Triterpenic acids in table olives

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Food Chemistry 118 (2010) 670–674 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Triterpenic acids in table olives Concepción Romero, Aranzazu García, Eduardo Medina, Mª Victoria Ruíz-Méndez, Antonio de Castro, Manuel Brenes * Instituto de la Grasa (CSIC), Avda. Padre García Tejero 4, 41012-Seville, Spain a r t i c l e i n f o Article history: Received 12 March 2009 Received in revised form 6 May 2009 Accepted 13 May 2009 Keywords: Table olives Maslinic acid Oleanolic acid Triterpenic acid, variety a b s t r a c t An experimental investigation was carried out for the first time on the triterpenic acids in table olives. Maslinic acid was found in a higher concentration than oleanolic acid in the flesh of 17, unprocessed olive varieties, with the Picual and the Manzanilla varieties showing the highest and almost the lowest contents, respectively. The level of triterpenic acids in several types of commercial black and green olives ranged from 460 to 1470 mg/kg fruit, which represents a much higher value than reported for virgin olive oils. In fact, the NaOH treatment employed to debitter black and green olives reduced the concentrations of these substances in the flesh because of their solubilisation into alkaline solutions. Thus, natural black olives, which are not treated with NaOH, showed a higher concentration than 2000 mg/kg in the olive flesh. These results will contribute to the reevaluation of table olives from a nutritional and functional point of view because of the promising bioactivity properties attributed to olive triterpenic acids. Published by Elsevier Ltd. 1. Introduction Triterpenic acids are widespread in plants in the form of free acids or aglycones for triterpenoid saponins. Their extracts have been used for centuries in folk medicine as anti-inflammatory, anti-diabetes and hepatoprotective agents (Liu, 1995) and they have recently attracted interest in the scientific community because of their anti-carcinogenic activity (He & Liu, 2007; Struch, Jaeger, Schempp, Scheffler, & Martin, 2008) which makes them very attractive for use in cosmetics and healthcare products as functional compounds. Among the free triterpenic acids, oleanolic, betulinic, ursolic and maslinic are some of the most abundant in the plant kingdom. Olive fruits and leaves are especially rich in oleanolic and maslinic acids (Vázquez & Janer, 1969), and small amounts of ursolic and betulinic have also been occasionally described in olive products (Bianchi & Vlahov, 1994). Both oleanolic and maslinic acids are concentrated on the surface of olive leaves to form a physical barrier that prevents microbes from penetrating into the leaf (Kubo, Matsumoto, & Takase, 1985). They are also present in high concentrations in the epicarp of the fruit forming part of the waxes that cover them; and oleanolic acid has even been found in the endocarp, wood shell and seeds of olives (Bianchi & Vlahov, 1994). The presence of triterpenic acids in olive oil is gaining interest because of their anti-tumour activities (Rodríguez-Rodríguez, Herrera, Álvarez de Sotomayor, & Ruíz-Gutiérrez, 2007). However, the * Corresponding author. Tel.: +34 954690850; fax: +34 954691262. E-mail address: brenes@cica.es (M. Brenes). 0308-8146/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodchem.2009.05.037 concentration of these compounds in oils depends on the oil quality (Pérez-Camino & Cert, 1999): extra virgin olive oils with low acidity did not reach 200 mg/kg, virgin olive oils with acidity higher than 1% exceeded 300 mg/kg, and crude pomace olive oils had up to 10,000 mg/kg, although the latter oils have to be refined before consumption. In fact, an enrichment in triterpenic acids occurs in the crude pomace oil during the storage of the pomace paste (alpeorujo, García, Brenes, Dobarganes, Romero, & Ruíz-Méndez, 2008) but a significant loss in these substances is produced during the refining process of the oil, particularly when a neutralisation step is used (chemical refining, Pérez-Camino & Cert, 1999). In the case of table olives, very few data on these substances are available. Earlier, Gaviña and Viguera (1964) detected oleanolic and maslinic acids in the sodium hydroxide solutions (lye) generated during the processing of Spanish-style green olives and this was confirmed later by other researchers (Bianchi, 2003; Vázquez & Janer, 1969), but, to our knowledge, no more data have been published until now. Because of the recent interest surrounding the triterpenic acids of olives, a deeper understanding of their contents in commercial table olives, their changes during olive processing and their contents in table olive varieties is required. At the same time, the olive triterpenic acids, oleanolic and maslinic, have been attributed with anti-oxidant (Tsai & Yin, 2008), anti-hyperglycemic (Liu, Hongbin, Weigang, Dongyan, & Luyong, 2007; Sato et al., 2007), anti-microbial (Horiuchi et al., 2007) and anti-cancer activity (Juan, Planas, Ruíz-Gutiérrez, Daniel, & Wenzel, 2008; ReyesZurita, Rufino-Palomares, Lupiáñez, & Cascante, 2009) They have also been proposed to feed rainbow trout (Fernández-Navarro, de la Higuera, Lupiáñez, Peragón, & Amores, 2008) and to enrich Author's personal copy C. Romero et al. / Food Chemistry 118 (2010) 670–674 vegetable oils as new functional compounds (Guinda, Albi, PérezCamino, & Lanzón, 2004). Therefore, the aim of this research was to study the triterpenic acid contents of table olives as well as the wastewaters generated during their processing as a potential source of these high-value substances. 671 of the method, the pH of lyes and washing solutions was dropped below 3. Subsequently, triterpenic acids were extracted from the supernatant with ethyl acetate and they were not detected in these solutions. By contrast, the precipitate formed during pH correction was dissolved in methanol and it was analysed by HPLC. The amount of triterpenic acids in the precipitate was the same as found when lyes and washing solutions without any pH modification were analysed by using ethyl acetate as extracting solvent. 2. Materials and methods 2.6. Extraction of triterpenic acids from olive flesh 2.1. Samples Olive fruits of Ascolana, Domat, Aloreña, Arbequina, Morona, Conservolea, Gordal, Picual, Hojiblanca, Leccino, Koroneiki, Verdial, Picholine, Cacereña, Kalamata, Galega and Manzanilla varieties were hand-harvested with a green–yellow colour on the surface from two different orchards located in the provinces of Seville and Cordoba (Spain) during September–October. Commercial table olives were purchased in Spanish and Greek markets. Three samples of three different commercial brands were obtained for each table olive preparation. Twenty grams of destoned and cut olive fruits were triturated, and desiccated at 105 °C until weight stabilisation. Subsequently, 1 g of dry olives were mixed in a 10 ml centrifuge tube with 4 ml of methanol/ethanol (1:1, v/v) and vortexed for 1 min, centrifuged at 9500 rpm for 5 min at 20 °C, and the solvent was separated from the solid phase. This step was repeated six times, and the pooled solvent extract was vacuum evaporated. The residue was dissolved in 2 ml of methanol, which was filtered through 0.2 lm pore size and an aliquot (20 ll) was injected into the liquid chromatograph. 2.7. HPLC analysis of triterpenic acids: UV and MS detection 2.2. Processing Spanish-style green olives Olive fruits of Manzanilla, Hojiblanca and Gordal varieties with a green–yellow colour on the surface were put into PVC vessels. Five kilograms of fruits were covered with 4 l of 2% NaOH and maintained in the alkaline solution until the alkali penetrated 2/3 the way to the pit of the olives. Subsequently, olives were washed with tap water for 16 h and covered with brine (11% NaCl). All experiments were run in duplicate. 2.3. Processing natural black olives Olive fruits of the Manzanilla variety with a black colour on the surface were put into PVC vessels. Five kilograms of fruits were covered with an acidified brine (8% NaCl, 0.8% acetic acid) and maintained under anaerobic conditions by covering the surface of the brine with a floating cap. Experiments were run in duplicate. 2.4. Processing black olives Olive fruits of Manzanilla and Hojiblanca varieties with a green–yellow colour on the surface were preserved as commented above for natural black olives for 6 months. Subsequently, fruits were treated in cylindrical containers over two consecutive days with NaOH solutions (lye) of 1.5% and 1.2%, which were permitted to penetrate 1–2 mm into the flesh and to the pit, respectively. After each lye treatment, tap water was added every day to complete a 24 h cycle. On the third day a new washing cycle of 24 h was used. Subsequently, fruits were suspended in a ferrous gluconate solution (0.1%) for another 24 h. Air was continuously bubbled through the mixture of fruits and liquid. Experiments were run in duplicate. The chromatography system consisted of a Waters 717 plus auto sampler, a Waters 600E pump, and a Waters 996 diode array detector (Waters Inc., Mildford, MA, USA). A Spherisorb ODS-2 (5 lm, 25  46 mm i.d.; Waters Inc.) column was used. The temperature of the column was kept constant at 35 °C by using a Waters column heater. The mobile phase (methanol/acidified water with phosphoric acid at pH 3.0, 92:8, v/v) was delivered to the column at a flow rate of 0.8 ml/min and the eluate was monitored at 210 nm. Triterpenic acids (oleanolic and maslinic acids) were quantified using oleanolic acid (Sigma, St. Louis, MO, USA) as external standard. A similar response factor was assumed for both triterpenic acids on the basis of their similar UV spectra. The calibration equation, concentration (mg/l) = 0.0001  peak area, was calculated in a range of 0–3000 mg/l, and the determination coefficient was 0.996. An Alliance Waters chromatography system connected to UV and MS detectors was used to identify the compounds. The chromatography method was similar to that explained above and the mass spectra were acquired using a quadrupole mass analyser (ZMD4; Waters Inc.), equipped with a electrospray ionisation (ESI) probe and working in the negative mode. Cone voltage fragmentation was at 10 V, capillary voltage was 3 kV, desolvation temperature was 200 °C, source temperature was 120 °C, and extractor voltage was 12 V. Standards linolenic, linoleic and oleanolic acids were used to identify peaks 2–4, respectively. 2.8. Statistical analysis Statistica software version 6.0 was used for data processing (Statistica for Windows, Tulsa, OK, USA). Comparison between mean variables was made by the Duncan’s multiple range tests and the differences considered significant when p < 0.05. 2.5. Extraction of triterpenic acids from liquids An amount of 0.8 ml of sample (lyes, washing solutions and brines) was mixed with 2 ml of ethyl acetate in a 10 ml centrifuge tube. The mixture was vortexed for 1 min, centrifuged at 9500 rpm for 5 min at 20 °C, and the organic solvent was separated from the water phase. This step was repeated six times. Subsequently, the pooled organic ethyl acetate extract was vacuum evaporated and the residue dissolved in 0.8 ml of methanol, which was filtered through 0.2 lm pore size and an aliquot (20 ll) was injected into the liquid chromatograph. In order to check the exhaustiveness 3. Results and discussion Despite the recent relevance of olive triterpenic acids, there is no information on the amount of these substances in whole olive fruits. Some reports have indicated that they are concentrated in the epicarp of the fruits and contribute to more than 60% of the total waxes (Bianchi, 2003), with oleanolic and maslinic found in similar concentrations. In contrast, the amount of these substances in the mesocarp of olives is low and maslinic acid is the main com- Author's personal copy 672 C. Romero et al. / Food Chemistry 118 (2010) 670–674 ponent. Nevertheless, the triterpenic acid contents in whole olive fruits have never been quantified, particularly in those intended for table olives. In this study, oleanolic and maslinic acids were extracted from the olive fruits, which were previously dried to avoid water interference during the analysis, with a mixture of methanol/ethanol (1:1), a similar method to that employed for ‘‘alpeorujo” olive oil (García et al., 2008). The chromatographic system used was also the same developed to analyse triterpenic acids in ‘‘alpeorujo olive oil” and a similar chromatographic profile was obtained. A good separation of maslinic and oleanolic acid was achieved (Fig. 1), and the presence of peaks corresponding to linoleic and linolenic acids along with the absence of other triterpenic acids were confirmed by HPLC-MS. Compounds corresponding to these four peaks had molecular masses of 472, 278, 280 and 456 uma. Peak 1 had a molecular mass similar to that of maslinic acid and its fragmentation pattern was the same as that of oleanolic acid (peak 4). Besides, UV spectra of peaks 1 and 4 were similar. Based on all these results and literature data, peak 1 was assigned to maslinic acid. The raw fruits of the varieties studied showed important differences in their triterpenic acid contents (Fig. 2). The sum of maslinic and oleanolic acids ranged from 1500 to 3000 mg/kg olive flesh, with the concentration of maslinic being higher than that of oleanolic acid for all the studied varieties. Surprisingly, olives of the Manzanilla variety, which is the main variety processed for table olives, practically had the lowest content in triterpenic acids, lower than other important table olive varieties such as Hojiblanca, Kalamata, Gordal or Conservolea. Indeed, the Manzanilla olive variety is rich in phenolic compounds (Romero et al., 2004) and triterpenic alcohols (López-López, Montaño, Ruíz-Méndez, & Garrido-Fernández, 2008) but this is not the case for maslinic and oleanolic acids. In contrast, the Picual variety, which is seldom used for table olives but it is the most important variety for olive oil worldwide, showed the highest content in maslinic and oleanolic acids. Concerning the evolution of triterpenic acids during olive processing, Figs. 3 and 4 reflect the concentration of these substances in the different solutions generated during the two main table olive preparation methods: Spanish-style green olives and black olives. Triterpenic acids were extracted from the olive solutions (lyes, washing waters and brines) with ethyl acetate without any pH correction because it is well-known that the solubility of triterpenic acids is very low in aqueous media at pH below neutrality, and it increases at alkaline pH (Jäger, Winkler, Pfüller, & Scheffler, 2007), which is in accordance with results obtained in this study. Results revealed that the alkaline treatment gave rise to the solubilisation of maslinic and oleanolic acids into the lyes and the washing solutions. It must be said that the pH of the lyes and washing water was in most cases higher than 8. Thus, maslinic acid was found in a range of 800–1000 mg/l in the lyes of the 0.90 1: Maslinic acid (R=OH) 1 4: Oleanolic acid (R=H) Absorbance 0.70 COOH R HO 0.50 2 Concentration (mg/l) Manzanilla olives 1200 1000 Maslinic acid Oleanolic acid 800 600 400 200 0 4 NaOH solution Washing solution Brine (1 day) 0.30 Brine (4 days) 3 0.10 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Minutes Fig. 1. HPLC chromatogram at 210 nm of the triterpenic acid extract of a Spanishstyle green olives sample. Peaks: (1) maslinic acid; (2) linolenic acid; (3) linoleic acid; and (4) oleanolic acid. Concentration (mg/l) 5.00 1200 1000 Hojiblanca olives Maslinic acid Oleanolic acid 800 600 400 200 0 4000 NaOH solution Washing solution Brine (1 day) Maslinic acid Brine (4 days) Oleanolic acid 3000 2500 2000 1500 1000 500 Pi Pi cua ch l ol in G e al e K or ga o A ne rb ik eq i u A ina lo r H oj eñ ib a la nc G a C on or se da rv l o Le lea cc i M no o K ron al am a C ac ata er eñ Ve a r A dia sc l M ola an na za ni l D la om at 0 Fig. 2. Concentration in triterpenic acids of several raw olive varieties intended for table olives. Bars indicate the standard deviation of two samples obtained from different orchards. 1200 Concentration (mg/l) mg /kg olive flesh 3500 1000 Gordal olives Maslinic acid Oleanolic acid 800 600 400 200 0 NaOH solution Washing solution Brine (1 day) Brine (4 days) Fig. 3. Concentration in triterpenic acids of the different solutions generated during the processing of Spanish-style green olives of the Manzanilla, Hojiblanca and Gordal varieties. Bars mean the standard deviation. Author's personal copy C. Romero et al. / Food Chemistry 118 (2010) 670–674 Manzanilla olives Concentration (mg/l) 500 Maslinic acid Oleanolic acid 400 300 200 100 0 First NaOH First solution washing solution 500 Second NaOH solution Second washing solution Ferrous gluconate solution Hojiblanca olives Maslinic acid Concentration (mg/l) Third washing solution Oleanolic acid 400 300 200 100 0 First NaOH First solution washing solution Second NaOH solution Second washing solution Ferrous Third washing gluconate solution solution Fig. 4. Concentration in triterpenic acids of the different solutions generated during the processing of black olives of the Manzanilla and Hojiblanca varieties. Bars mean the standard deviation. Manzanilla, Gordal and Hojiblanca varieties processed according to the Spanish-style green method. The values are comparable to those reported by Bianchi (2003) for the lyes of Italian varieties, and maslinic was always detected in a higher concentration than oleanolic. Although the concentration of triterpenic acids was lower in the washing solutions than in the lyes, the ratio maslinic/oleanolic was similar for both types of olive solutions. A low concentration of triterpenic acids was found in brines during the first days of fermentation when the pH varied from 9 to 7 and they were not detected thereafter as fermentation progressed and pH dropped below neutrality. Hence, it seems that these substances precipitated at low pH. In fact, an inhibitory activity against the lactic acid bacteria growth by a precipitate formed during the acidification of brines and washing waters has been reported (De Castro, Romero, & Brenes, 2005). Because triterpenic acids possess anti-microbial activity (Horiuchi et al., 2007) and are insoluble in aqueous media at acid pH, it can be supposed that these substances are components of this precipitate and could 673 contribute to the anti-lactic acid bacteria activity of it. Moreover, it must be taken into account that Jäger et al. (2007) have reported that the solubility of triterpenic acids in plant extracts was higher than expected due to their interaction with large molecules or aggregates. On the other hand, the most concentrated solutions in triterpenic acids of all the wastewaters generated during the processing of black olives were again the lyes, followed by the washing waters and the ferrous gluconate solutions, all of them having alkaline pH (Fig. 4). These streams, as well as those generated during the processing of green olives, represent a big environmental problem for the olive factories, and our results disclosed them as a good source of these new potential functional compounds, especially the lyes and the washing waters. Likewise, maslinic and oleanolic acids were never found in the brines of natural black olives (data not shown) since they were not treated with NaOH, and were initially acidified with acetic acid up to pH 4. Therefore, this acidic medium did not allow the solubilisation of the triterpenic acids into the brines. Olives and olive oil are recognised as healthy foods from a nutritional point of view because of their content in monounsaturated fatty acids, anti-oxidant phenolic compounds and others. Triterpenic acids could contribute to the good perception that consumer have of these products, although it is necessary to know their amounts in these foods. There are few data on the concentration of triterpenic acids in edible fruits (He & Liu, 2007; Cui et al., 2006; Frighetto, Welendorf, Nigro, Frighetto, & Siani, 2008), some on olive oil (Pérez-Camino & Cert, 1999) and none on table olives. The data presented in Table 1 reflect the amount of maslinic and oleanolic acids in the main trade olive preparations. As could be expected from the data in Fig. 2–4, maslinic acid was always found in a higher concentration than oleanolic acid and olives non-treated with alkali (turning colour olives, natural black olives) were richer in these substances than those elaborated as green and black olives. Additionally, the pitting and stuffing steps did not significantly affect the concentration of triterpenic acids in olives as opposite to the decrease in hydrophilic phenolic compounds (Romero et al., 2004). Regarding olive varieties, differences were also found, with the Hojiblanca variety having a higher amount of triterpenic acids than the Manzanilla. However, the concentration of triterpenic acids in commercial table olives was more affected by the alkaline treatment and further washing cycles than other variables. Thus, turning colour olives and natural black olives had an amount of triterpenic acids as high as 1000–2000 mg/kg olive flesh, much higher than reported for virgin olive oils (Pérez-Camino & Cert, 1999). Indeed, green and black olives also had a higher concentration in these substances than olive oils. Table 1 Content in triterpenic acids (mg/kg olive flesh) of commercial table olives. Cultivar Presentation Maslinic acid Oleanolic acid Manzanilla Manzanilla Manzanilla Hojiblanca Gordal Manzanilla Manzanilla Hojiblanca Cacereña Manzanilla Kalamata Plain green olives Pitted green olives Green olives stuffed with pimento Plain green olives Plain green olives Plain black olives Pitted black olives Plain black olives Plain black olives Plain turning colour olives Plain natural black olives 384.1 ± 50.0 a 497.3 ± 87.8 ac 355.5 ± 88.4 a 904.7 ± 259.6 b 414.2 ± 89.3 a 287.1 ± 66.6 a 290.7 ± 129.1 a 506.8 ± 232.5 ab 295.1 ± 203.9 a 824.9 ± 179.5 bc 1318.4 ± 401.0 d 202.6 ± 57.3 a 330.0 ± 185.7 a 191.2 ± 89.7 a 565.2 ± 107.1 bc 294.3 ± 4.5 a 178.8 ± 43.7 a 169.7 ± 121.0 a 364.5 ± 242.3 ac 185.1 ± 121.1 a 274.2 ± 61.4 a 841.4 ± 162.9 b Each value is the mean ± standard deviation of three samples. Letters within columns designate statistically significant differences (p < 0.05) according to the Duncańs New Multiple Range Test. Triterpenic acids were extracted from olives as described in Section 2.6. Author's personal copy 674 C. Romero et al. / Food Chemistry 118 (2010) 670–674 4. Conclusions On the basis of our results, it can be stated that table olives are a food product rich in triterpenic acids, in particular maslinic acid. However, the NaOH treatment used to debitter the fruits leads to the solubilisation of these substances into the alkaline solutions and, therefore, to significant losses in the final product. Likewise, the alkaline wastewaters have been revealed as a good source of these valuable triterpenic acids. These results may contribute to the revaluation of table olives in view of the promising bioactivity properties attributed to olive triterpenic acids (Juan et al., 2008; Reyes-Zurita et al., 2009). Acknowledgements This study was supported by a grant from the Spanish-Government and the European Union FEDER Funds (Projects AGL-200601552 and AGL-2007-63-647). The authors thank Virginia Martín for technical assistance. References Bianchi, G., & Vlahov, G. (1994). Composition of lipid classes in the morphologically parts of the olive fruit, cv. Coratina (Olea europaea Linn.). Fat Science Technology, 96, 72–77. Bianchi, G. (2003). Lipids and phenols in table olives. European Journal of Lipids Science and Technology, 105, 229–242. Cui, T., Li, J., Kayahara, H., Ma, L., Wu, L., & Nakamura, K. (2006). Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by highperformance liquid chromatography. Journal of Agricultural and Food Chemistry, 54, 4574–4581. De Castro, A., Romero, C., & Brenes, M. (2005). A new polymer inhibitor of lactobacillus growth in table olives. European Food Research and Technology, 221, 192–196. Fernández-Navarro, M., de la Higuera, V., Lupiáñez, M., Peragón, J. A., & Amores, J. (2008). Maslinic acid added to the diet increases growth and protein-turnover rates in the white muscle of rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 147, 158–167. Frighetto, R. T. S., Welendorf, R. M., Nigro, E. N., Frighetto, N., & Siani, A. C. (2008). Isolation of ursolic acid from apple peels by high speed counter-current chromatography. Food Chemistry, 106, 767–771. García, A., Brenes, M., Dobarganes, M. C., Romero, C., & Ruíz-Méndez, Mª. V. (2008). Enrichment of pomace olive oil in triterpenic acids during storage of Alpeorujo olive paste. European Journal of Lipid Science and Technology, 110, 1136–1141. Gaviña, F., & Viguera, J. Mª. (1964). Transformaciones químicas de la aceituna durante el aderezo. VII. Identificación de un dihidroxitriterpénico asilado de la lejía residual. Grasas y Aceites, 15, 143–146. Guinda, A., Albi, T., Pérez-Camino, M. C., & Lanzón, A. (2004). Suplementation of oils with oleanolic acid from the olive leaf (Olea europaea). European Journal of Lipid Science and Technology, 106, 22–26. He, X. J., & Liu, R. H. (2007). Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for applés anticancer activity. Journal of Agricultural and Food Chemistry, 55, 4366–4370. Horiuchi, K., Shiota, S., Hatano, T., Yoshida, T., Kuroda, T., & Tsuchiya, T. (2007). Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant Enterococci (VRE). Biological and Pharmaceutical Bulletin, 30, 1147–1149. Jäger, S., Winkler, K., Pfüller, U., & Scheffler, A. (2007). Solubility studies of oleanolic acid and betulinic acid in aqueous solutions and plant extracts of Viscum album L.. Planta Medica, 73, 157–162. Juan, M. E., Planas, J. M., Ruíz-Gutiérrez, V., Daniel, H., & Wenzel, U. (2008). Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells. British Journal of Nutrition, 100, 36–43. Kubo, I., Matsumoto, A., & Takase, I. (1985). A multichemical defense mechanism of bitter olive Olea europaea (Oleaceae). Is oleuropein a phytoalexin precursor? Journal of Chemical Ecology, 11, 251–263. Liu, J. (1995). Pharmacology of oleanolic acid and ursolic acid. Journal of Ethnopharmacology, 49, 57–68. Liu, J., Hongbin, S., Weigang, D., Dongyan, M., & Luyong, Z. (2007). Maslinic acid reduces blood glucose in KK-Ay mice. Biological and Pharmaceutical Bulletin, 30, 2075–2078. López-López, A., Montaño, A., Ruíz-Méndez, M. V., & Garrido-Fernández, A. (2008). Sterols, fatty alcohols, and triterpenic alcohols in commercial table olives. Journal of the American Oil Chemistś Society, 85, 253–262. Pérez-Camino, M. C., & Cert, A. (1999). Quantitative determination of hydroxy pentacyclic triterpene acids in vegetable oils. Journal of Agricultural and Food Chemistry, 47, 1558–1562. Reyes-Zurita, F. J., Rufino-Palomares, E. E., Lupiáñez, J. A., & Cascante, M. (2009). Maslinic acid, a natural triterpene from Olea europaea L. induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway. Cancer Letters, 273, 44–54. Rodríguez-Rodríguez, R., Herrera, Mª. D., Álvarez de Sotomayor, M., & RuízGutiérrez, V. (2007). Pomace olive oil improves endothelial function in spontaneously hypertensive rats by increasing endothelial nitric oxide synthase expressión. American Journal of Hypertension, 20, 728–734. Romero, C., Brenes, M., Yousfi, K., García, P., García, A., & Garrido, A. (2004). Effect of cultivar and processing method on the contents of polyphenols in table olives. Journal of Agricultural and Food Chemistry, 52, 479–484. Sato, H., Genet, C., Strehle, A., Thomas, C., Lobstein, A., Wagner, A., Mioskowski, C., Auwerx, J., & Saladin, R. (2007). Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea. Biochemical and Biophysical Research Communications, 362, 793–798. Struch, C. M., Jaeger, S., Schempp, C. M., Scheffler, A., & Martin, S. F. (2008). Solubilized triterpenes from mistletoe show anti-tumor effects on skin-derived cell lines. Planta Medica, 74, 1130. Tsai, S. J., & Yin, M. C. (2008). Antioxidative and anti-inflammatory protection of oleanolic acid and ursolic acid in PC12 cells. Journal of Food Science, 73, H174–H178. Vázquez, A., & Janer, Mª. L. (1969). Triterpenic acids from olive tree. Grasas y Aceites, 20, 133–138.