Food Chemistry 125 (2011) 652659

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The in vitro and in vivo antioxidant properties of seabuckthorn (Hippophae

rhamnoides L.) seed oil
Hung-Chih Ting a,1, Yu-Wen Hsu b,1, Chia-Fang Tsai c,1, Fung-Jou Lu a, Ming-Chih Chou a, Wen-Kang Chen d,
a
Institute of Medicine, College of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwan
b
School of Applied Chemistry, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwan
c
School of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwan
d
National Tainan Institute of Nursing, No. 78, Sec. 2, Minzu Rd., Tainan City, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: The antioxidant capacity of seabuckthorn (Hippophae rhamnoides L.) seed oil was investigated with a
Received 12 April 2010 number of established in vitro assays and in an in vivo study of carbon tetrachloride (CCl4)-induced oxi-
Received in revised form 15 July 2010 dative stress in mice. The results showed that DPPH radical scavenging activity, ferrous ion chelating
Accepted 14 September 2010
activity, reducing power and inhibition of lipid peroxidation activity all increased with increasing con-
centrations of seabuckthorn seed oil. Moreover, the EC50 values of seabuckthorn seed oil from the hydro-
gen peroxide, superoxide radical, hydroxyl radical scavenging assays were 2.63, 2.16 and 0.77 mg/ml,
Keywords:
respectively. In the in vivo study, seabuckthorn seed oil inhibited the toxicity of CCl4, as seen from the
Antioxidant
Hippophae rhamnoides L.
signicantly increased activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione
In vitro peroxidase and glutathione reductase. The GSH content in the liver was also increased, whereas hepatic
In vivo malondialdehyde was reduced. Taken together, these results clearly indicate that seabuckthorn seed oil
Seabuckthorn seed oil has signicant potential as a natural antioxidant agent.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Reactive oxygen species (ROS) including superoxide anions,
hydrogen peroxide (H2O2) and hydroxyl radicals are known to play
multiple important roles in the oxidative damage that is closely re-
lated to cardiovascular disease, cancer, liver disease and other
chronic and inammatory diseases (Halliwell, 1997). Several lines
of evidence from both epidemiological and experimental studies
have demonstrated that natural antioxidants, such as carotenoids,
tocopherols and avonoids, can effectively prevent and cure oxida-
tive stress-related diseases (Vitaglione, Morisco, Caporaso, &
Fogliano, 2004; Willett et al., 1984). Therefore, growing attention
has been paid to phytochemicals that have been characterised as
natural antioxidants.
Seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae) is
widely distributed throughout Eurasia and is found in India, China,
Nepal, Russia, Britain, Germany, Finland and France. Seabuckthorn
is a thorny deciduous bush, and it is a popular medicinal plant that
has traditionally been used in its raw form as a health food and
nutritional supplement (Yang & Kallio, 2002). The bark, leaves, ber-
ries and seeds of seabuckthorn are well known for their medicinal
Corresponding author. Tel.: +886 6 2110550; fax: +886 6 24110659.
E-mail address: yuwen@csmu.edu.tw (W.-K. Chen).
1
These authors contributed equally to this work.
properties, and all parts of the plant contain high concentrations of
various bioactive substances. Seabuckthorn leaf extracts contain
plentiful avonoids, which have been reported to have signicant
antioxidant, anti-inammatory and hepatoprotective activities
(Geetha, Sai Ram, Singh, Ilavazhagan, & Sawhney, 2002; Geetha
et al., 2008). Yang and Kallio (2002) reported that seabuckthorn
berries are rich in antioxidant substances, such as tocopherols,
avonoids and carotenoids that can improve the immune function
and can also suppress certain risk factors for cardiovascular dis-
ease. Furthermore, Upadhyay et al. (2009) showed that seabuck-
thorn seed oil is safe and has a positive impact on human health.
Seabuckthorn seed oil also contains large quantities of unsaturated
fatty acids, tocopherols, carotenoids and avonoids, which are
known to have signicant anti-bacterial, anti-atherogenic and car-
dioprotective activities (Basu et al., 2007; Negi, Chauhan, Sadia,
Rohinishree, & Ramteke, 2005). In various experimental models,
seabuckthorn seed oil has also been reported to protect against hy-
poxia-induced transvascular uid leakage and carbon tetrachloride
(CCl4)-induced toxicity in the liver (Hsu, Tsai, Chen, & Lu, 2009;
Purushothaman et al., 2008).
Although seabuckthorn seed oil contains abundant phyto-
chemicals, only limited information is available about its antioxi-
dant properties. Therefore, the present study was designed to
investigate the antioxidant properties of seabuckthorn seed oil
in vitro and in vivo.

0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.09.057
 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659
2. Materials and methods
2.1. Chemicals
Silymarin, gallic acid, thiobarbituric acid (TBA), 1,1-diphenyl-2-
picrylhydrazyl (DPPH), linoleic acid and dimethyl sulfoxide
(DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). All other chemicals and reagents used were analytical grade.
2.2. Seabuckthorn seed oil
The preparation of seabuckthorn (H. rhamnoides L.) seed oil from
China Beijing Tongrentang Group Co., Ltd. (Taipei City, Taiwan) was
collected exclusively by supercritical carbon dioxide extraction
from seabuckthorn seeds. The quality of seabuckthorn seed oil
was described and provided by the company. According to the
company-provided data, the nal content of the seabuckthorn seed
oil was 98.29% crude fat, 0.93% crude protein and 0.78% total
carbohydrate.
2.3. Determination of chemical composition
Fatty acid composition in the seabuckthorn seed oil were mea-
sured by gas chromatography with a ame ionisation detector (GC-
FID) using a Perkin Elmer Autosystem Gas Chromatograph (Model
N611-9000) as described previously (Morrison & Smith, 1964).
Tocopherols in the seabuckthorn seed oil were estimated using
an high performance liquid chromatography (HPLC) system (JASCO
International Co., Ltd., Japan) comprising a Jasco PU-2080 Plus
intelligent pump, a Jasco MD-2015 plus multiwavelength detector
and a C18 column (250  4.6 mm, 5 lm, Luna) (phenomenex,
USA), as described previously (Cunha, Amaral, Fernandes, &
Oliveira, 2006). Beta-carotene was estimated using the HPLC
system as described previously (Chen, Chuang, Lin, & Chiu, 1993).
2.4. In vitro antioxidant potential of seabuckthorn seed oil
2.4.1. DPPH radical scavenging activity
The method was based on that reported by Epsin, Soler-Rivas,
and Wichers (2000) with some modications. The reaction mixture
contained 200 ll of 0.1 mM DPPH and 30 ll of dimethyl sulphox-
ide containing seabuckthorn seed oil at different concentrations
(0.9218.30 mg/ml). The mixture was shaken vigorously and then
left to stand at room temperature for 60 min in the dark. Absor-
bance was measured by a UV/vis spectrophotometer (JASCO V-
500 series, JASCO International Co., Ltd., Japan) at 517 nm. In this
assay, lower absorbance indicates stronger scavenging activity.
The effective concentration at which 50% of the DPPH radicals were
scavenged (denoted by EC50; units: mg sample/ml) was obtained
by plotting the scavenging activity against the sample concentra-
tion. All the tests were performed in triplicate, and the mean values
were plotted.
2.4.2. Ferrous chelating ability
The chelation of ferrous ions by seabuckthorn seed oil was esti-
mated as in Dinis, Maderia, and Almeida (1994), with some modi-
cations. Briey, 0.94 ml of seabuckthorn seed oil in ethanol at
different doses was added to 0.02 ml of 2 mM FeCl2. The reaction
was initiated by the addition 0.04 ml of 5 mM ferrozine, after
which the mixture was shaken vigorously and left standing at
room temperature for 10 min. After equilibrium had been reached,
the absorbance of the solution at 562 nm was measured with a UV/
vis spectrophotometer. All the tests were performed in triplicate,
and the mean values were plotted.
653
2.4.3. Reducing power
Reducing power was measured according to the method re-
ported by Hu, Lin, Lu, Chou, and Yang (2008). Aliquots of seabuck-
thorn seed oil solution (0.5 ml) at different concentrations were
mixed with 0.5 ml of 0.2 M sodium phosphate buffer (pH 6.6)
and 0.5 ml of 1% K3Fe(CN)6, followed by a 20-min incubation at
50 C. After adding 0.5 ml of 10% trichloroacetic acid, the mixture
was centrifuged at 3750 g for 10 min. The upper layer of solution
(0.5 ml) was mixed with 0.5 ml methanol and 0.1 ml of 0.1% ferric
chloride for 10 min, and the absorbance was determined at 700 nm
with a UV/vis spectrophotometer (higher absorbance indicated
stronger reducing power). All tests were performed in triplicate,
and the mean values were plotted.
2.4.4. Total antioxidant activity determination using the ferric
thiocyanate method (inhibition of lipid peroxidation)
The reaction mixture contained 1.5 ml of 0.2 M phosphate buf-
fer (pH 7.0), 1.0 ml of 0.02 M linoleic acid in ethanol and various
amounts of seabuckthorn seed oil (0.603.00 mg/ml) in test tubes
(5 ml volume) and were well shaken to form emulsion. The tubes
were sealed tightly with silicon rubber caps and kept at 40 C in
the dark. At regular intervals, aliquots of the reaction mixtures
were withdrawn with a micro-syringe. The oxidation of each sam-
ple was measured using a slight modication of the ferric thiocya-
nate method (Mitsuda, Yasumodo, & Iwami, 1966). A 0.6-ml
aliquot of the reaction mixture was mixed with 0.7 ml of 75% eth-
anol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 0.02 M
ferrous chloride solution in 3.5% HCl. After 3 min, the absorbance
of the coloured solution at 500 nm was measured with a UV/vis
spectrophotometer. All tests were performed in triplicate, and
the mean values were plotted.
2.4.5. Hydrogen peroxide scavenging activity
The reduction of hydrogen peroxide was determined as de-
scribed by Yoshiki, Yamanaka, Satake, and Okubo (1999) with
some modications. To test hydrogen peroxide scavenging ability,
a reaction mixture consisting of H2O2, seabuckthorn seed oil and
acetaldehyde was added to phosphate-buffered saline (PBS) in
the stainless steel container of a chemiluminescence analysis sys-
tem (CLA 2100, Tokyo Electronic Industries, Tokyo, Japan), and
the chemiluminescence intensity was recorded. The total chemilu-
minescence intensity was calculated as the integration of the area
under the curve minus the background level. Gallic acid was used
as the reference compound. All the tests were performed in tripli-
cate, and the mean values were plotted.
2.4.6. Superoxide anion scavenging activity
Superoxide radicals were generated by the xanthinexanthine
oxidase system as described previously (Oosthuizen, Engelbrecht,
Lambrechts, Greyling, & Levy, 1997), with some modications.
Briey, xanthine oxidase (EC 1.1.3.22; grade I, from buttermilk,
0.25U; one unit converts 1 lmol of xanthine to uric acid per min
at pH 7.5 at 25 C), lucigenin, xanthine and various concentrations
of seabuckthorn seed oil were added to PBS, pH 7.4, in the stainless
steel container of a chemiluminescence analysis system, and the
chemiluminescence intensity was measured. Superoxide dismu-
tase (SOD) was used as the reference compound. All tests were per-
formed in triplicate, and the mean values were plotted.
2.4.7. Hydroxyl radical scavenging activity
Hydroxyl radicals were generated by the addition of ferrous
iron to the buffer solution (Yldz & Demiryrek, 1998). Freshly
prepared FeSO4 (in 0.9% NaCl), luminol and various amounts of sea-
buckthorn seed oil were added to PBS, pH 7.4, in a stainless steel
container, and the chemiluminescence intensity was measured.
The total chemiluminescence intensity was calculated by
654 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659

integrating the area under the curve and subtracting the back- 2.6. Statistical analysis
ground level. DMSO was used as the reference compound. All the
tests were performed in triplicate, and the mean values were All values are expressed as the means SD. Comparison be-
plotted. tween any two groups was evaluated using one-way analysis of
variance (ANOVA) followed by Tukeys multiple comparison tests.
2.5. In vivo antioxidant properties of seabuckthorn seed oil Statistically signicant differences between groups were dened
as p < 0.05.
2.5.1. Animals
Male ICR mice (20 2 g) were obtained from the Animal
3. Results and discussion
Department of BioLASCO, Taiwan, and were quarantined and al-
lowed to acclimate to the laboratory for a week prior to experi-
The generation of excessive ROS, beyond the antioxidant de-
mentation. The animals were handled under standard laboratory
fence capacity of a biological system, can cause oxidative stress,
conditions with a 12-h light/dark cycle in a temperature of
which plays a role in degenerative and pathological events
25 2 C and a relative humidity of 55 5% controlled room. The
(Halliwell, 1997). Several studies have suggested that natural
basal diet used in these studies, PMI Nutrition International, LLC,
antioxidants are helpful in treating diseases that are mediated by
Certied Rodent LabDiet 5001, is a certied feed with appropriate
oxidative stress (Vitaglione et al., 2004). In the present study, we
analyses performed by the manufacturer and provided to WIL Re-
investigated the in vitro and in vivo antioxidant effects of seabuck-
search Laboratories, LLC. Food and water were available ad libitum.
thorn seed oil on the production of ROS with several different
Our Institutional Animal Care and Use Committee approved all
assays. In addition, we also analysed the chemical composition of
protocols for the animal study, and the animals were cared for in
seabuckthorn seed oil.
accordance with the institutional ethical guidelines.

2.5.2. Treatment 3.1. Chemical composition in seabuckthorn seed oil

The animals were randomly divided into seven groups of 10
mice each. Group I served as the control and was given normal sal- Table 1 shows chemical composition in seabuckthorn seed oil.
ine daily for a period of 8 weeks. Group II served as the vehicle con- The amount of total fatty acid in seabuckthorn seed oil was
trol and was given olive oil daily for a period of 8 weeks. Oxidative 641.2 g/kg, and the weight percent of the total saturated fatty acid
stress was induced in Groups III, IV, V, VI and VII by oral adminis- and the total unsaturated fatty acid were 12.2% and 85.3%, respec-
tration of 1 ml/kg body weight of CCl4 (20% CCl4 in olive oil) twice a tively. The major components of fatty acid in seabuckthorn seed oil
week for a period of 8 weeks. Group III served as the CCl4 control. were linoleic acid, linolenic acid and oleic acid. Further, the content
Group IV served as the positive control and was given silymarin of a-tocopherol and c-tocopherol were 111 and 46.1 mg/100 g,
(200 mg/kg, orally) daily for a period of 8 weeks. Groups V, VI respectively; they were 93.8% of total tocopherols in seabuckthorn
and VII were given seabuckthorn seed oil dissolved in olive oil at seed oil and were the major tocopherols in seabuckthorn seed oil.
doses of 0.26, 1.30 and 2.60 mg/kg, respectively, by mouth daily The contents of b-tocopherol and d-tocopherol in seabuckthorn
for a period of 8 weeks. At the end of the experiment, the animals seed oil were 6.71 and 3.69 mg/100 g, respectively, while the total
were sacriced by cervical dislocation. Liver samples were dis- carotenoid was 12.6 mg/100 g. Basu et al. (2007) observed that
sected out, washed immediately with ice-cold saline to remove seabuckthorn seed oil was rich in polyunsaturated fatty acids like
as much blood as possible, and immediately stored at 70 C until oleic acid, linoleic acid and linolenic acid. Negi et al. (2005) pointed
further analysis. out that seabuckthorn seed oil contained abundant unsaturated
fatty acids, tocopherols and carotenoids. Arimboor, Venugopalan,
Sarinkumar, Arumughan, and Sawhney (2006) reported that a-
2.5.3. Measurement of antioxidant enzyme activities and GSH in liver
tocopherol and c-tocopherol were the major tocopherols in sea-
homogenate
buckthorn seed oil whether using hexane or supercritical carbon
Liver homogenates were prepared in cold TrisHCl (5 mM con-
dioxide extraction.
taining 2 mM EDTA, pH 7.4) using a homogenizer. Unbroken cells
and cell debris were removed by centrifugation at 10,000 rpm for
10 min at 4 C. The supernatant was used immediately for SOD,
Table 1
catalase, glutathione peroxidase (GPx), glutathione reductase
Chemical composition of seabuckthorn seed oil.
(GRd) and glutathione (GSH) assays. The activities of all of these
enzymes were determined according to the instructions provided Composition Results
with the Randox Laboratories Ltd. kit. (A) Fatty acid composition (wt%)
(a) Total saturated fatty acid 12.2
Myristiv acid (C14:0) 0.08
2.5.4. Measurement of lipid peroxidation Palmitic acid (C16:0) 9.99
The quantitative measurement of lipid peroxidation was done Stearic acid (C18:0) 1.97
by determining the concentration of thiobarbituric acid reactive Arachidic acid (C20:0) 0.14
(b) Total unsaturated fatty acid 85.3
substances (TBARS) in the liver according to the method published
Palmtoleic acid (C16:1) 3.46
by Buege and Aust (1978). The amount of malondialdehyde (MDA) Oleic acid (C18:1) 21.2
formed was quantitated by reaction with thiobarbituric acid (TBA), Linoleic acid (C18:2) 34.6
and used as an index of lipid peroxidation. Briey, samples were Linolenic acid (C18:3) 26.0
mixed with TBA reagent (0.375% TBA and 15% trichloroacetic acid (c) Others fatty acid 2.48

in 0.25 N hydrochloric acid). The reaction mixtures were placed in (B) Total tocopherols (mg/100 g) 168
a boiling water bath for 30 min and centrifuged at 1811g for 5 min. a-Tocopherol 111
b-Tocopherol 6.71
The supernatant was collected, and its absorbance was measured
c-Tocopherol 46.1
at 535 nm. The results were expressed as nmoles/mg protein d-Tocopherol 3.69
using the molar extinction coefcient of the chromophore
(C) Total carotenoids (mg/100 g) 12.6
(1.56  10 5 M 1 cm 1).
 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659 655

3.2. In vitro antioxidant properties

A
3.2.1. DPPH radical scavenging activity
The DPPH radical scavenging assay is regularly used for the rela-
tively rapid evaluation of the antioxidant activity. DPPH is a stable
free radical, even at room temperature, and shows strong absor-
bance at 517 nm. The DPPH radical accepts an electron or hydrogen
radical to become a stable diamagnetic molecule with a different col-
our. Thus, the degree of its discolouration from purple to yellow is
attributed to the hydrogen donating ability of the added compound,
which is indicative of its radical scavenging potential (Epsin et al.,
2000). In the present study, the DPPH radical scavenging ability of
seabuckthorn seed oil was calculated in a dose-dependent manner
(Fig. 1A). Seabuckthorn seed oil proved to be an effective scavenger
of DPPH radicals. At concentrations ranging from 0.92 to 18.3 mg/ml,
the DPPH radical scavenging ability of seabuckthorn seed oil was
measured at 1392%. The EC50 value of seabuckthorn seed oil in B
the DPPH radical scavenging assay was 7.37 mg/ml; solutions of
trolox (the reference compound used in this test) at the same con-
centrations had EC50 value of <0.92 mg/ml. Recently, our group dem-
onstrated that the EC50 values of scavenging DPPH radicals of
carotenoid standards, of all-trans forms of zeaxanthin, lutein, b-car-
otene and a-carotene, were 22.8, 23.0, 24.2 and 24.5 mg/ml, respec-
tively (Hu et al., 2008). Lower EC50 value indicates a higher DPPH free
radical scavenging activity. By contrast, the present results showed
that the activity of scavenging DPPH radicals by seabuckthorn seed
oil is higher than that of all-trans forms of zeaxanthin, lutein, b-car-
otene and a-carotene but lower than that of trolox. Therefore, sea-
buckthorn seed oil is an outstanding free radical inhibitor and acts
as a primary antioxidant. These results conrm those of other stud-
ies that reported the inhibition of DPPH radicals by the solvent ex-
tracts of seabuckthorn seed (Negi et al., 2005).

3.2.2. Ferrous chelating ability C

The hydroxyl radicals are the most reactive species of ROS and
are formed by the Fenton reaction of H2O2 with ferrous iron or the
HaberWeiss reaction of the superoxide anion with H2O2. Ferrous
ions play an important role in the catalysis reaction of hydroxyl
radical formation (Dinis et al., 1994). Several studies have demon-
strated that the scavenging of hydroxyl radicals by antioxidants
were effective mainly via metal ion chelation. Therefore, the esti-
mation of metal ion chelating ability is important for appraising
the free radical scavenging activity of natural antioxidants (Halli-
well & Gutteridge, 1990). In the present study, the ferrous ion che-
lating ability was determined by the reduction of absorbance at
562 nm; this red colour is quantitatively formed by the reaction
of ferrozine with ferrous ions. Fig. 1B shows the ferrous chelating
abilities of seabuckthorn seed oil, and are compared with that of
EDTA and trolox as reference compounds. At concentrations
ranging from 0.92 to 18.3 mg/ml, the ferrous chelating ability of Fig. 1. The antioxidant capacity of seabuckthorn seed oil was investigated with a
number of established in vitro assays. (A) DPPH radical scavenging assay, (B) ferrous
seabuckthorn seed oil was measured at 7.7438.5%. The seabuck-
ion chelating assay and (C) reducing power measurement. Each value is expressed
thorn seed oil exhibited much stronger metal chelating activity as mean SD (n = 3).
than trolox, which was found to have weak chelating ability for fer-
rous ions at the same concentration range. However, the metal
chelating activity of seabuckthorn seed oil was signicantly lower atom (Dorman, Peltoketo, Hiltunen, & Tikkanen, 2003). Natural
than that of EDTA, which had the strongest chelating capacity, and antioxidants are believed to break free radical chain reactions by
reached 90.4% at concentration of 0.92 mg/ml. As described above, donating an electron or hydrogen atom to free radicals, so, antiox-
the ferrous chelating ability may be related to antioxidant activity idant activities should be reected in the reducing power. In this
and may modify the antioxidant activity by inuencing other reac- assay, the yellow test solution becomes green or blue when the
tions. These results suggest that seabuckthorn seed oil has a bene- presence of reducers converts the Fe3+/ferricyanide complex to
cial effect on ferrous chelating ability and may thus exert its ferrous form. Thus, higher absorbance at 700 nm indicates
protection against oxidative damage. greater reducing power (Duh, 1998). The reducing power of sea-
buckthorn seed oil and trolox, as the reference compounds, were
3.2.3. Reducing power presented in Fig. 1C. As described in Fig. 1C, reducing power
The reducing power assays are usually used to evaluate the was increased with increasing concentrations of seabuckthorn
capacity of natural antioxidants to donate an electron or hydrogen seed oil and the reference compounds. All spectrophotometric
656 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659
measurements were repeated twice with at least three replicates.
At concentrations of 0.42, 0.83, 2.08, 4.18 and 8.32 mg/ml, the
reducing power of seabuckthorn seed oil was 0.11, 0.12, 0.17,
0.23 and 0.37, respectively, while solutions of trolox at the same
concentrations had reducing power values of 0.82, 1.55, 1.70,
1.69 and 1.69, respectively. These results showed that seabuck-
thorn seed oil has moderate electron donating abilities, which
may be involved in its antioxidant activity.
3.2.4. Inhibition of lipid peroxidation
The ferric thiocyanate method is often used to measure perox-
ide formation in the initial stages of lipid peroxidation. Increased
lipid peroxidation is generally believed to be an important under-
lying cause of oxidative stress initiation upon various tissue inju-
ries, cell death or the progression of many acute and chronic
diseases (Halliwell, 1997). In this assay, the inhibition of lipid per-
oxidation was determined through the formation of a red ferric
thiocyanate, which can be detected at 500 nm by spectrophotom-
etry. Fig. 2 shows the results of seabuckthorn seed oil inhibition
of linoleic acid peroxidation. At 72 h, seabuckthorn seed oil at
doses of 0.60, 1.20, 1.80, 2.40 and 3.00 mg/ml inhibited lipid perox-
idation in the linoleic acid system by 2.43%, 13.9%, 31.9%, 49.6% and
65.8%, respectively. The reference compound, trolox (3.00 mg/ml),
inhibited lipid peroxidation at 6, 24, 48 and 72 h by 40.0%, 39.0%,
27.6% and 18.4%, respectively. These results showed that the sea-
buckthorn seed oil had signicantly higher inhibition of lipid per-
oxidation than trolox.
Several studies have reported that seabuckthorn seed oil con-
tains high concentrations of a-tocopherol, c-tocopherol, b-tocotri-
enol, carotenoids and avonoids, which are known to have
signicant biological activities (Basu et al., 2007; Kallio, Yang,
Peippo, Tahvonen, & Pan, 2002; Negi et al., 2005; Yang, Karlsson,
Oksman, & Kallio, 2001). In the present study, we demonstrated
that the major antioxidants in seabuckthorn seed oil included
a-tocopherol, b-tocotrienol, c-tocopherol, d-tocotrienol and
carotenoids (Table 1). Haraguchi (2001) reported that various
types of phytochemicals, including tocopherols and carotenoids,
are effective in preventing lipid peroxidation. Krinsky (1989) has
also shown that b-carotene protects liposomes against lipid auto-
oxidation mediated by superoxide and hydroxyl radicals as well
as against lipid peroxidation and lysis caused by Fe2+-generated
radicals. Therefore, the tocotrienols and carotenoids enriched sea-
buckthorn seed oil might be responsible for their signicant activ-
ities against lipid peroxidation.
3.2.5. H2O2, superoxide anion radicals and hydroxyl radicals
scavenging activities
Superoxide anion radicals are normally formed in cellular oxi-
dation reactions that can produce hydrogen peroxide and hydroxyl
100 Seabuckthorn seed oil 0.60 mg/mL
Seabuckthorn seed oil 1.20 mg/mL
Seabuckthorn seed oil 1.80 mg/mL
80 Seabuckthorn seed oil 2.40 mg/mL
Seabuckthorn seed oil 3.00 mg/mL
Trolox 3.0 mg/mL
60
40
20
0
0 12 24 36 48 60
Time (hour)
Fig. 2. The lipid peroxidation inhibition of seabuckthorn seed oil by the ferric
thiocyanate method. Each value is expressed as mean SD (n = 3).
radical through dismutation and other chemical reactions. Hydro-
gen peroxide can also disassociate to form hydroxyl radicals
in vivo. Hydroxyl radicals, the most toxic reactive oxygen metabo-
lites, are mainly derived from the interaction of superoxide anion
radicals, hydrogen peroxide and iron salts in the HaberWeiss
and Fenton reactions. These ROS are capable of binding to DNA,
proteins or lipids, leading to membrane lipid peroxidation and -
nally cell necrosis (Halliwell & Gutteridge, 1990). Therefore, ROS
scavengers are of great importance. In this study, chemical lumi-
nescence reaction systems were used to determine the superoxide
anion radical, hydrogen peroxide and hydroxyl radical scavenging
capacities of seabuckthorn seed oil and reference compounds.
The chemiluminescence assays are very sensitive and convenient
methods for determining antioxidant activities. Seabuckthorn seed
oil (at doses of 0.76, 1.53, 3.05, 7.63 and 15.3 mg/ml) proved to be
an effective scavenger of ROS, scavenging 32.182.7%, 20.389.4%
and 49.690.4% of H2O2, superoxide anion radicals and hydroxyl
radicals, respectively. The EC50 values of seabuckthorn seed oil in
the hydrogen peroxide, superoxide radical and hydroxyl radical
scavenging assays were 2.63, 2.16 and 0.77 mg/ml, respectively.
Gallic acid, SOD and DMSO were used as reference compounds
for scavenging H2O2, superoxide anion radicals and hydroxyl radi-
cals with EC50 values of 12.5 mg/ml, 0.31 units and 11.7 mg/ml,
respectively (Table 2). Recently, our co-author used the same
methods to demonstrate that the EC50 values of lutein standard
in the hydrogen peroxide, superoxide radical, hydroxyl radical
scavenging assays, were 6.23, 7.69 and 98.7 mg/ml, respectively,
while solutions of a zeaxanthin standard at the same condition
had EC50 values of 8.38, 7.52 and 85.6, respectively. With these
results, this study conrmed that seabuckthorn seed oil was more
effective in inhibiting H2O2, superoxide anion radicals and hydro-
xyl radicals than lutein and zeaxanthin, which are well-known
antioxidants of xanthophylls.
3.3. In vivo antioxidant potential
3.3.1. Effect of seabuckthorn seed oil on antioxidant enzyme activities
in mice
Several studies reported that antioxidant enzymes, such as SOD,
catalase, GPx and GRd protect against oxidative stress-induced
damage (Halliwell & Gutteridge, 1990). SOD is an exceedingly
effective antioxidant enzyme that converts dismutated superoxide
anions into H2O2. Catalase is a hemoprotein present in all aerobic
cells that metabolizes H2O2 to oxygen and water. GPx catalyses
the reduction of H2O2 and hydroperoxides to nontoxic products
and also helps to detoxify xenobiotics in the liver. GRd is a cyto-
solic hepatic enzyme that is involved in the conjugating reaction
between xenobiotic compounds and GSH (Szymonik-Lesiuk et al.,
2003). In the present study, SOD, catalase, GPx, and GRd were mea-
sured as an index of the antioxidant status of tissues. The results
shown in Fig. 3 reveal a signicant reduction of liver SOD, catalase,
Table 2
The EC50 values of seabuckthorn seed oil and reference compounds in hydrogen
peroxide, superoxide radical and hydroxyl radical scavenging assays.
EC50a
H2O2 Superoxide anion Hydroxyl radicals
Seabuckthorn seed oil 2.63 0.08 2.16 0.11 0.77 0.05
(mg/ml)
Gallic acid (mg/ml) 12.5 0.78
SOD (unit) 0.31 0.03
72 DMSO (mg/ml) 11.7 0.72
Each value is expressed as mean SD (n = 3).
a
EC50 means the effective concentration of sample that can decrease the ROS
concentration by 50%.
 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659
Fig. 3. Effects of seabuckthorn seed oil on liver antioxidant enzymes in CCl4 intoxicated mice. (A) SOD and catalase. (B) GSH-Px and GSH-Rd. Values are the mean SEM for 10
mice; ap < 0.05 compare with normal control; bp < 0.05 compare with CCl4-treated control group.
and GRd activity in CCl4-treated mice compared to vehicle-control
mice (p < 0.05), indicating that CCl4 damaged the enzymatic anti-
oxidant defence system. In contrast, signicant increases in SOD,
catalase, GPx, and GRd activities were observed in the seabuck-
thorn seed oil-treated groups at doses of 0.26, 1.30 and 2.60 mg/
kg, respectively, compared to the CCl4-treated control group
(p < 0.05). Administration of silymarin, the positive control used
in this test, signicantly increased SOD, catalase, GPx and GRd
activities relative to CCl4 treatment alone (p < 0.05).
Szymonik-Lesiuk et al. (2003) indicated that antioxidant en-
zymes are inactivated by lipid peroxides or free radicals, which re-
sults in decreased activities of these enzymes, as seen by the CCl4
toxicity. On the other hand, the activities of antioxidant enzymes
are important to protect cells or organs against oxidative damage.
The results of the present study indicate that SOD, catalase, GPx
and GRd activities were signicantly elevated by the administra-
tion of seabuckthorn seed oil to CCl4-treated mice, suggesting that
seabuckthorn seed oil has the ability to protect antioxidant en-
zyme activities in the CCl4-damaged liver. Similar results from
our previous research conrmed that seabuckthorn seed oil played
a protective role in the reduction of oxidative stress, and restored
the activities of enzymes in the antioxidant defence system, such
as SOD, catalase, GPx and GRd (Hsu et al., 2009).
3.3.2. Effects of seabuckthorn seed oil on GSH level
GSH acts as a non-enzymatic antioxidant that decreases H2O2,
hydroperoxide and xenobiotic toxicity. GSH is readily oxidised to
glutathione disulphide (GSSG) upon reaction with xenobiotic com-
657
pounds, which may then cause a decrease in GSH levels. GSSG is
either rapidly reduced by GRd and NADPH or utilised in the endo-
plasmic reticulum to aid protein-folding processes. Eventually,
GSSG is recycled by the protein disulphide isomerase to form
GSH. Because of these recycling mechanisms, GSH is an extremely
efcient intracellular buffer for oxidative stress (Cantin et al.,
2007). Therefore, it appears that GSH conjugation is essential to de-
crease the toxic effects of CCl4. In the present study, the hepatic
GSH content was signicantly decreased in CCl4-treated mice com-
pared with control vehicle-treated mice (Fig. 4). GSH levels in the
seabuckthorn seed oil treated group at all test doses were signi-
cantly higher than those of the CCl4-treated control group, suggest-
ing that seabuckthorn seed oil could protect against the CCl4-
induced depletion of hepatic GSH. A similar observation was also
reported by Cheng et al. (1994). They indicated that seabuckthorn
seed oil markedly inhibited the acetaminophen-induced depletion
of hepatic GSH in mice.
3.3.3. Effect of seabuckthorn seed oil on lipid peroxidation
MDA is widely used as a marker of free radical-mediated lipid
peroxidation injury. MDA is the major reactive aldehyde that ap-
pears during the peroxidation of polyunsaturated fatty acids in bio-
logical membranes. An increase in the MDA levels in the liver
imply enhanced peroxidation leading to tissue damage and the for-
mation of excess free radicals that overwhelm the antioxidant de-
fence mechanisms (Buege & Aust, 1978). In this assay, TBA reacts
with MDA to form a pink chromogenic adduct that can be detected
spectrophotometrically at 532 nm. The results of the MDA assays
658 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659

8
b b
b b
b
6 a

0
Normal control Vehicle control CCl4 control Silymarin (200 Seabuckthorn Seabuckthorn Seabuckthorn
(1 mL/kg) mg/kg) + CCl4 seed oil (0.26 seed oil (1.30 seed oil (2.60
mg/kg) + CCl4 mg/kg) + CCl4 mg/kg) + CCl4

Fig. 4. Effects of seabuckthorn seed oil on liver GSH in CCl4 intoxicated mice. Values are the mean SEM for 10 mice; ap < 0.05 compare with normal control; bp < 0.05
compare with CCl4-treated control group.

10 a
9
8
7 ab
6
5 b
b b
4 b
ab
3
2
1
0
Normal control Vehicle control CCl4 control Silymarin (200 Seabuckthorn Seabuckthorn Seabuckthorn
(1 mL/kg) mg/kg) + CCl4 seed oil (0.26 seed oil (1.30 seed oil (2.60
mg/kg) + CCl4 mg/kg) + CCl4 mg/kg) + CCl4

Fig. 5. Effects of seabuckthorn seed oil on liver MDATBA in CCl4 intoxicated mice. Values are the mean SEM for ten mice; ap < 0.05 compare with normal control; bp < 0.05
compare with CCl4-treated control group.

in the present study are shown in Fig. 5. The MDATBA levels in the
CCl4-treated group were signicantly higher than those in the
vehicle-control group (p < 0.05), and treatment with seabuckthorn
seed oil signicantly reversed these changes. The administration of
seabuckthorn seed oil caused a signicant decrease in the MDA
TBA levels compared to the CCl4-treated group (p < 0.05), suggest-
ing that seabuckthorn seed oil can protect against CCl4-induced li-
pid peroxidation in mice. Silymarin also inhibited the elevation of
the MDATBA levels upon CCl4 administration (Fig. 5). These re-
sults are consistent with previous research, which conrmed that
the seabuckthorn seed oil could protect against MDATBA forma-
tion in the liver induced by acetaminophen, CCl4 and ethyl alcohol
(Cheng, 1992).
4. Conclusions
The results of this study demonstrated that seabuckthorn seed
oil contained abundant unsaturated fatty acids, tocopherols and
carotenoids, which are known to have signicant antioxidant
activities. Furthermore, we also found that seabuckthorn seed oil
exhibited remarkable antioxidant activity on the DPPH radical
scavenging activity, ferrous ion chelating activity, reducing power
and inhibition of lipid peroxidation activity, as well as high scav-
enging activities of superoxide anion radical, hydrogen peroxide
and hydroxyl radical. The seabuckthorn seed oil also showed
strong inhibition of oxidative damage induced by CCl4 on mice, in-
creased the activities of SOD, catalase, GPx, GRd and GSH content,
and decreased the MDATBA content in liver. The in vitro and
in vivo elevations of antioxidant activities by seabuckthorn seed
oil may be due to the presence of carotenoids and tocopherols. This
phenomenon appears to be mediated primarily by the ability of
carotenoids to quench excited sensitizer molecules and singlet
oxygen (Krinsky, 1989). Furthermore, Basu et al. (2007) showed
that a-tocopherol, an efcient antioxidant, is the major form of
vitamin E in seabuckthorn seed oil, and previous studies in our
group also found that dietary seabuckthorn seed oil supplementa-
tion of CCl4-treated mice resulted in a signicant reduction of lipid
peroxidation and enhancement of the antioxidant defence system
(Hsu et al., 2009). Therefore, the carotenoids and tocopherols con-
tent of seabuckthorn seed oil are reected by its in vitro and in vivo
antioxidant properties. Based on the assays presented here, it can
be concluded that seabuckthorn seed oil is an accessible source
of natural antioxidants that provides the expected health benets.
References
Arimboor, R., Venugopalan, W., Sarinkumar, K., Arumughan, C., & Sawhney, R. C.
(2006). Integrated processing of fresh Indian sea buckthorn (Hippophae
rhamnoides) berries and chemical evaluation of products. Journal of the Science
of Food and Agriculture, 86, 23452353.
 H.-C. Ting et al. / Food Chemistry 125 (2011) 652659
Basu, M., Prasad, R., Jayamurthy, P., Pal, K., Arumughan, C., & Sawhney, R. C. (2007).
Anti-atherogenic effects of seabuckthorn (Hippophaea rhamnoides) seed oil.
Phytomedicine, 14, 770777.
Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. In Sidney Fleischer
& Leister Packer (Eds.). Methods in enzymology (Vol. 52, pp. 302310). New
York, San Francisco, London: Academic Press.
Cantin, A. M., White, T. B., Cross, C. E., Forman, H. J., Sokol, R. J., & Borowitz, D.
(2007). Antioxidants in cystic brosis conclusions from the CF antioxidant
workshop, Bethesda, Maryland, November 1112, 2003. Free Radical Biology and
Medicine, 42, 1531.
Chen, B. H., Chuang, J. R., Lin, J. H., & Chiu, C. P. (1993). Quantication of provitamin
A compounds in chinese vegetables by high-performance liquid
chromatography. Journal of Food Protection, 56, 5154.
Cheng, T. J. (1992). Protective action of seed oil of Hippophae rhamnoides L. (HR)
against experimental live injury in mice. Zhongghua Yu Fang Yi Xue Za Zhi, 26,
227229 [in Chinese].
Cheng, T. J., Pu, J. K., Wu, L. W., Ma, Z. R., Cao, Z., & Li, T. J. (1994). An preliminary
study on hepato-protective action of seed oil of Hippophae rhamnoides L. (HR)
and mechanism of the action. Zhongguo Zhong Yao Za Zhi, 19, 367370 (in
Chinese).
Cunha, S. C., Amaral, J. S., Fernandes, J. O., & Oliveira, M. B. P. P. (2006).
Quantication of tocopherols and tocotrienols in Portuguese olive oils using
HPLC with three different detection systems. Journal of Agricultural and Food
Chemistry, 54, 33513356.
Dinis, T. C. P., Maderia, V. M., & Almeida, L. M. (1994). Action of phenolic derivatives
(acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane
lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry
and Biophysics, 315, 161169.
Dorman, H. J. D., Peltoketo, A., Hiltunen, R., & Tikkanen, M. J. (2003).
Characterization of the antioxidant properties of deodorised aqueous extracts
from selected Lamiaceae herbs. Food Chemistry, 83(2), 255262.
Duh, P. D. (1998). Antioxidant activity of burdock (Arctium lappa Linne): Its
scavenging effect on free-radical and active oxygen. Journal of the American Oil
Chemists Society, 75(4), 455461.
Epsin, J. C., Soler-Rivas, C., & Wichers, H. J. (2000). Characterization of the total free
radical scavenger capacity of vegetable oils and oil fractions using 2,2-diphenyl-
2-picrylhydrazyl radical. Journal of Agricultural and Food Chemistry, 48, 648656.
Geetha, S., Jayamurthy, P., Pal, K., Pandey, S., Kumar, R., & Sawhney, R. C. (2008).
Hepatoprotective effects of sea buckthorn (Hippophae rhamnoides L.) against
carbon tetrachloride induced liver injury in rats. Journal of the Science of Food
and Agriculture, 88, 15921597.
Geetha, S., Sai Ram, M., Singh, V., Ilavazhagan, G., & Sawhney, R. C. (2002). Anti-
oxidant and immunomodulatory properties of seabuckthorn (Hippophae
rhamnoides): An in-vitro study. Journal of Ethnopharmacology, 79, 373378.
Halliwell, B. (1997). Antioxidants and human disease: A general introduction.
Nutrition Reviews, 55, 4452.
Halliwell, B., & Gutteridge, J. M. C. (1990). Role of free radicals and catalytic metal
irons in human disease: An overview. Methods in Enzymology, 186, 5985.
Haraguchi, H. (2001). Antioxidative plant constituents. In Bioactive compounds from
natural sources, rst published (pp. 338377). New York: Taylor and Francis Inc.
Hsu, Y. W., Tsai, C. F., Chen, W. K., & Lu, F. J. (2009). Protective effects of
seabuckthorn (Hippophae rhamnoides L.) seed oil against carbon tetrachloride-
induced hepatotoxicity in mice. Food and Chemical Toxicology, 47, 22812288.
659
Hu, C. C., Lin, J. T., Lu, F. J., Chou, F. P., & Yang, D. J. (2008). Determination of
carotenoids in Dunaliella salina cultivated in Taiwan and antioxidant capacity of
the algal carotenoid extract. Food Chemistry, 109, 439446.
Kallio, H., Yang, B., Peippo, P., Tahvonen, R., & Pan, R. (2002). Triacylglycerols,
glycerophospholipids, tocopherols, and tocotrienols in berries and seeds of two
subspecies (ssp. sinensis and ssp. mongolica) of sea buckthorn (Hippopha
rhamnoides). Journal of Agricultural and Food Chemistry, 50, 30043009.
Krinsky, N. I. (1989). Antioxidant functions of carotenoids. Free Radical Biology and
Medicine, 7, 617635.
Mitsuda, H., Yasumodo, K., & Iwami, K. (1966). Antioxidative action of indole
compounds during the autoxidation of linoleic acid. Nihon Eiyo Shokuryo Gakkai
Shi, 19, 210214.
Morrison, W. R., & Smith, L. M. (1964). Preparation of Fatty acid methyl esters and
dimethyl acetals from lipid with borontriuoroide methanol. Journal of Lipid
Research, 5, 600605.
Negi, P. S., Chauhan, A. S., Sadia, G. A., Rohinishree, Y. S., & Ramteke, R. S. (2005).
Antioxidant and antibacterial activities of various seabuckthorn (Hippophae
rhamnoides L.) seed extracts. Food Chemistry, 92, 119124.
Oosthuizen, M. M., Engelbrecht, M. E., Lambrechts, H., Greyling, D., & Levy, R. D.
(1997). The effect of pH on chemiluminescence of different probes exposed to
superoxide and singlet oxygen generators. Journal of Bioluminescence and
Chemiluminescence, 12, 277284.
Purushothaman, J., Suryakumar, G., Shukla, D., Malhotra, A. S., Kasiganesan, H.,
Kumar, R., et al. (2008). Modulatory effects of seabuckthorn (Hippophae
rhamnoides L.) in hypobaric hypoxia induced cerebral vascular injury. Brain
Research Bulletin, 77, 246252.
Szymonik-Lesiuk, S., Czechowska, G., Stryjecka-Zimmer, M., Slomka, M., Madro, A.,
Celinski, K., et al. (2003). Catalase, superoxide dismutase, and glutathione
peroxidase activities in various rat tissues after carbon tetrachloride
intoxication. Journal of HepatoBiliaryPancreatic Surgery, 10, 309315.
Upadhyay, N. K., Kumar, R., Mandotra, S. K., Meena, R. N., Siddiqui, M. S., Sawhney, R.
C., et al. (2009). Safety and healing efcacy of sea buckthorn (Hippophae
rhamnoides L.) seed oil on burn wounds in rats. Food and Chemical Toxicology, 47,
11461153.
Vitaglione, P., Morisco, F., Caporaso, N., & Fogliano, V. (2004). Dietary antioxidant
compounds and liver health. Critical Reviews in Food Science and Nutrition, 44,
575586.
Willett, W. C., Polk, B. F., Underwood, B. A., Stampfer, M. J., Pressel, S., Rosner, B.,
et al. (1984). Relation of serum vitamins A and E and carotenoids to the risk of
cancer. The New England Journal of Medicine, 310, 430434.
Yang, B., & Kallio, H. (2002). Composition and physiological effects of seabuckthorn
(Hippophae) lipids. Trends in Food Science and Technology, 13, 160167.
Yang, B., Karlsson, R., Oksman, P., & Kallio, H. (2001). Phytosterols in sea buckthorn
(Hippopha rhamnoides L.) berries: Identication and effects of different origins
and harvesting times. Journal of Agricultural and Food Chemistry, 49, 5620
5629.
Yldz, G., & Demiryrek, A. T. (1998). Ferrous iron-induced luminol
chemiluminescence: A method for hydroxyl radical study. Journal of
Pharmacological and Toxicological Methods, 39, 179184.
Yoshiki, Y., Yamanaka, T., Satake, K., & Okubo, K. (1999). Chemiluminescence
properties of soybean protein fraction in the hydroperoxide and hydrogen
donor system. Luminescence, 14, 315319.