Oxidization Resistance in Vivo for Raspberry Flavone

doi:10.4236/eng.2013.510B093

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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

Email: *[email protected]

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

200.41 ± 2.56

417.06 ± 4.56

19.17 ± 0.39

The Positive control 92.20 ± 1.37

Δ#

208.71 ± 11.47

426.67 ± 6.09

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

197.51 ± 1.86 288.01 ± 9.19

17.81 ± 0.46

The Positive control 95.43 ± 1.65

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.

REFERENCES

[1] J. Y. Cha and Y. S. Cho, “Effect of Hesperetin, a Citrus

Flavonoid, on the Liver Triacy lglycerol C ontent and Pho-

sphatidate Phosphohydrolase Activity in Orotic Acid-Fed

Cha J. Y, Rats,” Plant Foods for Human Nutrition, Vol.

56, 2001, pp. 349 -358.

http://dx.doi.org/10.1023/A:1011884200848

[2] H. Koyuncu, B. Beerkarda and F. Baykut, “Preventive Ef-

fect of Hesperidin against Inflammation in Mouse Skin

Caused by Tumor Promoter,” Anticancer Research, Vol.

19, 2008, pp. 3237-3241.

[3] K. Kawaguchi, S. Kikuchi, R. Hasunuma, et al., “Sup-

pression of Infection-Induced Endotoxin Shock in Mice

by a Citrus Flavanone Naringi n, ” Planta Medica, Vol. 70 ,

2004, pp. 17-22. http://dx.doi.org/10.1055/s-2004-815449

[4] E. A. Kurowska and J. A. Manthey, “Hypolipidemic Ef-

fects and Absorption of Citrus Polymethoxylated Flavon-

es in Hamsters with Diet-Induced Hypercholesterolemia,”

Journal of Agricultural and Food Chemistry, Vol. 52,

2007, pp. 2879-2886. http://dx.doi.org/10.1021/jf035354z

[5] Q. F. Zhang and Z. R. Zhang, “Antioxidant Activity of

Rhizoma Smilacis Glabrae Extracts and Its Key Consti-

tuent Astilbin,” Food Chemistry, Vol. 115, 2008, pp. 297-

303. http://dx.doi.org/10.1016/j.foodchem.2008.11.053

[6] A. Ardestani and R. Yazdanpast, “Antioxidant and Free

Radical Scavenging Potential of Achillea Santolina Ex-

tracts,” Food Chemistry, Vol. 104, 2007, pp. 21-29.

http://dx.doi.org/10.1016/j.foodchem.2006.10.066

[7] L. R. Della, R. Loggia and S. Sosa, “Anti-Inflammatory

Activity of Ginkgobiloba Flavonoids,” Planta Medicine,

Vol. 59, 2006, p. A588.