Research Article
Received: 8 July 2011,
Revised: 18 October 2011,
Accepted: 25 October 2011
Published online in Wiley Online Library: 30 December 2011
(wileyonlinelibrary.com) DOI 10.1002/pca.1378
Towards an Efficient Protocol for the
Determination of Triterpenic Acids in Olive
Fruit: A Comparative Study of Drying and
Extraction Methods
Vlasios Goulas* and George A. Manganaris
ABSTRACT:
Introduction – Triterpenic acids, such as maslinic acid and oleanolic acid, are commonly found in olive fruits and have been
associated with many health benefits. The drying and extraction methods, as well as the solvents used, are critical factors in
the determination of their concentration in plant tissues. Thus, there is an emerging need for standardisation of an efficient
extraction protocol that determines triterpenic acid content in olive fruits.
Objective – To evaluate common extraction methods of triterpenic acids from olive fruits and to determine the effect of the
drying method on their content in order to propose an optimum protocol for their quantification.
Methodology – The efficacy of different drying and extraction methods was evaluated through the quantification of maslinic
acid and oleanolic acid contents using the reversed-phase HPLC technique.
Results – Data showed that ultrasonic assisted extraction with ethanol or a mixture of ethanol:methanol (1:1, v/v) resulted in
the recovery of significantly higher amounts of triterpenic acids than other methods used. The drying method also affected
the estimated triterpenic acid content; frozen or lyophilised olive fruit material gave higher yields of triterpenic acids
compared with air-dried material at both 35 C and 105 C.
Conclusion – This study provides a rapid and low-cost extraction method, i.e. ultrasonic assisted extraction with an eco-friendly
solvent such as ethanol, from frozen or lyophilised olive fruit for the accurate determination of the triterpenic acid content in
olive fruit. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: freeze-drying; Soxtec extraction; ultrasonic assisted extraction; maslinic acid; oleanolic acid; Olea europaea
Introduction
444
Olive (Olea europaea L.) fruit is a key ingredient of a Mediterranean
diet, and its disease-preventing effects have been attributed to its
fatty acid profile, as well as the presence of a plethora of bioactive
components, such as tocopherols, phospholipids, biophenols and
triterpenic acids (Trichopoulou and Dilis, 2007; Goulas et al., 2009;
Allouche et al., 2010). Olive fruit is a particularly rich source of
maslinic and oleanolic acids, while ursolic and betulinic acids have
been reported at lower amounts (Romero et al., 2010). These
compounds are present at high concentrations in the epicarp of
the fruit, forming part of the waxes that cover them; oleanolic acid
has also been found in the endocarp, the wood shell and the olive
seed (Bianchi and Vlahov, 1994). Triterpenic acids are a group of
phytochemicals that has been studied extensively for their
pharmacological properties, because pentacyclic terpene acids
present potent anti-oxidant, anti-inflammatory, anti-tumour and
anti-microbial activity (Li et al., 2009; Kontogianni et al., 2009;
Guinda et al., 2010). In addition, the anti-proliferative activity of
triterpenic acids against cancer lines has been also documented
(Petronellia et al., 2009).
The simultaneous determination of triterpenic acids using chromatographic and spectroscopic methods has been thoroughly
studied over recent years, mainly because of their similar chemical
structure (Wojciak-Kosior, 2007; Zacchina et al., 2009; Kontogianni
et al., 2009; Li et al., 2011a). On the other hand, the recovery of
Phytochem. Anal. 2012, 23, 444–449
triterpenic acids from olive fruit has been poorly investigated,
although it is a crucial step of the overall analytical process that
leads to the quantification of the target phytochemical. The significance of extraction in developing analytical methodologies for
accurate estimation of bioactive compounds in functional foods
has been highlighted (Luthria, 2006). A plethora of extraction
methods to recover triterpenic acids (e.g. Soxhlet extraction, heat
reflux extraction, ultrasonic assisted extraction, microwave assisted
extraction) have been described from diverse plant tissues, such as
herbs, fruits, olive leaves and olive mill wastes (Table 1). Romero
et al. (2010) extracted oleanolic acid and maslinic acid from table
olives using an exhaustive solid–liquid extraction with a mixture
of methanol:ethanol (1:1, v/v) and maceration for 1 h; ethanol
has also been suggested as an appropriate solvent for extraction
(Guinda et al., 2010). Overall, the extraction of pentacyclic terpene
acids is usually performed with ethyl acetate, methanol and ethanol; in addition the use of apolar organic solvents such as petroleum ether and dichloromethane has been also described (Banik
and Pandey, 2008; Schneider et al., 2009; Tarvainen et al., 2010).
* Correspondence to: V. Goulas, Cyprus University of Technology, Department
of Agricultural Sciences, Biotechnology and Food Science, 3603 Lemesos,
Cyprus. E-mail: vlasios.goulas@cut.ac.cy
Cyprus University of Technology, Department of Agricultural Sciences,
Biotechnology and Food Science, 3603 Lemesos, Cyprus
Copyright © 2011 John Wiley & Sons, Ltd.
Determination of Triterpenic Acid Content
Table 1. Extraction methods and solvents used to recover triterpenic acids from diverse plant tissues
Extraction
Solid–liquid extraction
Ultrasonic assisted
extraction
Extraction at 40 C
Soxhlet extraction
Solvent
Methanol, 70% methanol,
ethyl acetate
Methanol:ethanol (1:1, v/v)
Ethanol
Ethanol
Ethanol
Methanol
Methanol
Ethanol, methanol, chloroform,
hydroalcoholic mixtures
Ethanol
Methanol–ethyl acetate mixtures
Ethyl acetate
Methanol, ethanol, 70% ethanol,
diethyl ether
Methanol
The objective of the current study was to compare four
different methods, commonly used for extracting triterpenic
acids, and to evaluate their efficacy in the extraction of maslinic
acid and oleanolic acid from olive fruit tissues. To the best of our
knowledge this is the first attempt to determine the efficacy of
different types of drying procedures of olive fruits on their
triterpenic acid content. This study is expected to provide
researchers with information on an efficient extraction method
for studies on triterpenic acid composition of olive fruits.
Experimental
Chemicals and reagents
Standards of oleanolic acid and maslinic acid were purchased from
Sigma-Aldrich (St Louis, MO, USA). Methanol, ethanol and ethyl acetate
of analytical grade were provided by Scharlau (Barcelona, Spain). Water
(HPLC grade), methanol (HPLC grade) and ortho-pshophoric acid were
also obtained from Sigma-Aldrich.
Fruit material
Olive fruits (Olea europaea L., cvs ‘Picual’ and ‘Kalamon’) were harvested
at commercial maturity stage from a commercial orchard (Arakapas,
Lemesos, Cyprus), based on size uniformity and external colour. The
maturity of fruits was determined by their moisture, oil content and total
anthocyanins (data not shown). ‘Kalamon’ is destined for fresh consumption as table olives, while ‘Picual’ is mainly destined for oil production.
Subsequently, olive fruit were destoned manually and the pulp was
immediately frozen in liquid N2 and stored at 80 C until needed.
Drying processes
Phytochem. Anal. 2012, 23, 444–449
References
Medicinal plants
Janicsak et al., 2003
Pomace olive oil, olive fruits
Olive fruits and leaves
Prunellae spica herbal extract
Persimmon fruits
Chinese fruits
Chaenomeles sinensis fruits
Leaves of Sweria plants
Garcia et al., 2008,
Romero et al., 2010
Guinda et al., 2010
Lee et al., 2009,
Zhou et al., 2010,
Li et al., 2011b
Fang et al., 2010
Li et al., 2011a
Balm leaves
Lantana camara roots
Punica granatum flowers,
olive leaves and aromatic plants,
olive oil wastes
Leaves and seeds of great plantain
Herodez et al., 2003
Banik and Pandey, 2008
Wang et al., 2006,
Kontogianni et al., 2009,
Parra et al., 2010
Tarvainen et al., 2010
Chaenomeles sinensis fruits
Fang et al., 2010
loss of dried samples was recorded at time intervals and all samples were
dried until constant weight. Then, the dried samples were extracted by
ultrasonic assisted extraction with ethanol in order to evaluate the effect
of the drying method on triterpenic acid content.
Extraction methods
Frozen olive fruit material was triturated and extracted with four
common methods: (i) solid–liquid extraction, (ii) ultrasonic assisted
extraction, (iii) extraction at 40 C and (iv) Soxtec extraction. All extraction
methods were tested using ethyl acetate, methanol, ethanol and
methanol:ethanol (1:1, v/v) as solvents.
Solid–liquid extraction. Olive fruit (~0.5 g) was mixed into a 20 mL
centrifuge tube with 4 mL of solvent, agitated for 1 min and centrifuged
(9500 rpm, 5 min, 20 C). The procedure was repeated six times, and the
pooled solvent extract was evaporated to dryness and subsequently
was dissolved into 10 mL of methanol, as analytically described by
Romero et al. (2010).
Ultrasonic assisted extraction. Approximately 0.5 g of olive fruit was
placed with 20 mL of solvent into 50 mL centrifuge tube. The mixture
was left for 30 min in an ultrasonic bath (UCI-50, 35 KHz, Raypa-R. Espinar,
S.L., Terrassa, Barcelona, Spain). Then, the mixture was centrifuged
(9500 rpm, 5 min, 20 C) and the solvent extract was evaporated
to dryness. The residue was dissolved into 10 mL of methanol. This
extraction method has been suggested to extract triterpenic acids from
different plant tissues (Prunellae spica herb, roots, stems and leaves of
Sweria species; Lee et al., 2009; Li et al., 2011a).
Extraction at 40 C. Approximately 0.5 g of olive fruit was mixed with
27.5 mL of solvent into a 50 mL centrifuge tube. The sample mixture was
placed for 60 min into a water bath at 40 C. The extraction was
performed in a temperature-controlled water bath (Sonorex digitec
compact bath DT-100, Bandelin electronic GmbH, Berlin, Germany).
Then, the mixture was centrifuged (9500 rpm, 5 min, 20 C) and the
solvent extract was evaporated to dryness. The residue was dissolved
into 10 mL of methanol. This procedure has been optimised to recover
oleanolic acid from Lantana camara roots (Banik and Pandey, 2008).
Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/pca
445
Olive fruit tissue was dried with the following methods: (i) no drying
(designated as frozen material); (ii) drying in a convection oven (Venticell
111, MMM Group, Munich, Germany) at 35 C; (iii) drying in a convection
oven at 105 C; and (iv) freeze-drying using a laboratory-scale freeze
dryer (Christ Alpha 1-4 LD plus, SciQuip Ltd, Shropshire, UK). Moisture
Tissue
V. Goulas and G. A. Manganaris
Soxtec extraction. The extraction of triterpenic acids from olive
fruits was carried out on a semi-automated Soxtec system 2055 (FossW
Analytical, Hilleroed, Denmark). Two grams of each sample were
weighed into extraction cellulose thimbles and 80 mL of solvent were
transferred into each Soxtec extraction cup. Triterpenic acids were
extracted by setting the unit at the boiling position for 30 min and then
at the rising position for 1 h (Chukwumah, et al., 2007). The solvent
extracts were allowed to cool down at room temperature and
evaporated to dryness in a rotary evaporator at 35 C. The residue was
dissolved into 10 mL of methanol. Soxhlet extraction has been used to
extract triterpenic acids from an array of plant tissues (Kontogianni
et al., 2009; Parra et al., 2010; Tarvainen et al., 2010).
Determination of triterpenic acids
A Waters series HPLC equipped with vacuum degasser, binary pump,
autosampler, thermostatted column compartment, dual l absorbance
detector and Empower software (Waters Corporation, Milford, Ireland)
for data collection and analysis was used. After filtration on Millipore
paper (0.22 mm), 20 mL of each extract were injected on a Discovery
C18-column (250 mm 4.6 mm i.d., 5 mm). The temperature of the
column was kept constant at 35 C using a Waters column heater.
The mobile phase (methanol–water acidified with ortho-phosphoric
acid at pH 3.0, 92: 8, v/v) was introduced to the column at a flow rate
0.8 mL/min and the eluate was monitored at 210 nm. A calibration
curve was established by injecting the standards three times at six
different concentrations, in the range of 50 to 3000 oleanolic acid
mg/L. Maslinic acid and oleanolic acid were quantified using oleanolic
acid as a standard, because a similar response factor was assumed for
both triterpenic acids (Garcia et al., 2008; Romero et al., 2010). The
regression coefficient value was 0.9994 for oleanolic acid.
Statistical analysis
Statistical analysis was carried out using the software package SPSS v18.0
(SPSS Inc., Chicago, USA) and the comparison of averages of each
treatment was based on the analysis of variance (one-way ANOVA)
according to Duncan’s multiple range test at a significance level 5%
(p ≤ 0.05). Results were presented as average standard error of six
replications per sample.
Results and Discussion
The effect of solvent and extraction method
The efficacy of different solvents (ethyl acetate, methanol, ethanol and mixture methanol:ethanol) using different extraction
methods for the recovery of triterpenic acids from olive fruits
was investigated. The selection of the most suitable solvent for
extracting the analytes of interest from the sample matrix is a
fundamental step in the development of an extraction method.
Fruit material from two olive cultivars (‘Kalamon’ and ‘Picual’)
were used to compare different extraction methods, because
the oil content may affect the solubilization of triterpenic acids
in the solvents. Reverse-phase (RP) HPLC was performed for
the determination of oleanolic and maslinic acids, as described
previously (Garcia et al., 2008; Romero et al., 2010). Considering
the similar chemical structure of triterpenic acids, a satisfactory
chromatographic separation of maslinic acid and oleanolic acid
was achieved (Fig. 1). On the other hand, ursolic acid, an isomer
of oleanolic acid, was not detected in olive fruits in line with a
previous study (Romero et al., 2010).
The efficiency of exhaustive solid–liquid extraction was determined initially and the total triterpenic acids ranged from 2015
up to 2372 mg/kg fresh weight (FW) for ‘Kalamon’ fruit and from
2232 up to 2545 mg/kg FW for ‘Picual’ fruit (Fig. 2). The chromatograms also revealed that maslinic acid was the major triterpenic acid in olive fruits (Fig. 1). Results showed that the methanol:
ethanol mixture (1:1, v/v) was the most efficient for the recovery
of triterpenic acids, followed by ethyl acetate, while the extraction ability of methanol and ethanol was statistically lower. The
same trend was also observed for the recovery of individual
triterpenic acids (Fig. 2).
Ultrasonic assisted extraction has become a popular alternative method to extract different classes of phytochemicals in
food, herbs and other natural products. Results demonstrated
that ultrasonic assisted extraction was a more effective extraction method compared with conventional solid–liquid extraction. The use of ethanol or a mixture of ethanol and methanol
446
Figure 1. The HPLC chromatogram at 210 nm of the triterpenic acid extracts of olive fruits. Peaks: (1) maslinic acid; (2) oleanolic acid.
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Copyright © 2011 John Wiley & Sons, Ltd.
Phytochem. Anal. 2012, 23, 444–449
Determination of Triterpenic Acid Content
Figure 2. Triterpenic acid content of frozen (a) ‘Kalamon’ fruit and (b)
‘Picual’ fruit, subjected to different extraction methods and solvents applied. The symbols M, E, ME and EA represent the solvents methanol,
ethanol, methanol-ethanol mixture and ethyl acetate, respectively.
Phytochem. Anal. 2012, 23, 444–449
The effect of drying method
Based on the drying method applied, freeze-dried fruit material
had the higher triterpenic acid content followed by frozen material and oven dried material at both temperature regimes (35 C
and 105 C; Table 2). Interestingly, freeze-dried ‘Picual’ olive fruits
showed higher triterpenic acid content compared with frozenmaterial; this was not the case for ‘Kalamon’ fruit, as no
Copyright © 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/pca
447
as an extraction solvent is suggested as the most appropriate
solvent for the recovery of maslinic acid and oleanolic acid
(Fig. 2). Intriguingly, methanol showed the lowest extraction efficiency, although a previous study reported that the solubility
of oleanolic acid in methanol is higher compared with ethanol
and ethyl acetate at room temperature (Schneider et al., 2009).
It is known that the application of heating or ultrasound energy
affects the ability of solvents to extract phytochemicals. In
addition, it has been shown that ethanol had higher efficacy to
recover triterpenic acids from Swertia plants using ultrasonic
assisted extraction against methanol and hydro-ethanolic
mixtures (Li et al., 2011a).
The efficiency of solvents to recover triterpenic acids at 40 C
was also evaluated. The selection of the extraction temperature
was based on a previous optimisation study for oleanolic acid
extraction from Lantana camara roots, using response surface
methodology. Data indicated that the most efficient recovery
of triterpenic acids was performed using a mixture of methanol
and ethanol (1:1), while the yield of extraction using ethyl acetate was lower by 13.9% up to 16.3%. The solubilisation preference of triterpenic acids is affected by the temperature as this
was revealed when compared with the solid–liquid extraction
at room temperature (Fig. 2). This fact has been described previously for the recovery of other phytochemicals, such as gallic
acid and rosmarinic acid in different extraction temperatures
(Galanakis et al., in press). Furthermore, the synergistic effects
between mass transfer and heat transfer may also explain these
results (Mandal and Mandal, 2010).
Traditionally, Soxhlet has been the first choice for the extraction of triterpenic acids. In this study, a fully semi-automated
Soxtec system was used for rapid extractions of triterpenic acids
in olive fruit. This set-up is five times faster than conventional
Soxhlet extraction through rapid solvent extraction by lowering
the sample in the first step into the hot solvent (boiling step)
and then raising it above the solvent level (rinsing step). Furthermore, the solvent consumption is significantly lower, thus decreasing the cost of analysis. Results demonstrated that ethyl
acetate was the most suitable solvent for the recovery of triterpenic
acid content (2234 mg/kg FW for ‘Kalamon’ fruit and 2640 for mg/kg
FW ‘Picual’ fruit), and especially for the recovery of maslinic acid
(Fig. 2). On the other hand, the mixture of ethanol and methanol
and the methanol presented lower abilities to recover these
compounds (2028–2098 mg/kg FW for ‘Kalamon’ fruit and
2344–2405 mg/kg FW for ‘Picual’ fruit) than ethyl acetate and
ethanol (Fig. 2). A recent study on leaves and seeds of great
plantain has also reported that the use of diethyl ether dominated against methanol, ethanol and hydro-alcoholic mixtures
for the extraction of triterpenic acids (Tarvainen et al., 2010). In
addition, ethyl acetate has the advantage that it dissolves selectively all the triterpenic acids, but only little of other compounds that may interfere in further analysis.
The comparison of the different extraction methods revealed
that the ultrasonic assisted extraction with ethanol or methanolethanol mixture are the most efficient methods to extract triterpenic
acids from olive fruits. The ultrasonic assisted extraction gave
a higher yield at lower temperatures, had shorter extraction
time and in some cases better selectivity for over 50 vegetal
tissues (Toma et al., 2001). The efficiency of ultrasonication
can be explained by the fact that sonication simultaneously
enhances the hydration and fragmentation process, while
facilitating mass transfer of solutes to the extraction solvent
without significant decomposition of the solvent (Toma
et al., 2001). In general, the combination of extraction techniques with different solvents did not show great differences
(Fig. 2). However, the extraction of triterpenic acids from olive
fruit with solid–liquid extraction (methanol, ethanol), extraction
at 40 C (ethanol, ethyl acetate) and Soxtec extraction (methanol,
ethanol, ethanol and methanol mixture) should be avoided,
since they presented the lowest recovery of triterpenic acids.
Taking into consideration the extraction yield, the complexity
of extraction process, the production cost, the environmental
and safety issues, the ultrasonic assisted extraction with ethanol
is proposed for extraction of triterpenic acids from olive fruit.
The proposed methodology is fast, simple, requires small
volumes of green solvent (ethanol) and may be exploited for
other olive tissues and products, e.g. leaves, olive oil and olive
mill wastes.
V. Goulas and G. A. Manganaris
Table 2. Effect of drying method on maslinic acid, oleanolic acid and total triterpenic acids (sum of maslinic and oleanolic acid) of
olive fruits (cvs. ‘Picual’, ‘Kalamon’)*
Drying method
Content (mg/kg FW)
Maslinic acid
‘Picual’
Frozen
Freeze-drying
Oven-drying (35 C)
Oven-drying (105 C)
2447 19a
2524 16a
2252 29b
2083 64c
Oleanolic acid
‘Kalamon’
2100 17ab
2234 26a
1988 42bc
1905 85c
‘Picual’
1003 18b
1158 14a
987 54b
949 13b
‘Kalamon’
838 16c
933 13b
1026 10a
977 18ab
Total triterpenic acids
‘Picual’
3449 32b
3682 18a
3240 71c
3032 75d
‘Kalamon’
3076 34ab
3260 29a
2921 51bc
2743 93c
* Values within each column followed by the same letter are not statistically significant according to Duncan’s multiple range test
at a significance level of p < 0.05 (n = 6, standard error).
differences were found between the two drying methods.
Previous studies have also demonstrated the superiority of
freeze-drying compared with common drying methods in the
determination of phenolic compounds, saponins, carotenoids
and capsaicinoids in diverse plant material (Fuentes-Alventosa
et al., 2009; Rios and Gutierrez-Rosales, 2010; Topuz et al.,
2011). It is well known that the freeze-drying procedure
enhances the extraction efficiency since ice crystals formed
within the sample matrix can rupture cell structure, which allows
exit of cellular components and access of solvent (Zhang et al.,
2009). Overall, freeze-drying is proposed for analytical determination, while the use of frozen olive fruits to recover triterpenic
acids at the large scale is also recommended, since freeze-drying
is a high-cost procedure. On the other hand, oven drying at 35 C
and 105 C led to lower triterpenic acid content compared to
fresh olive fruit material. The amount decreased by 5.0% and
12.0% after oven drying (35 C) in ‘Kalamon’ and ‘Picual’ fruit,
respectively. The depletion of triterpenic acids was further
increased after oven drying at 105 C by 10.8% and 17.6% in
‘Kalamon’ and ‘Picual’ fruit, respectively. This reduction may be
attributed to the trapping of triterpenic acids into olive tissue
due to their dramatic shrinkage. The thermal degradation of triterpenic acids should be excluded, since triterpenic acid content
is usually determined with GC-MS methodologies (Hovaneissian
et al., 2008). The use of modern techniques such as vacuum
drying, low temperature drying, and heat pump assisted drying
may be investigated for the drying of olive fruits.
Summary
448
This study demonstrates that ultrasonic assisted extraction
improved the extraction of triterpenic acids in olive fruits
compared with conventional solid–liquid extraction, extraction
at 40 C and Soxtec extraction. The selection of solvents also
significantly affected the triterpenic acid content of olive fruit
extracts; ethanol or the methanol-ethanol mixture was the most
efficient solvent for ultrasonic extraction of triterpenic acids in
olive fruits. The oil content of olive fruits was not observed to affect the extractability of triterpenic acids. Furthermore, this work
shows the benefits of using frozen or lyophilised tissue to extract
triterpenic acids from olive fruits. The current study proposes a
simple, rapid, low-cost, eco-friendly and efficient protocol for
the extraction and subsequent determination of triterpenic acid
content. This methodology fulfills the need for a standardised
extraction protocol for the quantification of triterpenic acids
wileyonlinelibrary.com/journal/pca
in olive fruits. Finally, the optimisation of the parameters of each
extraction method using statistic methodologies such as response surface methodology and multi-linear regression presents a challenging future perspective.
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