Vol.1, No.3, 220-230 (2009)
Copyright © 2009 http://www.scirp.org/journal/HEALTH/
Openly accessible at
Optimisation of accelerated solvent extraction for
screening of the health benefits of plant food materials
Reginald Wibisono1, Jingli Zhang1,*, Zaid Saleh1, David E. Stevenson2, Nigel I. Joyce3
1The New Zealand Institute for Plant and Food Research Ltd, Auckland, New Zealand; Jingli.Zhang@plantandfood.co.nz
2The New Zealand Institute for Plant and Food Research Ltd, Hamilton, New Zealand
3The New Zealand Institute for Plant and Food Research Ltd, Lincoln, New Zealand
Received 9 September 2009; revised 14 September 2009; accepted 23 September 2009
The development of a rapid, robust and reliable
method for extracting plant food materials is
important for screening a wide range of plant
bioactives for their health benefits. In this study,
extractions of bioactive polyphenolic com-
pounds from fruits and vegetables were per-
formed using a pressurised solvent extraction
technique. Variables including solvent, extrac-
tion temperature and time, and number of ex-
traction cycles, were optimised to develop a
rapid and efficient extraction protocol. The re-
sulting extracts were then analysed for antioxi-
dant capacity, total phenolic content and com-
position. The optimal parameters found were
19:1 methanol/water (95% methanol) as solvent
and three extraction cycles, of 10 minutes at
40ºC or 2 minutes at 100ºC. High performance
liquid chromatography mass spectrometry did
not detect any difference in extract composition
between low and high temperatures. Extraction
at 100°C generally gave a moderately higher
yield of polyphenolics for some fruit and vege-
table extracts but appeared to reduce the anti-
oxidant activity particularly for turnip leaf, el-
derberry and sour cherry extracts as measured
by oxygen radical absorbance capacity assay.
We found that all 40°C extracts were better at
protecting cells from H2O2-induced cellular
damage than their 100°C counterparts. The 40°C
apple puree and elderberry extracts were about
2 fold and 1.7 fold more effective, respectively,
than extracts prepared at 100°C. Our results
demonstrated that pressurised solvent extrac-
tion technique with careful parameter selection
can be used as a quick method for screening the
health benefits of plant food materials.
Keywords: Antioxidant; Accelerated Solvent
Extraction; Cytoprotection; Polyphenolics
The consumption of fruits and vegetables is generally
accepted to improve health and wellbeing, as well as
reducing the risk of atherosclerotic heart disease [1],
neuronal degeneration [2], and cancer [3] by inhibiting
various stages of tumour initiation and proliferation [4].
These benefits are thought to be associated with the
presence of polyphenolic compounds in fruits and vege-
tables [5-7], such as those in apples, berries and green
leafy vegetables. Feng and co-workers [8] showed that
cyanidin-3-rutinoside could selectively kill leukemic cells
by inducing oxidative stress. They also suggested that this
compound could be used in cancer therapy, as it is widely
available in fruits such as black raspberry. Apple phenolic
compounds have also been shown to protect human
low-density lipoprotein LDL from being oxidised, which
may help to prevent cardiovascular disease [9].
The desire to extract nutrients and nutraceuticals from
plant materials as functional food ingredients has
prompted a continuing search for economically and
ecologically feasible extraction technologies. Traditional
solid-liquid extraction methods require a large quantity of
solvent and are time consuming. The large amount of
solvent used not only increases operating costs but also
causes additional environmental problems.
Accelerated solvent extraction (ASE) is a fully auto-
mated technique that uses common solvents to rapidly
extract solid and semisolid samples. ASE operates at
temperatures above the normal boiling point of most
solvents, using pressure to keep the solvents in liquid
form during the extraction process. Pressure allows the
extraction cell to be filled faster, helps to force liquid into
the pores and to keep the solvent liquid at operating
temperatures. ASE is considered as a potential alternative
technique to conventional atmospheric pressure methods,
for the extraction of polar compounds [10]. Compared
with conventional solvent extraction, there is a dramatic
decrease in the amount of solvent and extraction time
required for ASE [11]. The development of a rapid and
R. Wibisono et al. / HEALTH 1 (2009) 220-230
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robust extraction method is essential, particularly when
studying and comparing the phenolic composition of fruit
extracts, food material or other by product such as that
done by Spigno and colleagues [12] on grape marc. Re-
cently, ASE has been developed for extracting phyto-
chemicals from various fruit and vegetable samples
[13-15]. The ASE method is gaining in popularity, be-
cause of its practicality, speed and ability to process
samples automatically using different solvents, pressures
and temperatures under nitrogen. ASE extraction can be
carried out in minutes, compared with the hours required
for conventional methods such as Soxhlet extraction. A
rapid extraction maximises sample throughput and would
be expected to minimise phytochemical degradation [13].
The aim of the present paper is to verify the possibility
of using, instead of traditional extraction procedures,
ASE, in order to reduce time, cost of analysis, and waste
solvent. This technique uses conventional liquid solvents
at elevated temperatures and pressures to achieve quan-
titative extraction from solid and semisolid samples in a
short time and with a small amount of solvent. Here, we
report an optimised extraction protocol that is applicable
to a diverse range of fruit and vegetable materials, and
demonstrate that temperature is a critical extraction pa-
rameter, because increasing extraction temperature im-
proves extraction efficiency but appears to increase phy-
tochemical degradation only moderately. Careful choice
of conditions is at least as important with ASE as with
conventional extraction methods. We have also compared
three rapid assays for assessing polyphenolic or antioxi-
dant content of the extracts.
All extractions, colorimetric assays and high performance
liquid chromatography (HPLC) analyses of plant extracts
were carried out in duplicate. The results shown in this
study are reported as the mean values and were taken from
the result of two separate experiments.
2.1. Materials
Quercetin dihydrate was purchased from Acros Organic
(New Jersey, USA) and rutin trihydrate from Fluka,
BioChemika (Buchs, Switzerland). Annexin V-FITC and
binding buffer were obtained from BD Biosciences (San
Diego, CA). Folin-Ciocalteu reagent, sodium carbonate
and hydrogen peroxide were obtained from BDH
Chemicals (Poole, England, UK). All other chemicals
were purchased from Sigma-Aldrich Inc., (St. Louis,
olio]-1, 3-benzene disulfonate (WST-1 reagent) was ob-
tained from Roche (Basel, Switzerland). All solvents
used were of HPLC grade. Water used in experiments
was deionized (MilliQ). The human neuroblastoma SH-
SY5Y cells were obtained from the American Type Cul-
ture Collection (ATCC, Manassas, VA, USA).
2.2. Plant Materials
Apple puree (Malus x domestica, Pacific Beauty™
(Sciearly)) was selected for preliminary trials on extrac-
tion method development and optimisation. These apples
were harvested in February 2005 from a HortResearch
orchard in Hawke’s Bay, New Zealand. Five additional
local fruits and vegetables were selected for the main
study. These were: turnip leaf (Brassica campestris
‘Barkant’; 8.5 kg), elderberry (Sambucus nigra; 5.0 kg),
sour cherries (no stone; Prunus cerasus, ‘Fanal’; 5.0 kg),
swede leaf (Brassica napus ‘Aparima Gold’; 8.5 kg) and
apple puree (Malus x domestica, ‘Red Delicious’; 5.0 kg).
The elderberries were collected in March 2005 in Mosgiel
New Zealand, whereas sour cherries were collected in
April 2005 in Otematata, Waitaki Valley, New Zealand.
The turnip and swede leaf samples were collected in May
2005 from the Crop and Food Research Station in Gore,
New Zealand. ‘Red Delicious’ apples were harvested in
April 2005 from a HortResearch orchard in Hawke’s Bay,
New Zealand. After collection, the samples were imme-
diately frozen, freeze dried and milled using a domestic
coffee grinder (Coffee and spice grinder, Breville CG2B,
Australia), then stored in heat-sealed, gas-impermeable
foil bags under vacuum at -20°C until needed.
2.2.1. Preparation of Pacific Beauty™
(Sciearly) and ‘Red Delicious’
Apple Puree
Apple purees from both cultivars were prepared simi-
larly. Twenty-five apples (approximately 5.0 kg) were
selected randomly from the harvested batches. Apples
were sliced into quarters, cored and then stored in cold
water prior to blanching for approximately 6-7 min until
the core temperature reached 90ºC. The blanching step
was done in order to stop the browning process of the
apple slices before processing into puree could be done.
The blanched slices were crushed using a pilot-scale
screw press (Model 3600, Brown International Corpo-
ration, Covina, CA) to produce the puree, which was
freeze dried using a pilot-scale freeze dryer (W.G.G
Cuddon Ltd, New Zealand).
2.3. Accelerated Solvent Extraction (ASE)
Extraction of the plant materials was performed using an
accelerated solvent extractor (ASE300) unit (Dionex
Corp., Sunnyvale, CA). The ASE apparatus pumps
solvent into the extraction cell, pressurises it, holds the
solvent in the cell at a controlled temperature and time
(termed “static cycle”), and then drains it into a collec-
tion vessel. The high pressure appears to speed up
penetration of the solvent into the sample pores greatly,
which makes the extraction much faster and more effi-
cient than conventional atmospheric pressure methods.
R. Wibisono et al. / HEALTH 1 (2009) 220-230
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The high pressure also increases the boiling point of the
solvent, allowing the use of solvents at temperatures
above their atmospheric pressure boiling points. Sam-
ples for extraction were prepared by mixing the
freeze-dried sample powder (4 g) with an equal weight
of diatomaceous earth (Celite®), which was then packed
into the (34 mL) extraction cells. The use of Celite® was
found to be essential, particularly for samples that were
high in sugar, as they were hygroscopic and tended to
aggregate. It also allowed a more even distribution of the
sample within the cell [13].
2.4. Optimisation of Extraction Parameters
Preliminary trials were conducted using apple pomace
(Malus x domestica, Pacific Beauty™ (Sciearly) as a
model sample. Extraction conditions in the preliminary
trials were adopted from Alonso-Salces et al. [13] using 90
seconds purging of N2 and 60% flush volume. Parameters
tested were: choice of solvent (ethanol (EtOH); water; 100,
95, 80% aq. methanol (MeOH)), temperature (40, 100, 130
and 160°C), duration (2, 5, 10 or 15 minutes) and number
(1-4) of static cycles, allowing the use of solvents at tem-
peratures above their atmospheric pressure boiling points.
Extraction efficiency was evaluated using the Folin total
phenolics assay (Section 2.8). On this basis, the optimum
conditions chosen for the remaining experiments were 3
static cycles of either 10 minutes at 40ºC or 2 minutes at
100ºC, using 95% aq. MeOH as the solvent. The remaining
experiments were carried out to determine the better of the
two protocols arising from the preliminary study, and to
evaluate the quality of the extracts in more detail using
Liquid Chromatography Mass Spectrometry (LCMS) and
both chemical and cell-based antioxidant assays.
2.5. Processing of Extracts for Storage
About 70 mL of extract was produced from each sample
replicate. All extracts were made up to a standard volume
of 100 mL, and concentrated to 25 mL under a stream of
nitrogen gas using a RapidVap® concentrator unit (model
79100-01, LabConco Corporation, Kansas City, MO).
Samples were then aliquotted into freeze dryer vials (10 ml
Stopcock vial, Crown Scientific, Australia) and further
concentrated under vacuum using the centrifugal concen-
trator (model 78100-01, LabConco Corporation, Kansas
City, MO). Finally, extract aliquots were freeze dried (Tel-
star Cryodos-80, Telstar Industrial S.L., Spain) to remove
residual water and were stored under vacuum at -80ºC prior
to analyses.
2.6. HPLC-MS Analysis Procedure
Dried samples were prepared for analyses to make ap-
proximately 10 mg/mL into two 1.5 mL Eppendorf tubes
for each sample. One tube was extracted with 1 mL of
15% acetic acid in methanol for anthocyanin analysis
while the other tube was extracted with 1 mL of 85%
(v/v) methanol/water for analysis of other flavonoids and
phenolic acids. Samples were dissolved by vortex mix-
ing and then centrifuged at 20817 g for 10 minutes.
Samples were diluted as necessary to fall within a suit-
able linear dynamic range for the detectors used.
The LCMS system consisted of a Thermo Electron
Corporation (San Jose, CA) Finnigan Surveyor MS pump,
Finnigan MicroAS auto-sampler, Finnigan Surveyor PDA
detector and a ThermaSphere TS-130 column heater
(Phenomenex, Torrance, CA). The system was fitted with
a Synergi-HydroRP C18 column (250 x 2.1 mm, Phe-
nomenex). A 5 L aliquot of each sample was separated
with a mobile phase flowing at 250 μL/min, consisting of
0.1% formic acid in water (A) and 0.1% formic acid in
acetonitrile (B). A gradient was applied from 100% (A),
held for 5 minutes, to 50% (B) at 45 minutes, 80% (B) at
50 minutes, held for 5 minutes, then returned to the start-
ing conditions over 5 minutes before being re-equilibrated
for 10 minutes. The eluent was scanned by photodiode
array detector (PDA) from 160-600 nm and analysed by
ESI-MS (electrospray) in the positive mode for acetic
acid/methanol samples and negative mode for metha-
nol/water extracts. Parent masses from m/z 120-2000 were
selected for MS2 fragmentation, followed by the first and
second most intense ions from MS2 undergoing MS3, fol-
lowed by these two most abundant ions each being frag-
mented to the MS4. Parent ions were excluded for 15 sec-
onds after daughter ion fragmentation data collection.
Data were processed with the aid of Xcalibur®2.0-SUR1
(Thermo Electron corporation).
2.7. Total Phenolic Determination
Total phenolic content of the extracts was measured using
the Folin Ciocalteu colorimetric method [16], using
catechin as the standard. Results were then calculated as
mg catechin equivalent per gram of fresh sample. All
samples were analysed in duplicate.
2.8. FRAP Antioxidant Assay
The ferric-reducing antioxidant potential (FRAP) assay
was performed using a published method [17]. All sam-
ples were analyzed in triplicate and Trolox standards
between 25-250 μM and a blank were included in each
run. The antioxidant capacity of each extract was calcu-
lated as Trolox equivalents from the linear regression
formula obtained from a series of corresponding Trolox
standards. Finally results were converted to mol Trolox
equivalents per gram of fresh sample.
2.9. ORAC Antioxidant Assay (Hydrophilic)
The oxygen radical absorbance capacity (ORAC) assay
was performed using a published procedure [18]. All
samples were analyzed in triplicate and Trolox standards
between 50-180 μM and a blank was included in each run.
The antioxidant capacity of each extract was determined
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Table 1. Results from preliminary trials using Pacific BeautyTM (Sciearly) apple puree as a model to establish optimum extraction
conditions using an accelerated solvent extraction method (n=2). TPC (Total Polyphenolic Concentration) is expressed as mg catechin
eq./g FW. (FW: fresh weight).
tested Solvent Concentration T(°C)
Static time
(mg/g FW)
Solvent type
100 % water
1.19 ± 0.049
0.89 ± 0.022
0.71 ± 0.016
95 %
80 %
1.12 ± 0.057
1.40 ± 0.021
1.28 ± 0.028
95 %
80 %
0.87 ± 0.043
0.96 ± 0.013
0.94 ± 0.012
(°C) MeOH
95 %
95 %
95 %
95 %
1.28 ± 0.028
1.50 ± 0.021
1.50 ± 0.042
1.67 ± 0.028
Static time MeOH
95 %
95 %
95 %
95 %
95 %
1.20 ± 0.008
1.23 ± 0.042
1.18 ± 0.028
1.41 ± 0.021
1.53 ± 0.039
95 %
95 %
95 %
95 %
1st cycle
2nd cycle
3rd cycle
4th cycle
0.86 ± 0.028
0.25 ± 0.049
0.12 ± 0.028
0.006 ± 0.0005
Static cycle MeOH 95 %
95 %
95 %
95 %
1st cycle
2nd cycle
3rd cycle
4th cycle
1.20 ± 0.018
0.32 ± 0.042
0.14 ± 0.028
0.004 ± 0.0002
by comparing the area under the curve with that of a blank
sample and was calculated as Trolox equivalents. Results
were converted to mol Trolox equivalents per gram of
fresh sample.
2.10. Cytoprotection Assay
2.10.1. Cell Culture and Treatment of Human
Neuroblastoma SH-SY5Y Cells
The human neuroblastoma SH-SY5Y cells were cultured
in Dulbecco’s modified Eagle medium nutrient mixture
F12 ham (DMEM-F12) supplemented with 10% FBS at
37°C in humidified air with 5% CO2. SH-SY5Y cells
were used in the undifferentiated state. The SH-SY5Y
cells were plated in 24-well plates at a concentration of 2
105 cells/mL with or without various concentrations of
extracts. After 24 h of incubation, cells were harvested
and stained with both annexin V-FITC and propidium
iodide and subjected to flow cytometric analysis of cell
death as described previously [19,20]. The cell death
index (CDI) was calculated from the percentage of the
viable cells and damaged cells (both apoptotic and ne-
crotic). The cytoprotective effects of extracts were
measured by the inhibition of H2O2-induced total cell
death. The median effective concentration (EC50) values
were calculated through dose-response curves of the-
concentration of test extract against the percentage of
inhibition as described previously [19,20]. The cytotoxic
effects of extracts were assessed using WST-1 cell sur-
vival assays with the concentrations used in the cytopro-
tection experiments.
3.1. Preliminary Optimisation of ASE
Methanol was found to be the best solvent for extraction
of polyphenolic compounds from apple pomace (Table 1).
MeOH extracted approximately 25% more polyphenolics
than ethanol and 40% more than water. Addition of water
(up to 5%) to both MeOH and EtOH increased the ex-
traction efficiency by up to 20% and 9%, respectively.
Extraction temperatures between 40°C and 130°C did
not result in any detectable changes in the composition of
the extract, as determined by HPLC. Extraction at 160°C,
however, showed evidence of degradation in the HPLC
chromatogram. Some peaks diminished in size and new
ones appeared (data not shown). Hence, extraction tem-
peratures of 40°C and 100°C were chosen for comparison
in further experiments. The length and number of static
R. Wibisono et al. / HEALTH 1 (2009) 220-230
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Openly accessible at
Figure 1. Total phenolic content of Pacific Beauty apple pomace
tissue extracted by an accelerated solvent extraction method at
40ºC and 100°C for 10 and 2 minutes respectively using four
static cycles.
cycles influenced the extraction efficiency. The optimal
conditions were found to be 3 static cycles, either of 10
min at 40ºC or 2 min at 100°C (Figure 1, Table 1). Most
of the phenolic compounds (~90%) were extracted by the
first 2 cycles and most of the remaining material (~9%) by
the third cycle, as measured by the Folin-Ciocalteu assay.
A fourth cycle was found not to be necessary. Based on
the results above, the operating parameters used in the
remainder of this study were: solvent: 95% methanol,
temperature: 40ºC or 100ºC, purging time: 90 seconds,
flush volume: 60%, static time: 10 min for 40ºC extrac-
tion and 2 min for 100ºC extraction and 3 static cycles.
3.2. Identification of Phenolic Compounds
by LC-MS
Identification and quantification of the major compounds
found in the extracts was carried out by LC-MS (Table 2).
To help with interpreting the data, structural fragmenta-
tion characteristics reported by other researchers were
used [21, 22], together with in-house spectra based on
reference materials and previously interpreted com-
3.2.1. ‘Red Delicious’ Apple Puree (Malus X
Domestica ‘Red Delicious’)
Procyanidins made up a significant proportion of com-
pounds found in the apple puree extracts from both tem-
perature treatments (Table 2). This is in agreement with
studies by Giusti et al. [22] and Sudjaroen et al. [23].
Chlorogenic acid (the major individual compound), quer-
cetin glycosides and phloridzin were also abundant and have
also been previously reported to be present in apple [24].
3.2.2. Swede Leaf (Brassica Napus ‘Aparima Gold’)
The major compounds in swede leaf were several
kaempferol glycosides. Most of these were acylated with
sinapic or ferulic acid. The phenolic composition was
similar to that found by an earlier study [25]. A previous
study by Huang and colleagues [26] found some com-
pounds in the swede leaf samples (also known as rutabaga
in the US) that might be responsible for its antioxidant
property. Because of lack of reference compounds, they
were not able to identify them. However, we were able to
identify which compounds were present in the extract
analysed in the present study, and they were very similar
to those found in the turnip leaf extracts. The three most
abundant compounds in the swede leaf extract based on
the relative area from the total spectral scan were
kaempferol-3-(sinapoyl)-sophoroside-7-glucoside, kaem-
pferol-3-(feruloyl)-sophoroside-7-glucoside and kaempf-
erol-3-sophoroside-7-glucoside respectively.
3.2.3. Sour Cherry (Prunus Cerasus ‘Fanal’)
The major polyphenolic compounds found in the sour
cherry extracts were 3-p-coumaroyl and 5-caffeoyl
quinates (Table 2). Lesser amounts of rutin, quercetin and
isorhamnetin glycosides were also found. Clifford and
colleagues [27] reported the presence of chlorogenic acid
(3-caffeoyl quinate) and this is also confirmed by our
result. Procyanidins are also present in the extracts, in
good agreement with previous findings [23,28].
3.2.4. Turnip Leaf (Brassica Campestris ‘Barkant’)
Turnip leaf extracts from both high and low temperature
treatment had similar polyphenolic compositions (Table
2). The major components found in the extracts were
glycosylated flavonols such as quercetin, kaempferol and
isorhamnetin and to a lesser extent, sinapic acid deriva-
tives. The previously reported polyphenolic composition
of Brassica rapa (another turnip variety) [29], was similar
to that found here. Many of the same compounds were
also reported in Brassica oleracea (broccoli) by Vallejo et
al. [25]. The presence of sinapoyl-malate was previously
reported by Liang et al. [30]. Another prevalent com-
pound found in the turnip leaf extract was kaempferol-
3-sophoroside-7-glucoside, which was also confirmed by
Fernandes et al. [31].
3.2.5. Elderberry (Sambucus Nigra)
Anthocyanins were the major phenolic compounds in the
elderberry extracts (Table 2). Extracts from both tem-
perature treatments were again similar. High concentra-
tions of cyanidin sambubioside (the major individual co-
mponent), rutin and 5-caffeoyl quinate were found. These
compounds were previously reported by Piraud et al. [32]
and Milbury et al. [33].
3.3. Comparison of the Polyphenolic Profile
of Extracts
There were no detectable differences in the polyphenolic
R. Wibisono et al. / HEALTH 1 (2009) 220-230
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Table 2. Identification of some major phenolic compounds found in fruit and vegetable samples extracted by ASE
technique from λ=280nm chromatograms. Quantification was based on total spectral scan λ=250-600 nm and expressed
as μg/g FW sample.
Plant samplesPeak #Retention timeCompound μg/g FW sample μg/g FW sample
(n=2)(min) (shown from chromatograms at
=280nm)extracted at 40°Cextracted at 100°C
enic acid eq.Chloro
enic acid eq.
‘Red Delicious’ apple puree 221.1Chlorogenic acid112.2 ± 5.776.9 ± 5.5
(Malus x domestica ‘Red Delicious’)
lucoside eq. C
lucoside eq.
1 19.4Cyanidin-3-galactoside35.0 ± 2.632.8 ± 1.4
322.2Procyanidin-B2 (dimer)68.3 ± 4.351.4 ± 2.4
6 23.7Procyanidin-trimer64.8 ± 2.4 49.5 ± 1.7
724.2Procyanidin-tetramer36.6 ± 1.432.9 ±2.0
824.8Procyanidin-pentamer56.1 ± 3.144.1 ± 1.7
Epicatechin eq.Epicatechin eq.
423.1Epicatechin80.7 ± 3.270.9 ± 3.1
Rutin eq.Rutin eq.
926.7Quercetin-3-glucoside83.8 ± 1.562.9 ± 1.1
1028.6Quercetin-3-xyloside49.5 ± 1.939.5 ± 2.9
1128.9Quercetin rhamnoside41.9 ± 1.734.1 ± 0.9
Phloretin eq.Phloretin eq.
12 28.0Phloretin-2-O-xylo-glucoside23.1 ± 1.022.4 ± 0.9
1330.2Phloridzin51.0 ± 2.636.9 ± 1.7
Unknown Unknown
5 23.3Unknown----
Rutin eq.Rutin eq.
Swede leaf1519.7Kaempferol-3-sophoroside-7-glucoside513.6 ± 28.7583.0 ± 14.1
(Brassica napus ‘Aparima Gold’)1621.8Kaempferol-3-(sinapoyl)-sophoroside-7-sophoroside64.9 ± 2.175.2 ± 3.2
1722.1Kaempferol-3-(sinapoyl)-sophoroside-7-glucoside926.6 ± 261059.0 ± 15
1822.4Kaempferol-3-(feruloyl)-sophoroside-7-glucoside689.7 ± 28787.8 ± 20.5
1922.6Kaempferol-3-(coumaroyl)-sophoroside-7-glucoside257.1 ± 14.3301.8 ± 15
22 26.4
aempferol-3 -(4- dis i napoyl)-soph orot riosid e-7-sop horosid
53.1 ± 3.275.8 ± 4.3
2326.8Kaempferol-3-(4-disinapoyl)-sophorotrioside-7-glucoside77.9 ± 3.6103.6 ± 3.2
Sinapic acid eq.Sinapic acid eq.
2429.41,2-disinapoyl-gentiobiose33.2 ± 1.941.4 ± 3.1
2529.81,2-sinapoyl-feruloyl-gentibiose5.2 ± 0.36.9 ± 0.3
Unknown Unknown
14 18.8Unknown----
20 23.4Unknown----
21 25.0Unknown----
lucoside eq. C
lucoside eq.
Sour cherry2618.7Cyanidin-3-glucosyl-rutinoside21.3 ± 1.224.2 ± 1.4
(Prunus cerasus ‘Fanal’)3021.8Procyanidin-B2 (dimer)98.8 ± 8.795 ± 8.5
3123.7Procyanidin trimer30.3 ± 1.029.7 ± 1.1
ram not shown)3224.2Procyanidin tetramer18.8 ± 1.219.2 ± 0.7
Rutin eq.Rutin eq.
3326.0Quercetin-3-rutinoside13.9 ± 0.614.3 ± 0.5
3427.7Rutin76.9 ± 5.077.8 ± 3.3
3528.7Isorhamnetin-3-rutinoside17.6 ± 1.117.4 ± 1.5
enic acid eq.Chloro
enic acid eq.
2720.33-caffeoylquinic acid145.9 ± 9.6144.9 ± 9.1
2922.95-caffeoylquinic acid156.1 ± 12.0154.9 ± 13.0
p-Coumaric acid eq.p-Coumaric acid eq.
2822.23-O-p-coumaroylquinic acid1306.8 ± 59.01321.0 ± 55.0
Rutin eq.Rutin eq.
Turnip leaf3619.4Quercetin-3-sophoroside-7-glucoside26.8 ± 1.831.2 ± 1.4
(Brassica campestris ‘Barkant’)3820.7Kaempferol-3-methoxycafeoyl-sophoroside-7-glucoside49.2 ± 2.741.9 ± 2.6
3921.3Quercetin-3-glucoside-7-glucoside108.3 ± 3.2108.4 ± 5.2
(Chromatogram not shown)4021.5Quercetin-3-(sinapoyl)-sophoroside-7-glucoside111.9 ± 3.6150.5 ± 3.2
4120.0Kaempferol-3-sophoroside-7-glucoside132.1 ± 7.5169.4 ± 9.1
4223.9sorhamnetin-3,7-diglucoside2089.7 ± 46.11773.1 ± 49
4322.2Isorhamnetin-3-rutinoside370.0 ± 14.7472.2 ± 20
Sinapic acid eq.Sinapic acid eq.
4429.6Sinapoyl-malate155.8 ± 13.2236.1 ± 19.5
4529.21,2-disinapoyl-gentiobiose2.2 ± 0.111.9 ± 0.6
4630.01,2-sinapoyl-feruloyl-gentibiose3.6 ± 0.29.9 ± 0.4
Unknown Unknown
37 19.8Unknown-- --
lucoside eq. C
lucoside eq.
4722.2Cyanidin-sambubioside3907.0 ± 2353500.0 ± 182
ambucus ni
ra )Rutin eq.Rutin eq.
48 26.4Quercetin-3-glucoside26.1 ± 1.833.4 ± 2.5
ram not shown)4927.7Rutin1313.2 ± 47.01197.0 ± 49.1
enic acid eq.Chloro
enic acid eq.
5022.85-caffeoylquinic acid1296.0 ± 401033.0 ± 40
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R. Wibisono et al. / HEALTH 1 (2009) 220-230
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Figure 2. HPLC chromatograms at 280nm of Red delicious apple puree (I) and Swede leaf var. Aparima Gold
(II) from accelerated solvent extractions (ASE) at 40ºC and 100ºC. The sample concentration was 10mg/L.
Openly accessible at
R. Wibisono et al. / HEALTH 1 (2009) 220-230
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/
Figure 3. Total phenolic contentby Folin Ciocalteu mathod and antioxidant capacity (measured by FRAP (ferric re-
ducing antioxidant potential) and ORAC (oxygen radical absorbance capacity) methods for fruit and vegetable sam-
ples extracted by accelerated solvent extraction (ASE) at 40ºC and 100ºC. Results are expressed as μg catechin
equivalent or μmol Trolox equivalent /g fresh weight samples for total phenolic content and antioxidant capacity,
profiles obtained from ASE prepared using different
temperatures, for ‘Red Delicious’ apple puree or Swede
leaf extracts (Figure 2). The same result was obtained
from the other plant materials (data not shown). The only
noticeable difference was a 20-25% higher peak intensity
at 40ºC for the apple puree extract, indicating that the
lower temperature was, surprisingly, more efficient. If
there is increased degradation at 100ºC, it is apparently
evenly spread over all compounds. However, extraction
of swede leaf showed a different trend to that of apple
puree extract, as more phenolics seemed to be released
when extraction was carried out at the higher temperature
The polyphenolic composition and concentrations from
this study are in good agreement with the USDA database
of the flavonoid and proanthocyanidin content of foods
(http://www.ars.usda.gov/nutrientdata). As has been dis-
cussed by Kroon and Williamson [34] , the USDA has
now developed databases of polyphenolic compound
classes with recommended daily intake (RDI) values, as
well as for micronutrients such as vitamins and minerals.
Based on the USDA database, the major anthocyanidins
found in elderberry were found to be cyanidins, whereas
vegetables such as turnip have an appreciable amount of
flavonols such as kaempferols and quercetins. ASE ex-
traction appears to provide comparable extraction profiles
to manual extraction methods, such as that done by Ro-
mani et al. [29] on Brassica plants, and therefore appears
Openly accessible at
R. Wibisono et al. / HEALTH 1 (2009) 220-230
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/Openly accessible at
to be suitable for updating databases of food polyphenolic
content in the future.
3.4. Comparative Total Phenolic Content of
Total phenolic content as measured by the Folin-Ciocalteu
assay, combined with ASE, is a rapid and convenient means
to compare the overall polyphenolic antioxidant content of
different plant foods. Elderberry extract stands out, with a
total phenolic content 2-4 times higher than the other ex-
tracts tested (Figure 3).
Different plant materials behaved differently when ex-
tracted at different temperatures. Total phenolic content of
turnip leaf, elderberry and red delicious puree extracts
were slightly greater when the samples were extracted at
100ºC than at 40ºC, whereas contents in swede leaf and
sour cherry were slightly lower. This may indicate that
some polyphenolics from swede leaf and sour cherry are
more labile at a higher temperature than those in the other
materials extracted. These differences were, however,
minor, so on this basis the extraction temperature chosen
for comparison of different plant materials could be be-
tween 40°C and 100ºC.
3.5. Antioxidant Capacity of Plant Extracts
The chemical antioxidant activities of the extracts were
determined by both the FRAP and ORAC assays. The
effect of extraction temperature is more obvious in the
FRAP assay than in the total phenolic content assay. El-
derberry extract had the highest FRAP value and the trend
of the extracts was quite similar to that of total phenolic
content (Figure 3). It therefore appears that polyphenolics
play a major role in contributing to the metal ion-reducing
capacity in these extracts. The FRAP values of all samples
measured seemed to be higher when the extraction was
carried out at 100°C. Turnip leaf extract was found to have
the lowest antioxidant capacity by this assay.
Elderberry again had the highest value and the quali-
tative trend of other extracts was similar to the trend
identified in the total phenolic content and FRAP results
(Figure 3). However, the antioxidant capacities of the
elderberry and turnip extracts prepared at 100°C were
proportionately smaller than those of extracts prepared at
40ºC; the relationship to total phenolic content was much
weaker and extraction temperature had a considerable
effect. Elderberry and apple puree extracts had the highest
ORAC values overall, compared with those of other ex-
tracts. The ORAC value of elderberry was moderately
lower and turnip leaf ORAC value was reduced by about
half when comparison was made between the 100°C and
40°C treatments. Little difference was found between
ORAC values of sour cherry extracts prepared at low and
high temperatures. These differences are probably ex-
plained by both differences in lability of compounds with
high ORAC capacity and the presence of compounds
other than polyphenolics with significant ORAC capacity.
3.6. Cytoprotection Assay
3.6.1. Cytotoxicity of Extracts
The cytotoxicities of these extracts were tested on SH-SY5Y
cells at different concentrations for 24 hours using the
WST-1 assay. All extracts produced at either temperature
did not affect the viability of human neuroblastoma SH-
SY5Y cells within the concentration range used (data not
3.6.2. Cytoprotective Effects of Extracts
The inhibitory effects of test samples on cellular death
induced by H2O2 on human neuroblastoma SH-SY5Y
cells were determined.
For extracts at 40°C, elderberry had the highest pro-
tective capacity, followed by apple puree, sour cherry,
turnip and swede leaf extracts. For those extracted at
100°C, elderberry again had the highest protective ca-
pacity, followed by sour cherry, apple puree, turnip and
swede leaf extracts respectively. This ranking order was
the same as with the other assays. The trend of EC50
values was similar to that of the other assays (bearing in
mind that a low EC50 value indicates higher cytoprotec-
tive capacity; Figure 4). The only significant difference
from the other assays was that 40ºC extracts consistently
performed moderately better than the 100ºC extracts.
Based on the results reported here, it appears that there is
relatively little to choose between these four antioxidant
assays to combine with ASE as a means of rapidly com-
paring the antioxidant potential of plant foods. The results
of all four assays correlate very closely, with the possible
exception of the ORAC assay, which is qualitatively
similar but shows some quantitative differences.
Results indicated that different fruit and vegetable ex-
tracts prepared at low and high temperatures behaved
differently when analysed for their total phenolic contents
and antioxidant capacities. The polyphenolic composi-
tions of the ASE extracts in this study agree well with
those reported in literature on the same plant materials.
Elderberry extract was found to have the highest poly-
phenolic content and antioxidant capacity, followed by
sour cherry, red delicious apple puree and the two vege-
table samples. The four rapid assays used to evaluate the
extracts (Folin total phenolics, FRAP, ORAC and cyto-
protection), all ranked the five extracts similarly, which
possibly indicates that polyphenolics are the major anti-
oxidants in the extracts and are responsible for most of the
antioxidant capacity. The effect of extraction temperature
was more obvious in results from the ORAC antioxidant
assay, particularly for turnip and elderberry extracts.
However, all extracts prepared at 40°C performed better
in the cell-based antioxidant assay. This study has shown
R. Wibisono et al. / HEALTH 1 (2009) 220-230
SciRes Copyright © 2009 http://www.scirp.org/journal/HEALTH/
Openly accessible at
Figure 4. Protective effects (EC50) of extracts against H2O2-induced death of SH-SY5Y cells. After incubation, cells
were stained with both annexin V-FITC and PI and analyzed by flow cytometry. Results are expressed as EC50 values
with standard deviations from the mean of two separate determinations.
that accelerated solvent extraction, combined with HPLC,
antioxidant and cytotoxicity testing, is a useful tool for
rapid screening of plant extracts for comparison of the
abundance of compounds thought to be beneficial to
human health.
This work was funded by the Foundation for Research Science and
Technology (Wellness Foods Programme, Contract C06X0405). We
thank Joyce Au and Rosheila Vather for their contribution in doing the
antioxidant assays, Tom Orchiston for sample collection, Ms Teresa
Wegrzyn, Dr Denise Hunter and Dr Jeffrey Greenwood for checking and
the helpful discussions on the manuscript.
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