Food Chemistry 120 (2010) 1004–1010

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

Antiproliferative effects of sea buckthorn (Hippophae rhamnoides L.) extracts

on human colon and liver cancer cell lines
Carl Grey a,*, Cecilia Widén b, Patrick Adlercreutz a, Kimmo Rumpunen b, Rui-Dong Duan c
a
Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
b
Department of Plant Breeding and Biotechnology Balsgård, Swedish University of Agricultural Sciences, Fjälkestadsvägen 459, SE-291 94 Kristianstad, Sweden
c
Gastroenterology and Nutrition Laboratory, Biomedical Center, B11, Lund University, SE-221 84 Lund, Sweden

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

Article history: Sea buckthorn berries contain many bioactive compounds that have anticancer properties. To investigate
Received 11 August 2009 whether the antiproliferative effects could be associated with the presence of certain compounds, a
Received in revised form 6 October 2009 sequential extraction was performed. The extraction started with heptane followed by ethyl acetate, eth-
Accepted 20 November 2009
anol, and water. A second protocol using ethanol:water (1:1) was also used. The contents of the extracts
were determined and their effects on cell proliferation were investigated in both Caco-2 and Hep G2 cells.
The ethyl acetate fraction was exclusively found to contain high levels of ursolic acid, together with low
Keywords:
amounts of phenolics. The ethanol:water extracts contained high levels of phenolic compounds and pro-
Antiproliferation
Apoptosis
anthyocyanidin, but little ursolic acid. When the antiproliferative effects were examined, the strongest
Caco-2 cells inhibitory effect was found in the ethyl acetate extract for the Caco-2 cells and in the ethanol:water
Cell culture extract for the Hep G2 cells. The antiproliferative effects were in both cases dose-dependent and were
Hep G2 cells in the case of the ethyl acetate extract associated with an increase in apoptosis. The results obtained show
Isorhamnetin that the choice of extraction solvent is of considerable importance and that ursolic acid might be more
Phenols important than the polyphenols in inhibiting the cancer cell proliferation.
Proanthocyanidin Ó 2009 Elsevier Ltd. All rights reserved.
Rutin
Sea buckthorn
Ursolic acid

1. Introduction
Sea buckthorn (Hippophae L.) is a small Eurasian genus with
species of great economic potential due to the possible use of dif-
ferent parts of the plants: as nutritious food and fodder, as pharma-
ceutical and cosmetic ingredients, as a soil enhancer, and as a
source of energy. Today, there are seven recognized species in
the genus, of which Hippophae rhamnoides has the widest distribu-
tion (Swenson & Bartish, 2002). Thanks to Russian and European
plant breeders, domestication efforts based on native H. rhamno-
ides subsp. mongolica and H. rhamnoides subsp. rhamnoides have re-
sulted in access to several cultivars with both wide and specific
adaptation. This plant material forms the basis of the growing pro-
duction of sea buckthorn berries in Western Europe, especially in
the countries around the Baltic Sea.
Sea buckthorn berries are a rich source of many different bioac-
tive compounds that may contribute to the claimed and proven
health benefits of sea buckthorn juice and oil. Major bioactive com-
pounds that have already been identified in the berries are ascorbic
* Corresponding author. Tel.: +46 46 222 0858; fax: +46 46 222 4713.
E-mail address: carl.grey@biotek.lu.se (C. Grey).
acid (Tiitinen, Hakala, & Kallio, 2005; Tiitinen, Yang, Haraldsson,
Jonsdottir, & Kallio, 2006), carotenoids (Andersson, Olsson, Johans-
son, & Rumpunen, 2009), lipids (Kallio, Yang, Peippo, Tahvonen, &
Pan, 2002; Yang & Kallio, 2001, 2006), phenols (Arimboor, Kumar,
& Arumughan, 2008; Rosch, Bergmann, Knorr, & Kroh, 2003; Shar-
ma et al., 2008), phytoestrogens (Yang, Linko, Adlercreutz, & Kallio,
2006), phytosterols (Li, Beveridge, & Drover, 2007; Yang, Karlsson,
Oksman, & Kallio, 2001), tocopherols (Andersson, Rumpunen,
Johansson, & Olsson, 2008; Kallio, Yang, & Peippo, 2002), and triter-
penes (Cossuta, Simandi, Hohmann, Doleschall, & Keve, 2007), all
in significant amounts.
Sea buckthorn was used in medicine as much as 1000 years ago
as an effective herb to treat various conditions such as arthritis,
wounds, fever, pain, cough, stomach discomfort, and gynecological
diseases in India, China, Mongolia, Russia and the Middle East. Re-
cently, several biological effects of extracts of sea buckthorn have
been identified and some of them have been tested in clinical trials.
Oral administration of sea buckthorn oil in patients with atopic
dermatitis was found to improve the inflammation, and the effects
were correlated with the reduced formation of 4-leukotrienes and
increased formation of 5-leukotrienes from arachidonic acid (Yang
et al., 1999). Both animal studies and clinical trials have shown

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.11.039
 C. Grey et al. / Food Chemistry 120 (2010) 1004–1010
that seed oil of sea buckthorn has a protective effect against tissue
injury. It has been found to inhibit liver damage induced by CCl4 in
rats (Gao, Gu, Cheng, & Jiang, 2003) and to increase the survival
rate of mice treated with irradiation (Agrawala & Goel, 2002; Ku-
mar, Namita, & Goel, 2002). Cell culture studies have shown that
extract of sea buckthorn can inhibit cell proliferation or promote
apoptosis in cancer cells such as HT29 colon cancer cells, MCF-7
breast cancer cells, and HL-60 leukemia cells (Hibasami et al.,
2005; Olsson, Gustavsson, Andersson, Nilsson, & Duan, 2004). In
addition, sea buckthorn may also affect lipid metabolism, resulting
in decreasing LDL levels, increasing HDL levels, and decreasing
platelet aggregation, thus having an influence on the pathogenesis
of cardiovascular diseases (Eccleston et al., 2002; Johansson, Korte,
Yang, Stanley, & Kallio, 2000; Yang & Kallio, 2002). Apart from their
effects on mammalian cells, extracts from sea buckthorn inhibit
the growth of several strains of bacteria, particularly those that col-
onize the gastrointestinal tract including Helicobacter pylori (Li, Xu,
Zhang, Liu Jun, & Tan Ren, 2005).
While several biological effects of sea buckthorn have been re-
ported, the effects vary significantly depending on the type of ex-
tract used, i.e. on the composition of sea buckthorn extracts
(Yang & Kallio, 2002). Many bioactive compounds with possible
anticarcinogenic effects may be found within the groups of poly-
phenols and triterpens. Anticarcinogenic effects have previously
been demonstrated for isorhamnetin (Teng, Lu, Wang, Tao, &
Wei, 2006) and ursolic acid (Andersson, Liu, Nilsson, & Duan,
2003; Pathak et al., 2007). The objective of the present study was
(1) to compare the antiproliferative and apoptotic effects of differ-
ent sea buckthorn extracts on human colon cancer cells (the Caco-
2 cell line) and human liver cancer cells (the Hep G2 cell line), and
(2) to determine whether the antiproliferative activities of sea
buckthorn extracts are associated with the content of different bio-
active compounds.
2. Materials and methods
2.1. Chemicals and cells
Caco-2 and Hep G2 cells were purchased from the American Tis-
sue Culture Collection (Rockville, MD, USA). The Cell Death Detec-
tion kit and the Cell Proliferation Reagent, 4-[3-(4-lodophenyl)-2-
(4-nitrophenyl)-2H-5-tetrazoliol]-1,3-benzene disulfonate (WST-
1), were obtained from Roche Diagnostics GmbH (Mannheim, Ger-
many). The Folin–Ciocalteau reagent (85% ortho-phosphoric acid,
DMSO, methanol, acetonitrile and acetic acid) was obtained from
Merck (Darmstadt, Germany). Gallic acid, sodium carbonate, and
ursolic acid were from Sigma–Aldrich (Seelze, Germany). The stan-
dards used for HPLC analysis (procyanidin B2, isorhamnetin-3-O-
rutinoside, kaempferol-3-glucoside, isorhamnetin-3-O-rutinoside,
and rutin were purchased from Extrasynthese (Genay, France).
2.2. Plant materials
To evaluate the effects of different extraction solvents, whole
berries from the sea buckthorn cultivar H. rhamnoides ‘BHi
10726’ were coarsely ground while still frozen and then lyophilised
before extraction. To screen cultivars for ursolic acid content and to
evaluate the effects, berries from twenty-four cultivars and ad-
vanced selections of sea buckthorn were lyophilised. The seeds
were removed and the flesh with skin was ground in a laboratory
mill (Yellow line, A10; IKA-Werke, Staufen, Germany) before
extraction. Three cultivars of sea buckthorn with different ursolic
acid content in the berries were then chosen for further tests on
cell cultures: low (H. rhamnoides ‘BHi 10941’), medium (H. rhamno-
1005
ides ‘BHi 10726’), and high ursolic acid content (H. rhamnoides
‘Podaruk Sadu’).
2.3. Preparation of extracts
Two protocols were used to extract the sea buckthorn berries.
The first protocol was a sequential extraction using four different
solvents of different polarity, starting with the most unpolar. The
solvents were used in the following order: n-heptane, ethyl ace-
tate, 96% ethanol, and water. The second protocol contained one
solvent mixture, ethanol:acidified water (1:1), the water phase
containing 50 mM H3PO4. Lyophilised sea buckthorn berries with
or without seeds (5 g) were put in a 500 ml E-bottle. Solvent
(50 ml) was added, the bottle was capped, and the extraction
was carried out at ambient temperature in the dark, with gentle
shaking for 24 h. The extract was filtered, the solvent of the filtrate
was removed using a rotary evaporator and the remaining filtrate
was stored at 20 °C. Before the proliferation and apoptosis exper-
iments, the concentrated extracts were dissolved in 2 ml DMSO,
except the heptane and water fractions. The concentrated heptane
extract was already liquid and therefore used as it was. The dried
water extract was dissolved in water and filter-sterilised before it
was used in the cell experiments. In the first protocol, the berries
(on the filter) were returned to the flask before the next solvent
was added and the extraction proceeded in the same manner as
just described.
2.4. Analysis of phenols
The content of total phenols was determined according to the
Folin–Ciocalteau method (Singleton, Orthofer, & Lamuela-Raven-
tos, 1999). The extracts were mixed with Folin–Ciocalteau reagent,
15% Na2CO3, and water. The absorbance was measured at 765 nm
after 120 min using a Shimadzu spectrophotometer (UV-2101PC).
Gallic acid was used as a standard and the total amount of pheno-
lics was expressed as lg of gallic acid equivalents per gramme dry
weight.
Selected phenolic compounds were analysed on a Shimadzu
HPLC system equipped with a diode-array detector according to
a method slightly modified from Schieber, Keller, and Carle
(2001) and Tsao, Yang, Christopher, Zhu, and Zhu (2003). The elu-
ent consisted of solvent A (0.952% acetic acid, 4.76% acetonitrile)
and solvent B (95% acetonitrile, 5% methanol). The binary gradient
was as follows: 2% B (0–2 min), 15% B (2–8 min), 21% B (8–28 min),
80% B (28–37 min), and 2% B (37–40 min). A Phenomenex Synergi
4l Hydro-RP 80A column (250  4.6 mm) and a guard C18 precol-
umn were used. Evaluation of data was carried out with Shimadzu
Class-VP software (version 6.13 SP2). Retention times and spectral
data were obtained and compared with those of the external stan-
dards rutin, isorhamnetin-3-O-rutinoside, kaempferol-3-glucoside,
and isorhamnetin-3-O-rutinoside. Detection was carried out at
350 nm, the injection volume was 10 ll, and the flow rate was
1.0 ml/min.
2.5. Analysis of proanthocyanidins
Proanthocyanidins were analysed according to the method pub-
lished by Salminen et al. (2005). The eluent consisted of solvent A
(0.05 M H3PO4) and solvent B (acetonitrile). The binary gradient
was as follows: 2% B (0–3.5 min), 70% B (3.5–6 min), 95% B (6–
8.5 min), and 0% B (8.5–15 min). As the stationary phase a Super-
spher 100 RP-18 (75  4 mm) column and a C18 precolumn were
used. Detection was carried out at 240 and 510 nm, and data were
compared with those of procyanidin B2 as a standard. The injection
volume was 5 ll and the flow rate was 1.0 ml/min.
1006 C. Grey et al. / Food Chemistry 120 (2010) 1004–1010
2.6. Analysis of ursolic acid
The ursolic acid content of the samples was determined by the
method described by (Cui et al., 2006). The isocratic eluent con-
sisted of 92% methanol and 0.03% H3PO4. The separation was per-
formed using a Phenomenex Synergi 4l Hydro-RP 80A
(250  4.6 mm) column and a guard C18 precolumn. The injection
volume was 10 ll and the flow rate was 0.8 ml/min. Detection was
carried out at 210 nm against a standard of ursolic acid. Each sam-
ple was analysed in triplicate.
2.7. Cell culture
Monolayer cultures of Caco-2 cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) with 1 mM sodium pyruvate,
and Hep G2 cells were cultured in RMPI-1640 medium with L-glu-
tamine. Both media contained 100 IU/ml penicillin, 10 lg/mL
streptomycin, and 10% heat-inactivated FBS. The cells were grown
and maintained at 37 °C in an incubator containing 95% air and 5%
CO2. The cells were harvested with 0.05% trypsin/0.02% EDTA at
about 80% confluence, and subcultured in their respective media
before use. After 24 h incubation for attachment, the cells were
treated with medium containing sea buckthorn extracts or ursolic
acid, used as a positive control, at various concentrations for 48 h.
Sea buckthorn extracts and ursolic acid stock solution (80 mM)
were dissolved in DMSO. Medium containing 0–2% DMSO (depend-
ing on the experiment) was used as control.
2.8. Assay of cell viability
The cell viability was assayed by determination of the cleavage
of tetrazodium salt (WST-1) to formazan by mitochondrial dehy-
drogenases as described previously by (Olsson et al., 2004). The
amount of formazan formed is proportional to the number of via-
ble cells. Briefly, 2  104 cells in 200 ll medium were incubated
with sea buckthorn extracts or ursolic acid in a microtiter plate
as described above. The medium was then removed and the cells
were incubated with 110 ll medium containing 10 ll WST-1 re-
agent for about 1 h. The production of formazan was determined
spectrophotometrically at 415 nm against 655 nm as a back-
ground. The results were expressed as a percentage of the control.
The sample size for each concentration was six wells and most
experiments were repeated at least twice.
2.9. Assay of apoptosis
Apoptosis was detected using the Cell Death Detection ELISA
kit, which is based on a semiquantitative determination of the
enrichment of mono- and oligonucleosomes in the cytoplasm
(Liu et al., 2002). Briefly, 1  104 cells were seeded in a 96-well
microplate and incubated with sea buckthorn extracts or ursolic
acid for either 24 h for the Caco-2 cells or 48 h for the Hep G2 cells.
The cells were then lysed and centrifuged. The supernatant con-
Table 1
The levels of phenolic compounds and ursolic acid of 24 sea buckthorn cultivars, given as lg/g d.w. ± SD.
Extracta TP PC RT
Heptane 362 ± 9 – –
Ethyl acetate 1234 ± 50 998 ± 53 18.3 ± 0.4
Ethanol 2632 ± 18 2365 ± 68 79.6 ± 2.8
Water 456 ± 20 683 ± 50 6.2 ± 0.1
Ethanol:water (1) 3737 ± 56 2342 ± 148 79.8 ± 4.1
Ethanol:water (2) 4213 ± 42 6836 ± 525 96.2 ± 13.6
a
TP = total phenolics; PC = proanthocyanidine; RT = rutin; IH-3R = isorhamnetin-3-O-rutinoside; IH-3G = isorhamnetin-3-O-glucoside; KF-3G = kaempferol-3-glucoside;
UA = ursolic acid.
taining the cytoplasmic histone-associated DNA fragments was re-
acted with the anti-histone antibodies labelled with biotin, and
anti-DNA antibodies coupled to peroxidase, for 2 h. The substrates
of the peroxidase were added and color development was mea-
sured with a microplate reader (Bio-Rad, Stockholm, Sweden) at
415 nm against 490 nm as background. The specific enrichment
of mono- and oligonucleosomes released into the cytoplasm was
expressed as enrichment factor relative to the control. In both
the apoptosis and the proliferation assay, ursolic acid at 80 lM
was used as a positive control. Our previous study have shown that
at this concentration ursolic acid significantly inhibited cell prolif-
eration and induced apoptosis (Andersson et al., 2003).
2.10. Statistical analysis
The results of the proliferation experiments were expressed as
the mean ± the 95 % confidential interval. Each observation was re-
peated in six wells in one to five separate experiments. Confiden-
tial intervals were calculated using the Student’s-t distribution
and the two tailed t-test was used to determine significant results.
Outliers were rejected if they were found positive in Grubbs’ test at
a significance level of 5%.
3. Results and discussion
The levels of phenolic compounds and ursolic acid of different
extracts is given in Table 1. The heptane extract had the lowest
concentration of total phenols, and an initial screening also re-
vealed a very low content of ursolic acid (close to the detection
limit). Also, the water extract contained low amounts of the com-
pounds analysed. As expected, the extracts using ethanol were
generally rich in phenolic compounds. The ethanol:water extracts
(1 and 2) had consistently higher concentrations of phenolics than
the other extracts; finely ground berries were used in the second
extract protocol, which contained higher levels of most compounds
analysed than the first extraction method. The ethyl acetate extract
clearly had the highest level of ursolic acid – about ten times the
amount of the ethanol extract – but, on the other hand, quite
low concentrations of most phenolic compounds compared to
the ethanol extracts.
DMSO was used to dissolve all the extracts (except the water
and heptane fractions). In order to dissolve as much solids of the
evaporated extract as possible, a relatively high amount of DMSO
had to be used. Therefore, the effect on cell proliferation of DMSO
alone was investigated, before examining the biological effects of
the extracts. A slight inhibition by DMSO occurred in the Caco-2
cell line (p < 0.05 above 1%), whereas a significant (p < 0.05) in-
crease in the proliferation was observed in the Hep G2 cells
(Fig. 1). Thus, when DMSO concentrations above 0.5% are used, it
is also necessary to consider the effect of DMSO alone. The inhibi-
tion of cell proliferation found in Caco-2 cells by DMSO is in agree-
ment with the toxic properties of DMSO (Crawford & Braunwald,
1991). The stimulation of the Hep G2 cell proliferation might be
IH-3R IH-3G KF-3G UA
– – – –
149 ± 3 126 ± 2 4.5 ± 0.1 946 ± 17
490 ± 16 250 ± 8 8.9 ± 0.3 92 ± 2
31 ± 0.4 6±5 0.6 ± 0.02 4±2
498 ± 24 282 ± 13 9.9 ± 0.5 43 ± 6
549 ± 29 300 ± 16 11.8 ± 2.2 20 ± 16
 C. Grey et al. / Food Chemistry 120 (2010) 1004–1010 1007

160 the proliferation to a higher degree than the effect of DMSO alone
Caco-2 (**)
(*) at 1% and 2%. The second extract (ethanol:water (2)) using fine
140 Hep G2
ground berries was found to be more effective. However, the dose
dependency was quite different compared to that of the ethyl ace-
Cell proliferation (%)

120
tate extract, since the effect was rather sudden and no concentra-
100 (*) tion giving an intermediate inhibition was found. In addition, pure
(**)
ursolic acid used as positive control at 80 lM was found to strongly
80
suppress the cell proliferation to about 3% of the control (data not
60 shown); this was comparable to the effects of the 2% ethyl acetate
extracts that contained approximately 100 lM ursolic acid.
40
At this stage it was decided to continue with the extracts show-
20 ing the strongest inhibitory effects, the ethyl acetate fraction and
the two ethanol:water extracts. As shown in Fig. 3, these extracts
0 were tested on Hep G2 cells, a different cancer cell line In contrast
0.5 1 2
to the results with Caco-2 cells, the ethanol:water extracts inhib-
DMSO concentration % (v/v)
ited the proliferation more efficiently than the ethyl acetate frac-
Fig. 1. Effects of different concentrations of DMSO on proliferation of Hep G2 and tion, especially the second ethanol:water extract. Also, a more
Caco-2 cells. Pure medium was used as control. Error bars represent the 95% clear dose-dependant response was found for the ethanol:water
confidence intervals. () Indicate significance p < 0.05 and () indicate p < 0.01, extract up to 1%. But at 2% all of the extracts increased cell prolif-
compared to the control. eration. The reason for the weak effect of the ethyl acetate extracts
in Hep G2 cells may indicate that hepatic cells, which are glandular
cells, are less sensitive than the epithelial Caco-2 cells. The less
effective inhibition in cell proliferation induced by the etha-
160
nol:water extracts at 2% was probably due to the increase in cell
abcdf
140 abd
proliferation caused by the relatively high DMSO concentration
bc
abc (see Fig. 1). Ursolic acid was also included in these experiments,
120 bc abcef abcef Hep
Cell proliferation (%)

b and a concentration of 80 lM inhibited cell proliferation to about

b
EtAc
100 bcd bf 7%.
f EtOH
adef
bdef bdef
The results obtained above showed a strong inhibition of cell
adef bdef H2O
80 proliferation for the ethyl acetate and ethanol:water extracts. The
abdef EtOH:H20 (1)
acde abcde results are consistent with a previous study in which etha-
60 acde EtOH:H2O (2)
nol:water-derived sea buckthorn extracts were also found to inhi-
acde
40 bit proliferation of HT29 and MCF-7 cells, although the inhibition
acdf
acd was reached at higher concentrations of extract (Olsson et al.,
20
2004). The antiproliferation results for ursolic acid obtained here
0 for both Caco-2 and Hep G2 are also similar to the effects reported
0.25 0.5 1 2 in a previous study using 40 and 80 lM ursolic acid on HT29 cells
-20 (Andersson et al., 2003).
Extract concentration % (v/v)
To confirm whether the observed inhibition of cell proliferation
Fig. 2. Inhibition of proliferation of Caco-2 cells using different extracts as was associated with an apoptotic effect, the ethyl acetate and the
indicated. Pure culture medium was used as control. The dried extracts from 5 g
(d.w.) of berries were dissolved in 2 ml of DMSO and mixed with culture medium in
different ratios, shown as volume %. The dried heptane extract was used directly
and the water extract was dissolved in 2 ml of water. Error bars represent the 95% 100
confidence intervals and significant results within the same concentration are EtAc
c
indicated as ap < 0.05 vs Hep, bp < 0.05 vs EtAc, cp < 0.05 vs EtOH, dp < 0.05 vs H2O, 90 c
e
p < 0.05 vs EtOH:H2O (1), fp < 0.05 vs EtOH:H2O (2). EtOH:H2O (1)*
80
EtOH:H2O (2)*
Cell proliferation (%)

70
connected to an upregulated detoxifying function of the liver cells 60
induced by DMSO. Also, DMSO has previously been shown to stim- ab
50
ulate proliferation in fetal liver cells (Koyama, Ehashi, Ohshima, & bc

Miyoshi, 2009). 40
The effect of the different sea buckthorn extracts was first eval- 30
uated by measuring Caco-2 cell proliferation. As shown in Fig. 2, ac
20
the extracts were found to inhibit cell proliferation in the concen- ab

tration range of 0.25–2% (v/v), whereas no effect was observed be- 10

low 0.1% for any extracts (data not shown). In the sequential
0
extraction, the ethyl acetate fraction stood out as the most effec- 0.25 0.5 1 2
tive one, showing a clear dose-dependent inhibition. The degree Extract concentration % (v/v)
of the inhibition was much higher than that induced by DMSO
alone (Fig. 1), indicating a true positive effect that reflects the com- Fig. 3. Inhibition of proliferation of Hep G2 cells using different extracts as
position of the extract. The heptane and pure ethanol fractions also indicated. Pure culture medium was used as control. The dried extracts from 5 g
showed inhibitory effects, but both were much weaker and less (d.w.) of berries were dissolved in 2 ml of DMSO and mixed with culture medium in
different ratios, shown as volume %. Error bars represent the 95% confidence
dependent on the dose. The water fraction was the only extract intervals and significant results within the same concentration are indicated as
not found to have any significant effect. Using the second extrac- a
p < 0.05 vs EtAc, bp < 0.05 vs EtOH:H2O (1), cp < 0.05 vs EtOH:H2O (2). *Not tested at
tion protocol, the ethanol:water extracts were also found to inhibit 0.25%.
1008 C. Grey et al. / Food Chemistry 120 (2010) 1004–1010

ethanol:water extracts were analysed with an ELISA-based Cell
Death Detection assay, using Caco-2 and Hep G2 cells. The results
of two repeated experiments are shown in Fig. 4A and B. Ethyl ace-
tate extracts induced apoptosis in both Caco-2 and Hel G2 cells.
Pure ursolic acid (at 80 lM) was also effective (data not shown),
which is in agreement with the results obtained by Andersson
et al. using HT29 cells (Andersson et al., 2003). However, weaker
effects were observed for the ethanol:water extract. The apoptosis
results basically followed the proliferation results found for Caco-
2, since a strong inhibition of proliferation was observed for the
ethyl acetate extract and ursolic acid, whereas a concentration of
ethanol:water extracts (ethanol:water (1)) of 1% or less did not in-
hibit the growth. In contrast, the strong inhibition of proliferation
using ethanol:water extracts at 1% on Hep G2 cells could not be
correlated to an apoptotic effect. These results indicate that the
ethyl acetate extract has a composition that gives both antiprolif-
erative and proapoptotic effects, whereas the effects of the compo-
sitions in ethanol:water extract are mainly antiproliferative.
The major composition found in ethyl acetate fraction was urso-
lic acid, which is a type of triterpenoids and its anticancer effects
have been well documented (Pathak et al., 2007). Interestingly, in
line with the results presented here, Teng et al. found that when
fractioning sea buckthorn extracts, only the ethyl acetate fraction
contained active compounds that were responsible for cytotoxicity
in BEL-7402 cells (human hepatocellular carcinoma) (Teng et al.,
2006). Since their ethyl acetate fraction was rich in polyphenols,
they assumed that the polyphenols were responsible for the inhi-
bition of proliferation. However, the ursolic acid content was not
analysed in their study, and in the light of the results obtained
here, the importance of ursolic acid may have been overlooked.
Ursolic acid has been shown to have anticancer properties that
are mediated by multiple signaling pathways, leading to apoptosis
and inhibition of proliferation, invasion, and metastasis (Ikeda,
Murakami, & Ohigashi, 2008). It also inhibits STAT3 proteins (sig-
nal transducers and activators of transcription), which are impor-
tant in tumor cell survival, proliferation, and angiogenesis
(Aggarwal & Shishodia, 2006; Pathak et al., 2007). Isorhamnetin,
found in abundance in different glycosylated forms in sea buck-
thorn, has been shown in vitro to have antitumour activity in
BEL-7402 cells, giving about 50% inhibition of cell viability at
600 lM when incubated for 24 h (Teng et al., 2006). The concentra-
tions reached here were lower, where the combined concentration
of the different forms of isorhamnetin reached about 100 lM in the
2% mixtures of ethanol:water extracts.
Following screening of 24 cultivars, three sea buckthorn culti-
vars (‘BHi 10941’, ‘Podaruk Sadu’ and ‘BHi 10726’) were selected
A 90

80

Apoptosis rate 70
A 14
Cell proliferation (%)

BHi 10726
60
12
BHi 10941
50
10 Podaruk
Sadu
Enrichment factor

8 40

6 30

4 20

2 10

0 0
0.125 0.25 0.5
-2 Concentration ethyl acetate extract % (v/v)
0 0.5 1
Extract concentration % (v/v)
B 100
B 8
90
7 80

6 BHi 10726
Cell proliferation (%)

70
BHi 10941
Enrichment factor

5 60
Podaruk
4 50 Sadu

3 40

2 30

1 20

0 10
0 0.5 1
Extract concentration % (v/v) 0
0.125 0.25 0.5
Fig. 4. Apoptosis analysis combining two separate experiments of ethyl acetate Concentration ethyl acetate extract % (v/v)
extracts (N) and ethanol:water (j) using Caco-2 cells (A) or Hep G2 cells (B). The
increase in mono- and oligonucleosomes released into the cytoplasm (indicating Fig. 5. Inhibition of proliferation of Caco-2 cells (A) and Hep G2 cells (B) using ethyl
apoptosis) is expressed as an enrichment factor compared to the control containing acetate extracts of different sea buckthorn species. DMSO (0.5%) in pure medium
1% DMSO. Positive controls were included in each experiment. was used as control.
 C. Grey et al. / Food Chemistry 120 (2010) 1004–1010
Table 2
Composition of ethyl acetate extracts of three different sea buckthorn cultivars, given as lg/g d.w. ± SD.
Cultivar TPa PC
BHi 10941 754 ± 16 865 ± 48
Podaruk Sadu 789 ± 16 1129 ± 85
BHi 10726 886 ± 18 929 ± 116
a
TP = total phenolics; PC = proanthocyanidine; RT = rutin; IH-3R = isorhamnetin-3-O-rutinoside; IH-3G = isorhamnetin-3-O-glucoside; KF-3G = kaempferol-3-glucoside;
UA = ursolic acid.
and extracted according to protocol 1, in which the ethyl acetate
fractions were used to investigate whether any genotype-depen-
dent difference in cell proliferation could be detected. The extracts
were tested on both the Caco-2 and Hep G2 cell lines in relatively
low doses (up to 0.5%) to avoid the interference of DMSO. As shown
in Fig. 5A and B, all the extracts showed dose-dependant inhibitory
effect on the cell proliferation. The difference between the extracts
of the different cultivars was quite small. The ‘BHi 10941’ ethyl
acetate extract inhibited the growth somewhat more than the oth-
ers, whereas the ‘Podaruk Sadu’ extract was significantly (p < 0.01)
less effective than the ‘BHi 10726’ and ‘BHi 10941’ extracts on Hep
G2 cells. The content of these extracts is given in Table 2. The ef-
fects observed in Fig. 5 do not correlate with the concentrations
of polyphenols or ursolic acid found. The most likely reason is that
the content of ursolic acid was in the 8–50 lM range (calculated
from data in Table 2), and at these concentrations the effects of
pure ursolic acid have been found to be rather weak (Andersson
et al., 2003). Thus, the inhibitory effects at the relatively low ex-
tract concentrations used in Fig. 5 were not, at least solely, caused
by a single component, highlighting the potential advantage of
using whole extracts rather than using isolated compounds when
trying to assess the physiological relevance of fruits and berries
(Coates Emma et al., 2007; Lee, Lee, Surh, & Lee, 2003). Synergistic
effects in which combinations of phytochemicals show a greater
inhibition of proliferation of human cancer cells than purified sin-
gle compounds have previously been reported for different groups
of polyphenols (Mertens-Talcott, Talcott, & Percival, 2003; Seeram,
Adams, Hardy, & Heber, 2004).
4. Conclusions
Based on our results, it is worthy of note that when evaluating
berry extracts, the effects of important compounds such as ursolic
acid may not be apparent if only one extraction solvent, e.g. etha-
nol, is used. So far, most in vitro cancer cell studies involving berry
and fruit extracts have mostly focused on the polyphenols, which
are effectively extracted with alcohols. If a more accurate evalua-
tion of the potential health benefits is desired, several extracts with
different solvents should be tested – and perhaps also combina-
tions of them. In addition, the fact that the ethyl acetate fraction
was far more effective than both the ethanol fraction and the eth-
anol:water extract (protocol 2) in the present study indicates that
the phenolics are not the most potent antiproliferative compounds
present in sea buckthorn, a role that ursolic acid may possess
instead.
Acknowledgements
This project was supported by the Swedish Research Council for
the Environment, Agricultural Sciences, and Spatial Planning and
by the Albert Påhlsson Foundation. We thank Anders Ekholm,
Department of Plant Breeding and Biotechnology Balsgård, the
Swedish University of Agricultural Sciences, for providing skillful
support with the HPLC analyses.
1009
RT IH-3R IH-3G KF-3G UA
14.0 ± 0.6 201 ± 8 288 ± 12 9.9 ± 0.3 1307 ± 14
21.9 ± 0.7 209 ± 6 426 ± 13 11.1 ± 0.4 1903 ± 40
15.9 ± 0.6 227 ± 7 335 ± 10 11.9 ± 0.5 1213 ± 27
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