Optimisation of accelerated solvent extraction for screening of the health benefits of plant food materials

doi:10.4236/health.2009.13037

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Vol.1, No.3, 220-230 (2009)

doi:10.4236/health.2009.13037

SciRes

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

ABSTRACT

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

1. INTRODUCTION

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

221

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.

2. MATERIALS AND METHODS

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,

MO,

USA).4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetraz

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

222

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

R. Wibisono et al. / HEALTH 1 (2009) 220-230

223

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).

Parameter

tested Solvent Concentration T(°C)

Static time

(min)

Static

cycle

TPC

(mg/g FW)

Solvent type

MeOH

EtOH

Water

absolute

100 % water

1.19 ± 0.049

0.89 ± 0.022

0.71 ± 0.016

MeOH

absolute

95 %

80 %

1.12 ± 0.057

1.40 ± 0.021

1.28 ± 0.028

Solvent

concentration

EtOH

absolute

95 %

80 %

0.87 ± 0.043

0.96 ± 0.013

0.94 ± 0.012

Temperature

(°C) MeOH

95 %

100

130

160

1.28 ± 0.028

1.50 ± 0.021

1.50 ± 0.042

1.67 ± 0.028

Static time MeOH

95 %

100

1.20 ± 0.008

1.23 ± 0.042

1.18 ± 0.028

1.41 ± 0.021

1.53 ± 0.039

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 %

100

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. RESULTS AND DISCUSSIONS

3.1. Preliminary Optimisation of ASE

Conditions

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

224

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-

pounds.

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

225

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

Chloro

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’)

anidin-3-

lucoside eq. C

anidin-3-

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----

anidin-3-

lucoside eq. C

anidin-3-

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

(Chromato

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

Chloro

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-- --

anidin-3-

lucoside eq. C

anidin-3-

lucoside eq.

Elderberr

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

(Chromato

ram not shown)4927.7Rutin1313.2 ± 47.01197.0 ± 49.1

Chloro

enic acid eq.Chloro

enic acid eq.

5022.85-caffeoylquinic acid1296.0 ± 401033.0 ± 40

Openly accessible at

R. Wibisono et al. / HEALTH 1 (2009) 220-230

226

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

227

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,

respectively.

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

(100°C).

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

228

to be suitable for updating databases of food polyphenolic

content in the future.

3.4. Comparative Total Phenolic Content of

Extracts

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

shown).

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.

4. CONCLUSIONS

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

229

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.

5. ACKNOWLEDGMENTS

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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