SUPPLEMENTARY MATERIAL
Free radical scavenging activities of oligomeric proanthocyanidin from Rhodiola
rosea L. and its antioxidant effects in vivo
Qian Zhoua,b, Zhi-Ping Yina, Lei Maa, Wen Zhaoa,b＊, Hong-Wei Haoa and Hui-Ling Lia
a
Department of nutritional and food safety, College of Food Science and Technology, Agricultural
University of Hebei, Baoding 071001, P.R. China;
b
Engineering Technology Research Center for Agricultural Product Processing of Hebei, Baoding
071001, P.R. China
*Corresponding author: Tel.: +86 312 7528426; fax: +86 312 7528189.
E-mail address: 13582820221@163.com (W. Zhao)
Free radical scavenging activities of oligomeric proanthocyanidin from Rhodiola
rosea L. and its antioxidant effects in vivo
This study aimed to determine the antioxidant activity of oligomeric proanthocyanidin from Rhodiola
rosea L. (OPCRR). The free radical scavenging activities of OPCRR were determined by
1,1-diphenyl-2-picrylhydrazyl (DPPH·), hydroxyl radical (·OH), superoxide anion (·O2-), and showed
greater than that of vitamin c. The effects of OPCRR on the antioxidant enzymes activity and lipid
peroxide content in vivo were evaluated through three observation biomarkers, including superoxide
dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in serum, heart,
liver and brain tissues in mice. The OPCRR significantly enhanced the SOD and GSH-Px activities,
while reduced the MDA content in mice. These results indicated that the OPCRR has a great potential
to be a natural antioxidant due to the good antioxidant activities in vitro and in vivo.
Keywords: Rhodiola rosea L.; oligomeric proanthocyanidin; antioxidant activity; mice
Experimental details
1. Materials
Rhodiola rosea L. roots were collected from Xinjiang Province of China, identified by Guojiang Wu
Professor of Agricultural University of Hebei, and the voucher specimen number is No.20051003009.
All biochemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, USA). The
commercial kits for determination of glutathione peroxidase (GSH-Px), superoxide dismutase
(SOD), and malondialdehyde (MDA) were obtained from Jiancheng Bioengineering Institute
(Nanjing, China). All the other reagents were of analytical grade.
2. Animals
32 specific-pathogen-free (SPF-level) ICR mice, weighing 18-22 g, were purchased from Fukang
Biotechnology Co., Ltd. (Beijing, China) (license no.: SCXK-Beijing-2009-0004). The mice,
including 16 males and 16 females, were housed individually in stainless wire netting cages with ad
libitum access to standard rodent feed and water. The a room maintained on a 12 h light-dark cycle
with a temperature range of 21 to 23°C and a relative humidity of 55% to 60%. Animal welfare and
experimental procedures were in accordance with the guidelines for the care and use of laboratory
animals and the related ethical regulations. The research was approved by the Research Ethics
Committee of the College of Food Science and Technology in Agricultural University of Hebei
under number 102/2013.
3. Preparation of OPCRR
The OPCRR was prepared in our laboratory according to the previous method (Yin et al. 2011). The
optimized extraction process was as followed in brief. 1.0 g of powder Rhodiola rosea L. roots was
weighed and extracted in water at 28 °C for 30 min. The supernatant was dissolved in 75% ethanol to
obtain the elementary extract. The procedure was followed by washing with petroleum ether for two
times. The solution was freeze-dried and re-extracted by 50% ethylacetate to obtain crude extracts of
OPCRR. Finally, OPCRR was purified with macroporous absorption resin.
4. Determination of free radical scavenging activities of OPCRR in vitro
4.1. Scavenging activity on DPPH·
The DPPH· radical scavenging assay was used to determine the antioxidant activity of OPCRR
according to the previous described method (Karagozler et al. 2008) with minor modifications. In
brief, the OPCRR was weighed and dissolved in methanol to prepare a series of solutions at different
concentrations (10-100 µg/mL). 0.1 mL of different OPCRR concentrations was mixed with 3.9 ml
25µg/mL methanol solution of DPPH· and the reaction mixture was incubated at room temperature
for 30 min in the dark. The same concentrations of vitamin c were conducted as positive control. The
absorbance was measured at 517 nm using a Shimadzu UV-Vis spectrophotometer mini 1240. All
tests were performed in triplicate and averaged. The DPPH· free radical scavenging activity was
expressed as [DPPH·] scavenging rate, and calculated using the following formula:
[DPPH·] scavenging rate (%) = [(A0-A1) / A0] × 100
A0 is the absorbance of DPPH solution without OPCRR and A1 is the absorbance of DPPH
solution with OPCRR.
The IC50 concentration at which 50% DPPH· radical scavenging occurred was calculated from
the regression equations performed by different amounts of OPCRR.
4.2. Scavenging activity on ·OH
The ·OH radical scavenging activity was determined according to the described method with some
modifications (Smironoff & Cumbes 1989). The reaction mixture containing OPCRR at different
concentrations (0.5-8 mg/mL), 2mM EDTA-Fe, 3% H2O2 and 360 µg/mL crocus in 450mL 150mM
sodium phosphate buffer was incubated at 37°C for 30min and the content of ·OH was detected by
monitoring the absorbance at 520nm. Vitamin c was substituted for OPCRR as the control. The ·OH
scavenging effciency of OPCRR was estimated from the difference in absorbance with and without
addition of the OPCRR extract.
[·OH] scavenging rate (%) = A0 – (A1 – A2)/A0 × 100%
A0 is the absorbance of the reaction system without OPCRR; A1 is the absorbance of the reaction
system after addition of the extract; and A2 is the background absorbance of the extract.
The IC50 concentration at which 50% ·OH radical scavenging occurred was calculated from the
regression equations performed by different amounts of OPCRR.
4.3. Scavenging activity on ·O2–
The ·O2– radical scavenging capacity was determined according to the previous method (Mohsin et al.
2013). Different concentrations of OPCRR (0.4-2 mg/mL) was mixed in the NADH-NBT-PMS
system, which included 16 mM Tris-HCl buffer, 338 µM NADH, 72 µM NBT and 30 µM PMS. The
mixtures were incubated for 10 min at room temperature and measured for the absorbance at 560nm.
The OPCRR was substituted with vitamin c as the control. The ·O2– radical scavenging rate was
calculated using the following formula:
[·O2–] scavenging rate (%) = [1-A1/A0] × 100%
A0 is the absorbance of the control without the OPCRR; A1 is the absorbance of the reaction
system in the presence of OPCRR.
The IC50 concentration at which 50% ·O2– radical scavenging occurred was calculated from the
regression equations performed by different amounts of OPCRR.
5. Evaluation of antioxidant effects of OPCRR in vivo
5.1. Animal experimental design
The ICR mice were randomly divided into four groups (n = 8, four males and four females). Mice
were intragastrically administered at low, middle and high dose (100, 200 and 400 mg/kg body
weight of OPCRR) each day for 5 weeks. The control group was treated with distilled water instead.
On day 36, the animals were weighed and sacrificed under ether anesthetized after overnight fasting.
The tissue samples, including serum, heart, liver and brain, were immediately removed and washed
in physiological saline (pH=7.4). Then tissue samples were weighed and homogenized with 0.1
g/mL ice-cold isotonic physiological saline based on wet weight. The homogenate was centrifuged at
1000 g for 10 min at 4 °C to yield a low-speed supernatant which was for the following biochemical
analysis.
5.2. Measurement of biochemical parameters
The SOD and GSH-Px activities and MDA content in tissue samples were determined with the
corresponding commercial kits. SOD activity was determined using xanthine and xanthineoxidase
method by the inhibitory effect of SOD on the reduction of nitroblue tetrazolium (NBT) by
superoxide anion radicals, which are produced in the xanthine/xanthine oxidase system. GSH-Px
activity was evaluated by a spectrophotometric assay based on quantifying the rate of oxidation of
GSH to GSSG by H2O2 catalyzed by GSH-Px. Lipid peroxidation was estimated by the level of
malondialdehyde (MDA), which is an end product of lipid peroxidation. The MDA levels were
determined spectrophotometrically as previously described through the thiobarbituric acid reactive
substances (TBARS) assay (Ohkawa et al. 1979). All tests were conducted according to the
instructions of manufactures.
The absorbance measured for SOD, GSH-Px and MDA was 550 nm, 412 nm and 532 nm using a
Shimadzu UV-Vis spectrophotometer mini 1240, respectively.
6. Statistical analysis
The experimental data were calculated as arithmetic means ± standard deviations (SD). Statistical
analysis was performed using the SPSS17.0 software package. Data comparisons among treatments
were performed through analysis of Student’s t-tests. Significant differences between means were
evaluated by least significant difference (LSD) tests at p <0.05.
References
Karagozler AA, Erday B, Emek YC, Uggun DA. 2008. Antioxidant activity and proline content of
leaf extracts from Dorystoechas hastata. Food Chem. 111:400-407.
Mohsin S, Mahadevan R, Kurup MG. 2013. Free-radical-scavenging activity and antioxidant effect
of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr. 4:75-79.
Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric
acid reaction. Anal Biochem. 95:351-358.
Smironoff N, Cumbes QJ. 1989. Hydroxyl radical scavenging activity of compatible solutes.
Phytochemistry.28:1057-1060.
Yin ZP, Zhang BY, Chen HY, Wang SS, Zhao W. 2011. Study on separation and purification of
oligomeric proanthocyanidin from Rhodiola rosea. Fronties of Agriculture in China.
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Table S1: Effects of OPCRR on antioxidant status of mice in heart, liver and brain.
liver
heart
MDA
GSH-Px
SOD (U/mg pro)
MDA
SOD
GSH-Px
(nmol/mg
(U/g pro)
(nmol /mg
(U/mg pro)
GSH-Px
MDA
(U/g pro)
(nmol/mg pro)
SOD (U/mg pro)
(U/g pro)
pro)
Control group
brain
pro)
77.95 ± 17.42
60.64 ± 15.12
27.64 ± 9.14
69.11 ± 18.02
39.89 ± 6.40
9.15 ± 1.93
67.56 ± 15.65
27.02 ± 7.97
12.08 ± 2.56
79.58 ± 12.22
66.50 ± 8.28
20.11 ± 4.78
65.04 ± 4.31
47.4 ± 7.85
7.37 ± 0.68
85.98 ± 9.63
59.06 ± 5.81*
9.34 ± 0.37
106.39 ± 11.01*
76.00 ± 23.61
17.16 ± 1.99
78.15 ± 3.90
55.02 ± 5.90*
5.43 ± 1.66*
97.08 ± 12.09*
84.77 ± 5.81**
7.76 ± 1.50
113.12 ± 13.77*
87.83 ± 17.12*
12.98 ± 2.90*
75.06 ± 8.69
57.67 ± 5.11**
2.77 ± 0.99**
105.66 ± 16.67**
94.01 ± 13.09**
6.64 ± 0.94*
Low dose group
(100 mg/kg)
Middle dose group
(200 mg/kg)
High dose group
(400 mg/kg)
Values were expressed as mean ± SD (n=8).
*Asterisk represents significant effect (p <0.05) as compared with control group.
**Asterisks represent significant effect (p <0.01) as compared with control group.