen-
eration can be detected at 405 nm, so as to calculate CAT
activity. CAT kit used in experiments to detect was
A007-1 (Nanjing Jiancheng Bioengineering Institute). 1
umol H2O2 which was resolved by 1 mg tissue protein
was taken as a unit of activity [4], shown by U/ml.
The determination of SOD activity
The determination of SOD activity with the method of
xanthine oxidase Xanthine and xanthine oxidase reaction
system can produce superoxide anion free radical, which
oxidized hydroxylamine to form nitrite that demonstrated
to be purple when affected by chromogenic agent. The
absorbance was detected at 550 nm to calculate SOD
activity which was shown by U/mg protein. SOD kit used
in experiments was A001-1 (Nanjing Jiancheng Bioengi-
neering Institute).
The determination of GSH-Px activity
Glutathione peroxidase can promote H2O2 to react with
reduced type glutathione for generating oxidized gluta-
thione and water. The activity of glutathione peroxidase
can be shown by enzymatic reaction velocity. By mea-
suring the consumption of reduced type glutathione in
enzymatic reaction, enzyme activity can be obtained. In
reaction system, glutathione concentration decreased by
1 umol/L, which was an activity unit. In the experiment,
GSH-Px kit A005-1 was employed to determine [5]
(Nanjing Jiancheng Bioengineering Institute).
The determination of MDA content
The determination was by means of TBA (Thiobarbi-
turic Acid Test). MDA kit used in the experiments was
A003-1 (Nanjing Ji a ncheng B ioenginee ring Inst itute) .
The determination of total protein content in tis-
sues
By means of Coomassie brilliant blue protein deter-
mination kit A045-2 (Nanjing Jiancheng Bioengineering
Institute), the determination was conducted and shown
by mg/mL.
The statistical analysis
SPSS18.0 was used in statistical analysis, and th e data
was x ± SD, a = 0.05.
3. Results and Conclusion
According to the analysis of oxidization resistance activ-
ity data in vitro of raspberry flavone extraction, among
raspberry flavone crude extraction, raspberry flavone
extract and raspberry flavone purified matters, raspberry
flavone purified matters group has a greater ability in
scavenging hydroxyl, H2O2 and DPPH than the other two
ones. Therefore, raspberry flavone purified matters was
chosen to be the raw material of rats’ in vivo oxidization
resistance experiments to conduct analysis, while the re-
sults are as follows:
The results of rats’ liver oxidization resistance ac-
tivity
From Table 1, it can be seen that in rats’ liver tissues,
high dose group has the highest CAT, SOD and GSH-Px
activity (cholesterol and high dose raspberry flavon e pu-
rified matters were fed), being significantly different
from other four groups (p < 0.05); the second highest one
is low dose group (low dose raspberry flavone purified
matters and cholesterol were fed), which differs from
some groups remarkably. Between blank, positive and
negative groups, there is also some significance of dif-
ference, which is small. The high dose purified matters
group has an obvious higher MDA content than other
groups, being significantly different from other groups (p
Table 1. The determination result for big mouse liver CAT, SOD, GSH-Px, MDA.
Item
Treatment
CAT (U/mL) SOD (U/m) GSH-Px MDA (mol/mg)
The Blank control 45.57 ± 1.1 6 204.06 ± 1. 4 7 626.41 ± 5. 34 9.89 ± 0.4 3
The Negative control 53.58 ± 1.60Δ 212.25 ± 1.63Δ 638.70 ± 3.5 4 Δ 12.96 ± 0. 3 9Δ
The Positive control 59.09 ± 1.77Δ# 219. 0 6 ± 1.14Δ# 647.81 ± 3.85Δ# 7.23 ± 0. 2 9 Δ#
The low dose group 61.01 ± 1.55Δ# 229.92 ± 2.3 5Δ# 698.09 ± 4.76Δ# 6 .63 ± 0.08Δ#
The high dose g roup 69.19 ± 1.38Δ#☆◇ 243.75 ± 1.69Δ#☆◇ 718.25 ± 6.70Δ#☆◇ 4.21 ± 0.13Δ#☆◇
Note: “△” stands for the significant difference when compared with blank control; “#” stands for the significant difference compared with negative control; “☆”
stands f o r th e s i gnificant d i fference co mpar ed with p u r i fi ed matters of low dos e; “ ◇” s t an ds for the si gnificant difference compared wi t h purif i ed matt er s of low
dose.

J. X. SUN ET AL.
457
< 0.05), which demonstrates that high dose raspberry
flavone purified matters can increase the enzymes of oxi-
dization resistance and scavenging free radical in rats’
liver, benefiting removing the free radical in mice’s liv-
ers, increasing liver’ s oxidization resistance , which is ef-
fective in oxidization resistance.
The result of detecting rats’ kidney oxidization re-
sistance acti vity
Table 2 shows that, being similar to rats’ liver expe-
riments, high dose group has the highest CAT, SOD and
GSH-Px activity in rats’ kidney, being significantly dif-
ferent from other four groups (p < 0.05 ). The second one
is low dose group (low dose raspberry flavone purified
matters and cholesterol were fed), which differs from
some groups remarkably. The high dose purified matters
group has a obvious lower MDA content than other
groups, being significantly different from other groups (p
< 0.05), which demonstrates that high dose raspberry
flavone purified matters can increase the enzymes of
oxidization resistance and scavenging free radical in rats’
kidney, being propitious to remove the free radical in
mice’s kidneys, increasing kidney’s oxidization resistance,
which is effective in oxidization resistance. This experi-
mental result is consistent with the oxidization r esistance
of rats’ liver.
The result of detecting rats’ blood resistance activ-
ity
From the results of Table 3, CAT, SOD and GSH-Px
activity in rats’ blood, there is difference in the groups.
By comparison, the purified matters of high dose have an
obvious higher activity than other groups, being obvious-
ly different from others (p < 0.05). Between purified mat-
ters of low dose and other groups, there is some signifi-
cant difference, while the difference is not significant be-
tween the remaining groups. It demonstrates that after
feeding, raspberry flavone purified matters can markedly
increase the activity of free radical scavenging enzyme in
rats’ blood and strengthen the oxidization resistance of
rats’ blood. MDA, the decomposition product of lipid pe-
roxide, is higher in high dose purified mattes group than
in other groups, and there is significant difference, which
shows that high
The result of detecting rats’ skeleton muscular tis-
sue resistance activity
The result of Table 4 demonstrates to be consistent
with the experimental results above. CAT, SOD, GSH-
Table 2. The determination result for big mouse kidney CAT, SOD, GSH-Px, MDA.
Item
Treatment CAT (U/mL) SOD (U/m) GSH-Px MDA (mol/mg)
The Blank control 84.79 ± 0.9 3 203.47 ± 2. 9 9 409.40 ± 3. 78 18.47 ± 0. 8 6
The Negative control 91.41 ± 1.26Δ 214.30 ± 5.39Δ 423.44 ± 1.8 9 Δ 19.66 ± 0. 9 4Δ
The Positive control 97.36 ± 1.96
Δ#
209.71 ± 2.40 440.64 ± 2.19
Δ#
15.70 ± 0. 56
Δ#
The low dose group 105.59 ± 2.24Δ#☆ 227.02 ± 5.17Δ#☆ 453.67 ± 2.65Δ#☆ 13.20 ± 0.55Δ#☆
The high dose g roup 121.12 ± 1.45Δ#☆◇ 248.10 ± 4.47Δ#☆◇ 469.40 ± 1.05Δ#☆◇ 9.9 1 ± 0.65Δ#☆◇
Note: “△” stands for significant difference when compared with blank control; “#” stands for the significant difference compared with negative control; “☆”
stands for the significant difference compared with positive control group; “◇” stands for the significant difference compared with purified matters of low dose.
Table 3. The determination result for big mouse blood CAT, SOD, GSH-Px, MDA.
Item
Treatment
CAT (U/mL) SOD (U/m) GSH-Px MDA (mol/mg)
The Blank control 76.12 ± 2.61 187.39 ± 5.38 391.67 ± 2.53 18.48 ± 0.58
The Negative control 84.47 ± 2.50
Δ
200.41 ± 2.56
Δ
417.06 ± 4.56
Δ
19.17 ± 0.39
The Positive control 92.20 ± 1.37
Δ#
208.71 ± 11.47
Δ
426.67 ± 6.09
Δ
14.55 ± 0.63
Δ#
The low dose group 94.89 ± 0.79Δ#☆ 209.29 ± 1.03Δ 441.38 ± 5.71Δ#☆ 13.12 ± 0.96Δ#
The high dose g roup 109.55 ± 1.18Δ#☆◇ 232.47 ± 1.85Δ#☆◇ 458.98 ± 4.70Δ#☆◇ 10.86 ± 0.34Δ#☆◇
Note: “△” stands for significant difference when compared with blank control; “#” stands for the significant difference compared with negative control; “☆”
stands for the significant difference compared with positive control group; “◇” stands for the significant difference compared with purified matters of low dose.
Table 4. The CAT, SOD, GSH-P x, MDA determination result for big mouse skeleton muscular tissue.
Item
Treatment
CAT (U/mL) SOD (U/m) GSH-Px MDA (mol/mg)
The Blank control 84.04 ± 2.40 193.66 ± 4.30 303.85 ± 6.21 16.80 ± 0.63
The Negative control 90.51 ± 0.90
Δ
197.51 ± 1.86 288.01 ± 9.19
Δ
17.81 ± 0.46
The Positive control 95.43 ± 1.65
Δ
210.17 ± 2.85
Δ#
320.06 ± 1.54
Δ#
15.40 ± 0.57
#
The low dose group 106.66 ± 3.42Δ#☆ 225.77 ± 4.40Δ#☆ 330.94 ± 3.43Δ# 12.24 ± 0.26Δ#☆
The high dose g roup 122.34 ± 1.20Δ#☆◇ 239.74 ± 1.99Δ#☆◇ 350.62 ± 2.01Δ#☆◇ 10.17 ± 0.48Δ#☆◇
Note: “△” stands for significant difference when compared with blank control; “#” stands for the significant difference compared with negative control; “☆”
stands for the significant difference compared with positive control group; “◇” stands for the significant difference compared with purified matters of low dose.

J. X. SUN ET AL.
458
Px activity in rats’ musculus skeleti are significantly
higher in high dose raspberry flavone purified matters
than in other groups, while MDA is lower, being signifi-
cantly different from other groups, followed by low dose
raspeberry flavone purified matters. It shows that rasp-
berry flavone purified matters can obviously increase the
activity of free radical scavenging enzyme in rats’ mus-
culus skeleti, strengthening the oxidization resistance of
rats’ blood, decreasing lipid peroxidization and improv-
ing oxidization resistance.
4. DISCUSSION
1) In recent years, the relation of free radical and many
diseases has been increasingly aroused great attention.
The development of free radical biomedicine enables the
natural antioxidant, high efficient and low-toxic free rad-
ical scavenger to be the research hotspot of biological
chemistry and medicine and pharmacology [6]. Oxidiza-
tion resistance is considered to be the most important me-
chanism of flavonoid compound. The flavonoid com-
pound is well received in western countries, whose oxi-
dization resistance and other biologica l activities are also
gradual ly paid at t e ntion to by C hinese.
2) Dried fruit of raspberry is used as both food and
medicine, being effective in protecting liver and kidney
and preventing and resisting cancers [7]. One of the ma-
jor functional matters in raspberry is flavonoid com-
pound. The identified raspberry flavonoid compounds are
mainly kaempferol, quercetin and tiliroside, which are
special in structure and effective with more than 90% of
bioavailability when compared with most flavones.
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