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. wileyonlinelibrary.com/journal/pca 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. 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