全 文 ：※营养卫生 食品科学 2015, Vol.36, No.23 279
Antioxidant Effect of Perilla Oil on Ethanol-Induced Oxidative Stress in Mice
ZHANG Yang1, SUN Heping2, LIU Zhuo1, ZHOU Hongli1,*
(1. School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin 132022, China;
2. College of Life Sciences, Jilin University, Changchun 130012, China)
Abstract: With the aim of exploring the free radical scavenging potential of perilla oil (PO) as a functional food ingredient,
the antioxidant effect of PO on ethanol-induced oxidative stress in mice was investigated. The mice were subjected to oral
administration of PO at the doses of 50, 100, 200, and 400 mg/(kg·d) for 30 days and the antioxidant activity was evaluated
by the determination of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and protein carbonyl
(PC). The results showed that MDA contents in the normal control (NC) group, and 100, 200, and 400 mg/(kg·d) PO
groups were decreased by 46.08% (P < 0.01), 17.28% (P < 0.05), 25.12% (P < 0.05), and 48.16% (P < 0.01), respectively,
when compared with the model control (MC) group. SOD activity in 100 mg/(kg·d) PO group was significantly increased
(P < 0.01). GSH levels in 100 and 200 mg/(kg·d) PO groups were remarkably increased (P < 0.01). PC content in
100 mg/(kg·d) PO group was obviously decreased (P < 0.05). Therefore, due to its effect on MDA, SOD, GSH and PC, PO
has obvious antioxidant activity on ethanol-induced oxidative stress in mice, and 100 mg/(kg·d) may be an optimal dosage
for antioxidant effect. The antioxidant effect of PO is highly correlated with decreased accumulation of MDA and PC,
increased SOD activity, and increased GSH level.
Key words: perilla oil; antioxidant effect; ethanol-induced oxidative stress; functional food
紫苏油在乙醇诱导氧化损伤模型小鼠体内的抗氧化作用
张 扬1，孙和平2，刘 卓1，周鸿立1,*
（1.吉林化工学院化学与制药工程学院，吉林 吉林 132022；2.吉林大学生命科学学院，吉林 长春 130012）
摘  要：探索紫苏油作为抗氧化功能性食品的潜力，研究紫苏油在小鼠体内的抗氧化作用。给昆明小鼠每日灌胃
不同剂量的紫苏油，饲养30 d后，除空白对照组外，其他实验组灌胃乙醇造成小鼠氧化损伤模型，并对小鼠肝脏丙
二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）、蛋白质羰基（protein carbonyl，PC）含量和血
清超氧化物歧化酶（superoxide dismutase，SOD）活力进行测定。结果表明：与模型组比较，空白对照组，100、
200、400 mg/（kg•d）紫苏油剂量组小鼠肝脏的MDA含量分别降低了46.08%（P＜0.01）、17.28%（P＜0.05）、
25.12%（P＜0.05）、48.16%（P＜0.01）；100 mg/（kg•d）紫苏油剂量组小鼠血清的SOD活力极显著增强
（P＜0 . 0 1）；1 0 0、2 0 0 m g /（k g • d）紫苏油剂量组小鼠肝脏的G S H含量极显著升高（P＜0 . 0 1）；
100 mg/（kg•d）紫苏油剂量组小鼠肝脏的PC含量显著降低（P＜0.05）。因此，紫苏油在乙醇诱导氧化损伤模型小鼠
的体内具有抗氧化作用，其通过降低小鼠肝脏内MDA、PC含量，提高GSH含量，升高血清SOD活力发挥作用，其中
100 mg/（kg•d）为紫苏油推荐剂量，应进一步对其进行研究。
关键词：紫苏油；抗氧化作用；乙醇诱导氧化损伤；功能性食品
中图分类号：TQ641 文献标志码：A 文章编号：1002-6630（2015）23-0279-04
doi:10.7506/spkx1002-6630-201523051
收稿日期：2015-02-28
基金项目：吉林省科技厅重点项目（20130303050NY）；吉林化工学院科研项目（201350）
作者简介：张扬（1982—），男，讲师，博士，研究方向为活性天然产物开发。E-mail：zhangyhappy@126.com
*通信作者：周鸿立（1967—），女，教授，博士，研究方向为天然药用资源的生物有效成分分析与质量控制。E-mail：zhl67@126.com
Free radicals and other reactive oxygen species (ROS)
are constantly formed in body. Oxidative stress manifests
an imbalance between the systemic formation of ROS and
biological antioxidant defense[1]. Excessive ROS can cause
damages to DNA, proteins, and lipids[2]. Furthermore, ROS
can cause the disruption in normal cellular signaling to
280 2015, Vol.36, No.23 食品科学 ※营养卫生
induce human diseases, including cancer[3], Alzheimer’s
disease[4], depression[5], cardiovascular diseases[6], infection[7],
and chronic fatigue syndrome (CFS)[8]. Aging can also
be accelerated by oxidative stress[9]. Thus, the safety and
effective antioxidant agents remains ongoing.
Perilla oil (PO), an edible vegetable oil widely used in
Asia, is obtained by pressing the seeds of Perilla frutescens (L.)
Britt. PO is rich in palmitic acid, linoleic acid, α-linolenic
acid, octadecanoic acid, and arachidonic acid. The content
of polyunsaturated fatty acids in PO is up to 90%, and
approximately 50%–62% of PO consists of α-linolenic
acid[10-11]. These ingredients are essential for human beings,
and contribute to the potential of PO for the applications in
healthcare, such as serum lipid regulation[12], prevention of
coronary heart disease[13], anti-tumor[14], and regulation of
brain function[15].
In previous studies, the short-term toxicological
evaluation of PO has been conducted[16]. The results suggested
that dietary consumption of PO has no obvious adverse
effects, which provides the possibility for the development
of PO-based functional foods. In the present study, in order
to confirm the PO potential as functional foods for free
radical scavenging, the antioxidant effect of PO in mice was
evaluated. According to the relevant guiding principles of
functional food evaluation issued by China Food and Drug
Administration (CFDA)[17], 50% solution of ethanol was used
as an inducer for the model of oxidative stress.
1 Materials and Methods
1.1 Materials and reagents
Perilla oil (PO) was provided by Huangzhihua
Pharmaceutical Co. Ltd. (Changchun, China).
Reagent kits for the determination of malondialdehyde
(MDA, lot No. 20140706), superoxide dismutase (SOD, lot
No. 20140702), glutathione (GSH, lot No. 20140708), and
protein carbonyl (PC, lot No. 20140710) were purchased
from Jiancheng Biotechnology Co. Ltd. (Nanjing, China).
Other reagents and solvents were analytical grade and
obtained from Sigma Aldrich Chemical Co. Ltd. (St. Louis,
MO, USA).
1.2 Animals
Male Kunming mice (age: 6–8 weeks; body weight:
(20 ± 2) g) were provided by the Animal Research Institute of
Jilin University (Approval No. SCXK (Ji) 2011-0003). The
mice were housed at controlled temperature and humidity,
and acclimated to housing conditions for at least one week
before experiments. Animal experiments were carried
out in compliance with the Guide for the Care and Use of
Laboratory Animals of the National Research Council of
USA, 1996 and corresponding ethical regulations of Jilin
University.
1.3 Methods
1.3.1 Experimental design
Sixty Kunming male mice were randomly divided into
six groups with ten mice in each group. The mice in normal
control (NC) and model control (MC) groups were orally
administered with saline alone, and the treatment groups
were administered with PO at the doses of 50, 100, 200, and
400 mg/(kg·d) body weight respectively. All administrations
were conducted once a day for 30 consecutive days. On the
last day, after a 16-hour fasting period, all mice except for
the NC group were orally administered with a 50% solution
of ethanol at a dose of 12 mL/kg body weight to induce
oxidative stress[17].
1.3.2 Determination of biochemical indicators
After a period of 6 h, all mice were anesthetized with
chloroform, and approximately 1 mL of blood samples were
collected to determine SOD activity by previously reported
method[18]. Then, mice were killed and livers were dissected
to determine the contents of MDA, GSH, and PC[19].
1.4 Statistical analysis
Experimental data were expressed as ± s. Differences
among means were determined by a one-way analysis of
variance (ANOVA) followed by Tukey’s post-hoc test
using SPSS software version 19.0 (SPSS Inc, Chicago,
IL). Probability value (P) less than 0.05 was considered as
statistically significant difference.
2 Results and Analysis
2.1 Effect of PO on the decrease of MDA contents
In the body, free radicals can trigger the lipid
peroxidation process, and induce the peroxidation
o f po lyunsa tu ra t ed f a t t y ac id s i n ce l l s , such a s
4-hydroxynonenal, MDA, and 8-isoprostane F2α[20]. MDA
is one of the most common products. An increase in free
radicals causes overproduction of MDA[21]. As shown in
Fig.1, when compared with the MC group, MDA contents
in the NC group, and PO treatment groups at the doses of
100, 200, and 400 mg/(kg·d) were decreased by 46.08%
(P < 0.01), 17.28% (P < 0.05), 25.12% (P < 0.05), and 48.16%
※营养卫生 食品科学 2015, Vol.36, No.23 281
(P < 0.01), respectively. In addition, the correlation
coefficient between PO dose and MDA content reduction in
mice was 0.966, suggesting that the ideal animal model of
ethanol-induced oxidative stress was successfully established
and PO could reduce MDA levels in a linear (R2 = 0.966),
and dose-dependent manner.
**
**
*
*
M
D
A
c
on
te
nt
/(n
mo
l/m
L)
MC
gr
ou
p
NC
gr
ou
p 50
0
2
4
6
8
100
PO dose/(mg/(kg·d))
200 400
*. Difference was significant (P < 0.05) as compared with
the MC group; **. Difference was highly significant (P <
0.01) as compared with the MC group. The same in Fig.2-4.
Fig.1 Effect of PO on MDA content (x ± s, n = 10)
2.2 Effect of PO on the increase of SOD activity
SOD is an important antioxidant enzyme in vivo,
which can destroy the highly reactive superoxide radical
by converting it into less reactive hydrogen peroxide[22-23].
In the current study, the activity of SOD in the MC group
was significantly decreased than those in the NC group
(P < 0.05) (Fig.2). These data clearly revealed that 50%
ethanol could induce oxidative stress in mice. The PO
treatment groups originally exhibited increase in SOD activity
in a dose-dependent manner, and the SOD activity reached
the highest level ((190.32 ± 14.74) U/mL) at the dose of
100 mg/(kg·d), which was significantly higher than that in
the MC group ((155.13 ± 22.43) U/mL) (P < 0.01). However,
after that, the SOD activity was decreased as the increase of
PO dose. These results showed that the effect of PO on SOD
activity was not presented as a dose-dependent manner, and
the optimal dosage of PO for increasing SOD activity in mice
might be 100 mg/(kg·d).
**
*
SO
D
a
ct
iv
ity
/(U
/m
L)
MC
gr
ou
p
NC
gr
ou
p 50
0
50
100
150
200
250
100
PO dose/(mg/(kg·d))
200 400
Fig.2 Effect of PO on SOD activity (x ± s, n = 10)
2.3 Effect of PO on the increase of GSH contents
As a prototype antioxidant, GSH involves in cell
protection from the noxious effect of excessive oxidant stress
and prevents the damage to important cellular components
caused by free radicals[24]. As shown in Fig.3, the effect
of PO on GSH was similar to the observed SOD. The
contents of GSH in PO treatment groups were increased as
the dose increase from 50 to 100 mg/(kg·d), but decreased
as the further increase in dose from 100 to 200 mg/(kg·d).
Nevertheless, the contents of GSH were higher after PO
treatments when compared with the MC group, and PO at the
doses of 100 and 200 mg/(kg·d) could significantly accelerate
the production of GSH (P < 0.01). Besides, the content of
GSH in the NC group was also evidently higher than that
in the MC groups (P < 0.01). These results indicated that
PO could improve the content of GSH in well-established
ethanol-induced oxidative stress model in a certain dosage
range. In addition to the MC group, the contents of GSH in
100 mg/(kg·d) group ((73.1 ± 14.6) mg/g pro) was also much
higher than that in the NC group ((54.1 ± 7.48) mg/g pro).
**
**
**
G
SH
c
on
te
nt
/(m
g/g
pr
o)
MC
gr
ou
p
NC
gr
ou
p 50
0
20
40
60
100
80
100
PO dose/(mg/(kg·d))
200 400
Fig.3 Effect of PO on GSH content (x ± s, n = 10)
2.4 Effect of PO on the decrease of PC contents
* *
*
PC
c
on
te
nt
/(n
mo
l/m
g p
ro)
MC
gr
ou
p
NC
gr
ou
p 50
0
20
40
60
100
PO dose/(mg/(kg·d))
200 400
Fig.4 Effect of PO on PC content (x ± s, n = 10)
Carbonyls can directly react with protein or react with
other biomarcromolecules to generate reactive carbonyl
species, thereby reacting with proteins[25]. Hence, PC is a
biomarker of oxidative stress. As shown in Fig.4, PC level
in PO group at the dose of 100 mg/(kg·d) was significantly
lower than that in the MC group (P < 0.05). On the contrary,
282 2015, Vol.36, No.23 食品科学 ※营养卫生
as the increase of PO dose, the effect of PO on the reduction
of PC contents revealed an obvious decrease. At the dose
of 400 mg/(kg·d), the PC level was higher than that in the
MC group (P < 0.05). These results indicated that effect
of PO on PC level was not presented in a dose-dependent
manner, and the dose of 100 mg/(kg·d) might be the optimal
dose of PO for the reduction of PC.
3 Conclusions
PO has excellent antioxidant effect on ethanol-induced
oxidative stress in mice, but unsatisfactory dose-effect
relationship. Generally speaking, effect of PO on MDA
contents was in a linear, dose-dependent manner, other effect
including on SOD activity, GSH levels, and PC contents were
not in a dose-dependent manner. Therefore, based on the
comprehensive effects of PO on MDA, SOD, GSH, and PC,
PO at a dose of 100 mg/(kg·d) is a highly potential candidate
for the development of antioxidant functional foods due to its
functions for decreasing the accumulation of MDA and PC,
increasing SOD activity, and improving GSH level.
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