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Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation

  • Olga García-Martínez,

    Affiliation Faculty of Health Sciences, University of Granada, Avda de la Ilustración s/n, 18071, Granada, Spain

  • Elvira De Luna-Bertos,

    Affiliation Faculty of Health Sciences, University of Granada, Avda de la Ilustración s/n, 18071, Granada, Spain

  • Javier Ramos-Torrecillas,

    Affiliation Faculty of Health Sciences, University of Granada, Avda de la Ilustración s/n, 18071, Granada, Spain

  • Concepción Ruiz,

    Affiliation Faculty of Health Sciences, University of Granada, Avda de la Ilustración s/n, 18071, Granada, Spain

  • Egle Milia,

    Affiliation Faculty of Medicine and Surgery, University of Sassari, Piazza Universitá 21, 07100, Sassari, Italy

  • María Luisa Lorenzo,

    Affiliation Department of Nutrition and Food Science, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071, Granada, Spain

  • Brigida Jimenez,

    Affiliation Agricultural Research Training Centre, Ministry of Agriculture and Fisheries, Ctra. Cabra-Doña Mencía, Km. 2.5, 14940, Cabra, Córdoba, Spain

  • Araceli Sánchez-Ortiz,

    Affiliation Agricultural Research Training Centre, Ministry of Agriculture and Fisheries, Ctra. Bailen-Motril, 23620, Km. 18,5, Mengibar, Jaén, Spain

  • Ana Rivas

    amrivas@ugr.es

    Affiliation Department of Nutrition and Food Science, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071, Granada, Spain

Phenolic Compounds in Extra Virgin Olive Oil Stimulate Human Osteoblastic Cell Proliferation

  • Olga García-Martínez, 
  • Elvira De Luna-Bertos, 
  • Javier Ramos-Torrecillas, 
  • Concepción Ruiz, 
  • Egle Milia, 
  • María Luisa Lorenzo, 
  • Brigida Jimenez, 
  • Araceli Sánchez-Ortiz, 
  • Ana Rivas
PLOS
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Abstract

In this study, we aimed to clarify the effects of phenolic compounds and extracts from different extra virgin olive oil (EVOO) varieties obtained from fruits of different ripening stages on osteoblast cells (MG-63) proliferation. Cell proliferation was increased by hydroxytyrosol, luteolin, apigenin, p-coumaric, caffeic, and ferulic acids by approximately 11–16%, as compared with controls that were treated with one vehicle alone, while (+)-pinoresinol, oleuropein, sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation. All phenolic extracts stimulated MG-63 cell growth, and they induced higher cell proliferation rates than individual compounds. The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. A decline in osteoblast proliferation was observed in oils obtained from olive fruits collected at the end of the harvest period, as their total phenolic content decreases at this late stage. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e.osteoporosis) in adulthood and the elderly.

Introduction

Bone health is a major public health issue. Osteoporosis is a disease that affects many millions of people around the world and will take on increasing significance as people live longer and the world´s population continues to increase in number [1]. Although nutrition is only one of the many factors that influence bone mass and fragility fractures, it is of particular importance to bone health because it is modifiable.

In Europe, conspicuous differences are encountered in the severity of osteoporosis, the lowest incidence being reported in the Mediterranean area. Among the environmental factors underlying this difference is the traditional Mediterranean diet, rich in fruit and vegetables consumption, with a high intake of olive oil [2, 3]. The biological properties of the phenolic compounds found in Extra Virgin Olive Oil (EVOO) have been extensively studied [4]. However, despite the myriad of potential health benefits of olive oil phenolic compounds, there are only few data relating their possible preventive effect on osteoporosis [59]. It has been described that the consumption of olives, olive oil, and oleuropein and hydroxytyrosol–the main phenolic compounds in leaves and fruit oil respectively–can prevent the loss of bone mass in animal and cell models [7]. Much less is known about the effects of other olive oil phenolic compounds on bone health. Recently, we have demonstrated that the phenolic fractions of Sicilian EVOO induced osteoblast cell growth using the human MG-63 osteosarcoma cell line [9].

Osteoblasts are bone-forming cells derived from undifferentiated pluripotent mesenchymal cells. The formation of bone involves a complex series of events that include osteoprogenitor cell proliferation and differentiation, and eventually result in the formation of a mineralized extracellular matrix. Numerous cytokines, hormones, and growth factors control bone formation by regulating osteoblast cell proliferation and differentiation. It has been demonstrated that phenolic compounds may modulate osteoblast cell functions [10, 11]. However, to our knowledge, there is no information available on the ability of olive oil phenolic compounds or olive oil extracts to regulate cultured osteoblast proliferation.

The most important phenolic compounds that have been identified on EVOO may be divided into different groups such as phenolic acids, phenolic alcohols, secoiridoids, lignans, and flavones [12].The phenolic content of EVOO is influenced by several factors: the harvest date–during which the qualitative and quantitative composition of the phenolic substances undergoes sharp fluctuations-[13], industrial processing techniques, and storage and preservation methods [14]. Therefore, the aims of this study were, firstly, to investigate the effects of EVOO phenolic compounds on osteoblast cell growth, using the human MG-63 osteosarcoma cell line. Secondly, we investigated the influence of olive variety and ripening degree on the osteoblastic cell proliferation effect of EVOO phenolic extracts. To such purpose, we sampled four monovarietal EVOO obtained from fruits at three different ripening stages.

Material and Methods

Chemicals

Standards of apigenin, luteolin, hydroxytyrosol, tyrosol, vanillin, caffeic, p-coumaric, ferulic, sinapic and vanilic acids were purchased from Sigma-Aldrich (St. Louis, MO); (+)-pinoresinol was from Arbo-Nova (Turku, Finland), and oleuropein was acquired from Extrasynthese (Genay, France). Stock solutions of phenolic compounds were prepared in methanol and stored at -20°C. All the solvents used were of analytical or HPLC grade (Sigma-Aldrich). Water was of Milli-Q quality (Millipore Corp, Bedford, MA, USA).

Olives

Olive fruits were sampled from olive trees of the cultivars Picual, Arbequina, Picudo and Hojiblanca grown in the experimental farm at the Agricultural Research Training Centre in Cabra (Cordoba, Southern Spain). Ten adult 27 year-old olive trees of the varieties Picual and Hojiblanca and 16 year-old olive trees of the Arbequina and Picudo varieties, spaced 12x12 m2, were identified and carefully marked. Olive samples were hand-picked, at the beginning, middle, and end of the harvest from different olive varieties. The olive ripening index was determined according to the method proposed by the International Olive Oil Council (IOOC), based on olive skin and pulp colour [15]. Only healthy fruits without any kind of infection or physical damage were processed.

Oil Samples

Extra virgin olive oil (EVOO) samples were obtained using an Abencor analyzer (Abengoa S.A., Sevilla, Spain); this system reproduces the industrial process at laboratory scale and consists of three basic elements: hammer mill, thermobeater, and pulp centrifuge [16]. The EVOO obtained was decanted and stored in amber glass bottles at 4°C in darkness without headspace until analysis.

Sample Preparation

To isolate the phenolic fraction of olive oils, we used the method proposed by the IOOC [2, 17, 18]. Briefly, the analytical methodology combines olive oil extraction with methanol/water (80/20), ultrasonic bath for 15 min at ambient temperature and centrifuge at 5000 rev/min for 25 min. After that, an aliquot of the supernatant phase is filtered through a 5 ml plastic syringe using a Millex®-HV PVDF 0.45 μm filter (Millipore Corp, Billerica, MA, USA). Extractions were replicated three times, and phenolic extracts were stored at -20°C until analysis.

Total Phenol Measurements

Phenolic extracts were subsequently quantified by HPLC using an UV-Vis detector following IOOC’s methodology [17]. The content of total phenols is expressed in mg/kg of tyrosol. HPLC analyses were performed with a Varian ProStar (Walnut Creek, CA, USA), equipped with a binary pump delivery system, and a Varian 230 UV-Vis detector. A Waters Spherisorb® 5 mm ODS2 column, 5 μm, 4.6 cm x 200 mm (Marlborough, MA, USA) was used.

UPLC-TOF- MS Analyses of Individual Phenolic Compounds

We used ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS) analysis to detect and measure individual phenolic compounds in prepared phenolic extracts. The UPLC system consisted of an AcQuity TM UPLC equipped with a binary pump system (Waters, Milford, MA, USA) using an AcQuity UPLC TM BEH C18 column (1.7 μm, 100 mm x 2.1 mm i.d.) from Waters. The UPLC-TOF-MS methodology was describes elsewhere [18]. The identification of phenolic compounds was carried out by comparing both, retention times and MS spectral data from olive oil samples and standards.

Cell Culture

The human osteosarcoma cell line MG-63 was purchased from American Type Cultures Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Gibco Cell Culture Products, Carlsbad, CA) with 100 IU/ml penicillin (Lab Roger SA, Barcelona, Spain), 50 μg/ml gentamicin (Braum Medical SA, Jaen, Spain), 2.5 μg/ml amphotericin B (Sigma, St Louis, MO, USA), 1% glutamine (Sigma, St Louis, MO, USA), 2% HEPES (Sigma, St Louis, MO, USA), and supplemented with 10% fetal bovine serum (FBS) (Gibco, Paisley, UK). Cultures were kept at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were detached from the culture flask with a solution of 0.05% Trypsin (Sigma, St Louis, MO, USA) and 0.02% ethylenediaminetetraacetic acid (EDTA) (Sigma, St Louis, MO, USA) and then washed and suspended in complete culture medium with 10% FBS. Prior to the beginning of each experiment, all cells were grown in estrogen-free media (DMEM without red phenol) for at least 24 hours.

Cell Proliferation Assay

The cell proliferation methodology was described elsewhere [19, 20]. Briefly, proliferation was determined with an MTT method (Sigma-Aldrich Chemie). This is a colorimetric method that measures the chemical reduction of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into formazan, which is directly proportional to the number of viable cells in the tested culture. Osteoblasts were seed at 1 x 104 cells/ml per well into a 96-well plate (Falcom, Becton Dickinson Labware, New Jersey) in estrogen-free culture medium without FBS and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 for 24 hours. Then, the media was replaced with DMEM containing different concentrations of treatment compounds: phenolic compounds at 10−5, 10−6, 10−7, 10−8 and 10−9 M and prepared phenolic extracts dissolved in fresh culture medium at concentrations of 0.001, 0.0001 and 0.00001%. All experiments included cells incubated under the same conditions without treatment compounds and incubated cells with 0.001% of methanol as an internal control. Three separate experiments were performed for each treatment and at least every experiment was performed in triplicate. On completion of treatment, the media were replaced with DMEM, without phenol-red, containing 0.5 mg/ml MTT (Sigma, St Louis, MO, USA) and incubated for 4 hours. Cellular reduction of the tetrazolium ring of MTT resulted in the formation of a dark-purple water-insoluble deposit, formazan crystals. After incubation, the media was aspired and DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm with a spectrophotometer (Sunrise TM, Tecan, Männedorf, Switzerland). Cell proliferation percentages (% > control) were calculated as related to cell cultures treated with methanol alone (controls).

Statistical analysis

Statistical analysis was performed using SPSS (v. 15.0, Statistical Package for the Social Sciences, Chicago, IL). Numerical data on cell proliferation are expressed as the mean of three independent experiments; statistically significant differences were analyzed by Student´s t-test. Significance of differences at a 5% level among phenolic compounds means was determined by one-way ANOVA, using Tukey´s test. The results for phenolic compounds are the average of at least three repetitions. Principal components analysis (PCA) was applied to data. PCAs were carried out for all raw data processed with a cross-validation method. Data were auto-scaled before analysis.

Results and Discussion

Effect of olive oil phenolic compounds on MG-63 osteoblast cells proliferation

Nutritional and pharmacological factors are needed to prevent bone loss with aging. In this study, we aimed to clarify the potential effects of olive oil phenolic compounds on osteoblast proliferation. MG-63 osteoblast cells were cultured in the presence of phenolic-compound methanolic dilutions in fresh culture medium. The MG63 human osteosarcoma line was selected as osteoblast model in this study because it is the most widely used cell line in studies on the effects of drug or other treatments on the osteoblast [21, 22], although the limitations of studying a tumor line should be borne in mind, as the differentiation pattern of MG63 may differ from that of real osteoblasts. Twelve olive oil phenolic compounds were evaluated. The highest solvent concentration in culture media (0.001%, v/v methanol) had no significant effects on cell proliferation. Treatments with phenolic compounds (10−4 to 10-3M) decreased dramatically cell proliferation (data not shown), suggesting that higher concentrations of these compounds were cytotoxic in MG-63 cells.

There were remarkable differences in the ability of individual phenolic compounds to stimulate osteoblast proliferation (Table 1). Thus, concentrations as high as 10−6 M were needed to significantly increase cell proliferation when MG-63 cells were cultured in the presence of hydroxytyrosol. Exposure of osteoblast cells to hydroxytyrosol increased the number of cells by approximately 11% in 24 h, as compared with control cultures that were treated with one vehicle alone. Conversely, concentrations lower than 10-6M did not have any significant effect on cells. The underlying mechanism of the effect of hydroxytyrosol on MG-63 cells needs to be elucidated. However, despite of these results, we can not affirm that the polyphenols in general are mitogenic for osteoblasts. Moreover, other in vitro studies have shown that hydroxytyrosol stimulates osteoblastic cell activity. These studies have used another cell line (MC3T3 -E1 cells) and obtained similar results to ours with the MG63 cell line [5].

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Table 1. Proliferation of MG-63 cells with treatment of olive oil phenolic compounds.

https://doi.org/10.1371/journal.pone.0150045.t001

Among olive oil polyphenols, hydroxytyrosol has the strongest antioxidant effects [4]. Oxidative stress resulting in increased levels of intracellular reactive oxygen species has been reported to suppress bone metabolism. It has been recently demonstrated that hydroxytyrosol effectively decreases H2O2 levels in MC3T3-E1 osteoblastic cells [5]. In addition, H2O2 has been reported to suppress differentiation markers–such as alkaline phosphatase activity–, type I collagen gene expression, and osteoblastic cell mineralization [23].

Treatment of osteoblast cells with olive oil phenolic acids increased the number of cells approximately by 12–16% as compared with controls. The investigated phenolic acids affect MG-63 cells differently. Cell proliferation was increased by p-coumaric, caffeic, and ferulic acids by approximately 12–16%, as compared with controls in the range of 10−5 to 10−9 M, while sinapic, vanillic acid and derivative (vanillin) did not affect cell proliferation (Table 1). Proliferation increase was not dose-dependent, probably due to plateau reached at the lowest concentration. Researchers have shown the phenolic compounds in different vegetable species can modulate the functions of osteoblastic cells, including their proliferative capacity and maturation, by increasing alkaline phosphatase activity and the deposit of calcium ions in the extracellular matrix [5, 10]. The mechanisms proposed to underlie these changes in osteoblastic activity include the modification of osteoblast function by certain phenolic compounds via the modulation of different transcription factors, e.g., Cbfa1/Runx2, and bone morphogenetic proteins, e.g., osterix and osteocalcin; these are all essential molecules to induce osteoblast differentiation, which in turn may activate genes involved in the bone remodelling process [10, 24, 25]

The effect of phenolic acids on MG-63 proliferation could be added to the list of positive effects of these acids such us their anti-inflamatory, antioxidant, antimutagenic, anticarcinogenic, and body weight activities [26]. There is also growing evidence both in vitro and in vivo studies that natural phenolic acids may favorably affect the skeletal system. This effect has been attributed to the inhibition of bone resorption and stimulation of bone formation [27, 28]. In addition, Chen et al. [29] showed that phenolic acids derived from the breakdown of blueberries polyphenols appear in the serum following consumption and stimulate osteoblast differentiation through Wnt signaling, indicating their potential in the prevention of bone loss.

A possible mechanism of phenolic acids on osteoblast to be considered is their binding to estrogen receptors, which are found in osteoblastic cells [30]. The presence of 17 β-estradiol has been reported to induce a significant increase in osteoblastic cell proliferation, DNA and protein content, and alkaline phosphatase activity [31]. There are sparse experimental data that phenolic acids may have some in vitro estrogenic activity. Ferulic acid estrogen receptor-dependently stimulated proliferation of human breast cancer cells in a concentration and estrogen receptor-dependent manner [32]. On the other hand, caffeic acid has been reported to increase the proliferation of MCF7 cells [33]. In addition, there are also some data available on the in vivo estrogenic effects of phenolic acids. In ovariectomized rats, ferulic acid, caffeic acid and, to a lesser extent, p-coumaric acid increased serum estradiol levels [34].

The effect of oleuropein–a polyphenol belonging to the secoiridoid class found in olives and their derivatives–on human osteosarcoma MG-63 proliferation was also investigated. Oleuropein did not induce MG-63 osteosarcoma cell proliferation. To our knowledge, there are no data available on the effect of oleuropein on human osteoblasts. However, it has been described that the consumption of oleuropein can prevent the loss of bone mass in a rat model of bone mass loss associating ovariectomy and acute inflammation [35].It is interesting to note that this compound was not able to affect bone mineral density in ovariectomised rats when inflammation was not induced [7, 36], which suggests that oleuropein may exert its bone-sparing effect by modulating inflammation rather than acting directly on bone metabolism [37].

Flavonoids and Lignans–groups of phenolic compounds abundant in plants–are known to have many beneficial biological effects including anti-inflammatory, antioxidant and estrogenic activity. In this sense, (+)-pinoresinol–a major component in the lignan phenolic fraction of olive oil–did not have any effect on cell proliferation. On the contrary, in the rat osteosarcoma cell line UMR106, pinoresinol shows stimulating effects both on UMR 106 cell proliferation and alkaline phosphatase activity [38]. Luteolin and apigenin–which are olive oil flavonoids–increased cell proliferation by 11–15% in all tested concentrations. It has been shown that luteolin inhibited the bone resorptive activity of differentiated osteoclasts [39]; additionally, luteolin has an anabolic effect, as it increases collagen synthesis, alkaline phosphatase (ALP) activity, and osteocalcin secretion, and it inhibits 3-morpholinosydnonimine-stimulated production of proinflammatory mediators in osteoblastic MC3T3-E1 cells in vitro [40]. Also, oral administration of luteolin (5 and 20 mg/kg per day) to ovariectomised mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur [39]. It should be pointed out that the mechanism of action of flavonoids on bone has been partly attributed to estrogen action [41].

Identification and quantification of phenolic compounds in EVOO

We identified and quantified the phenolic compounds in phenolic extracts obtained from EVOOs Picual, Arbequina, Hojiblanca and Picudo used in MG-63 cell proliferation assays. Mass spectral data and retention time are reported for all identified compounds (Table 2). Table 3 shows the mean values for the total and individual phenolic compounds detected in oils of the varieties Picual, Hojiblanca, Arbequina, and Picudo at three different ripening stages. In all varieties, total phenol content decreased during ripening, in accord with the results obtained by other authors in different olive oil varieties [42]. The main phenolic compounds found in the EVOOs under study were tyrosol and hydroxytirosol. The highest amounts of both substances were found at the earliest harvest sample. Our results are in agreement with those of Martinez-Nieto et al. [13], which reported that tyrosol and hidroxytirosol concentrations decreased with increasing olive ripeness in Picual and Arbequina varieties. Caffeic acid was not detected in oils of the Picual, Arbequina and Picudo varieties; p-coumaric acid was not detected in Arbequina oils. The phenolic compounds vanillic acid and vanillin were common to all four varieties; only Hojiblanca oils obtained from olives collected at the end of the harvest did not contain vanillic acid.

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Table 3. Content of olive oil phenolic compounds (mg/Kg) with regards to olive cultivar and fruit ripening stage.

https://doi.org/10.1371/journal.pone.0150045.t003

As far as concentrations of flavones and lignans are concerned, luteolin, apigenin and (+)-pinoresinol were more abundant in Arbequina and Hojiblanca varieties. There are studies on the phenolic content of olive fruits that reported an increase in luteolin concentrations and a decrease in apigenin concentrations with increasing ripeness, which is in accordance with the changes found in flavonoid concentrations in this study [42].

Effect of olive oil phenolic compounds extract on MG-63 osteoblast cell proliferation

Recently, we have demonstrated the effects of VOO phenolic extracts obtained from Sicilian monovarietal olive oils on the proliferation of human osteosarcoma cells [9]. Treatment of osteoblast cells with Sicilian olive oil phenolic extracts increased the number of cells 13.77–30.98% compared with controls. In this work, the effect of EVOO phenolic extracts obtained from the most common four Spanish movarietal olive oils at three different ripening stages on the proliferation of human osteosarcoma cells was investigated. We hypothesized that the phenolic fractions of each EVOO variety may induce osteoblast proliferation differently. MG-63 cells were treated with various concentrations of extracts to determine the biological actions of complex multicomponent phenolic compounds. All extracts significantly stimulated MG-63 cell growth in a non-concentration dependent manner in this order of potencies: Picual> Hojiblanca> Picudo> Arbequina (Fig 1). The most effective EVOO phenolic extracts were those obtained from the Picual variety, as they significantly increased cell proliferation by 18–22%. Conversely, Arbequina phenolic extracts increased cell proliferation by 9–13%. It should be noted that Picual and Hojiblanca phenolic extracts–which have higher phenol concentrations–were more effective in inducing cell proliferation than the other varieties. Moreover, in all studied varieties there is a decrease in the ability to stimulate osteoblast proliferation in oils obtained from olive fruits collected at the end of the harvest, as total phenol content decreased in all four varieties.

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Fig 1. Effect of EVOO phenolic extracts on the growth of MG-63 cells as determined by MTT.

Cells were treated with various concentrations of EVOO phenolic extract or vehicle alone (control) for 24 h. Data are means, with standard error of the mean shown by vertical bars of three separate experiments and at least every experiment was performed in triplicate. Mean values were significantly different as compared with control groups: *p<0.05, **p<0.01.

https://doi.org/10.1371/journal.pone.0150045.g001

The increase of cell proliferation induced by phenolic extracts was higher than that obtained by individual phenolic compounds. These results indicate that phenolic compounds in EVOO may synergize together to increase MG-63 cell proliferation to a greater level than a single treatment using individual chemicals. Although the observed effects suggest that there may be a synergism among phenolic compounds in EVOO extracts, other substances present in this extract can also cause MG-63 cell proliferation. However, there are not studies available that address the effect of other olive oil bioactive compounds on osteoblastic cell proliferation. Further studies are required to identify other bioactive substances and the mechanisms by which EVOO phenolic extracts induce MG-63 cell proliferation.

To date, the most consistently followed approach to examine the potential relation between EVOO phenolic compounds and bone health was based on particular phenols. The analysis considering the effect of a few isolated compounds on bone health misses information regarding complex or cumulative correlations and interactions between these compounds contained in olive oil. In this regard it should be considered that in vitro studies on individual phenolic compounds should always consider the effect of olive oil active fractions that could answer some questions and promote the development of appropriate recommendations for overall dietary habits.

Although these experimental studies support the hypothesis of EVOO phenolic compounds exert a beneficial effect on bone, further studies assessing the in vivo accessibility of EVOO phenolic compounds to osteoblast cells should be performed. Accordingly, and as EVOO phenolic compounds metabolism is still unknown, it is necessary to perform further in vitro studies where the intestinal absorption and biotransformation of biocompounds are considered. While other studies have revealed that olive oil intake results in increased urine and plasma concentrations of tyrosol and hydroxytyrosol [43] more research should be conducted to determine the bioavailability of other olive oil phenolic compounds.

Statistical Analysis

Principal component analysis was performed to explore data distribution patterns and visualize potential relationships among the study variables. Fig 2 shows the bi-plot graph for the Scores (A) and Loadings (B) obtained. The first-dimension PC-1 (which accounted for 45% of total variance) and the second dimension PC-2 (which accounted for 22% of variance) allowed us to distinguish the samples by olive variety and ripening stage. Table 4 shows the loading of the variables for the Principal Component 1 and 2. The values with the greatest factor loading for PC1 were MG-63 proliferation, total phenols, hydroxytirosol and tyrosol. Likewise, the variables showing a greater factor loading of the PC2 component were caffeic acid and apigenin. Comparative analysis of the two PCA plots, scores, and loading plot indicated that MG-63 proliferation and total phenols were the main factors for identifying Picual oils obtained from early-harvested fruits, while vanillic acid is related to oils obtained from Picudo variety. Conversely, the variables caffeic acid, vanillin and oleuropein differentiate Hojiblanca oils. In addition, the phenolic compounds tyrosol, luteolin, pinoresinol and ferulic acid were mainly responsible for the grouping of EVOO obtained from the Arbequina variety.

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Fig 2. Bi-plot (A. Scores and B. Loadings) for the PCA model.

Samples are plotted by selected letters and numbers: Picual RI 0.37 (P1); Picual RI 2.75 (P2); Picual RI 4.67 (P3); Hojiblanca RI 0.51 (H1); Hojiblanca RI 1.78 (H2); Hojiblanca RI 4.44; Arbequina RI 0.48 (A1); Arbequina RI 2.92 (A2); Arbequina RI 3.67 (A3); Picudo RI 0.88 (Pi1); Picudo RI 1.74 (Pi2); Picudo RI 3.84 (Pi3). The capital letters A, B and C correspond with 0.001, 0.0001 and 0.0001% extract concentration in osteoblast culture medium.

https://doi.org/10.1371/journal.pone.0150045.g002

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Table 4. X loadings for the variables with respect to the Principal Component 1 and 2.

https://doi.org/10.1371/journal.pone.0150045.t004

Conclusion

This study concludes that phenolic compounds and extracts from different extra virgin olive oil varieties stimulate osteoblast cells (MG-63) proliferation. Further research on the signaling pathways of olive oil phenolic compounds involved in the processes and their metabolism should be carried out to develop new interventions and adjuvant therapies using EVOO for bone health (i.e. osteoporosis) in adulthood and the elderly.

Acknowledgments

We thank Jesús García and the Granada’s Laboratory of Food Analysis staff for their assistance on laboratory techniques.

Author Contributions

Conceived and designed the experiments: OGM CR AR. Performed the experiments: OGM AR EDLB JRT BJ EM. Analyzed the data: ASO MLL. Contributed reagents/materials/analysis tools: OGM AR CR. Wrote the paper: OGM AR CR.

References

  1. 1. Cashman K. Diet, nutrition and bone health. J Nutr. 2007; 137:2507–2512.
  2. 2. Rivas A, Romero A, Mariscal-Arcas M, Monteagudo C, Feriche B, Lorenzo ML et al. Mediterranean diet and bone mineral density in two age groups of women. Int J Food Sci Nutr. 2013; 64:155–161. pmid:22946650
  3. 3. Rivas A and Romero A., Adherence to Mediterranean Diet and bone health. Nutr Hosp. 2014; 29: 989–996. pmid:24951976
  4. 4. Visioli F, Bernardini E, Extra virgin olive oil's polyphenols: biological activities. Curr Pharm. Des. 2011; 17:786–804. pmid:21443485
  5. 5. Hagiwara K, Goto T, Araki M, Miyazaki H, and Hagiwara H.. Olive polyphenol hydroxytyrosol prevents bone loss. Eur J Pharmacol. 2011; 662:78–84. pmid:21539839
  6. 6. Fernández-Real JM, Bulló M, Moreno-Navarrete JM, Ricart W, Ros E, Estruch R et al. Mediterranean diet enriched with olive oil is associated with higher serum total osteocalcin levels in elderly men at high cardiovascular risk. J Clin Endocrinol Metab. 2012; 97:3792–3798. pmid:22855341
  7. 7. Puel C, Mardon J, Kati-Coulibaly S, Davicco MJ, Lebecque P, Obled C et al. Black Lucques olives prevented bone loss caused by ovariectomy and talc granulomatosis in rats. Br J Nutr. 2007; 97:1012–1020. pmid:17408530
  8. 8. Puel C, Mardon J, Agalias A, Davicco MJ, Lebecque P, Mazur A et al. Major phenolic compounds in olive oil modulate bone loss in an ovariectomy/inflammation experimental model. J Agric Food Chem. 2008; 56: 9417–9422 (2008). pmid:18800805
  9. 9. García-Martínez O, Mazzaglia G, Sánchez-Ortiz A, Ocaña-Peinado FM, Rivas A. Phenolic content of Sicilian virgin olive oils and their effect on MG-63 human osteoblastic cell proliferation. Grasas y Aceites 2014; 65.
  10. 10. Dai Z, Li Y, Quarles LD, Song T, Pan W, Zhou H et al. Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation. Phytomedicine 2007;14:806–814. pmid:17689939
  11. 11. Trzeciakiewicz A, Habauzit V, Horcajada MN.When nutrition interacts with osteoblast function: molecular mechanisms of polyphenols. Nutr Res Rev. 2009; 22:68–81. pmid:19243669
  12. 12. Bendini A, Cerretani L, Carrasco-Pancorbo A, Gómez-Caravaca AM, Segura-Carretero A, Fernández-Gutiérrez A, and Lercker G, Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. Molecules. 2007;12: 1679–1719. pmid:17960082
  13. 13. Martínez Nieto L, Hodaifa G, and Lozano-Peña J.L., Changes in phenolic compounds and Rancimat stability of olive oils from varieties of olives at different stages of ripeness. J. Sci. Food Agr. 2010; 90:2393–2398.
  14. 14. Cerretani L, Bendini A, Del Caro A, Piga A, Vacca V, Caboni MF et al. Preliminary characterisation of virgin olive oils obtained from different cultivars in Sardinia. Eur Food Res Technol. 2006; 222:354–361.
  15. 15. IOOC, 1984. Document n° 6. International Olive Oil Council, Madrid.
  16. 16. Martínez JM, Muñoz E, Alba J, Lanzon A. Informe sobre la utilización del analizador de rendimientos Abencor, Grasas y Aceites 1984; 26:379–385.
  17. 17. IOOC, 2009. Document COI/T.20/DOC. 29. International Olive Oil Council, Madrid.
  18. 18. Jiménez B, Sánchez-Ortiz A, Lorenzo ML, Rivas A. Influence of fruit ripening on agronomic parameters, quality indices, sensory attributes and phenolic compounds of Picudo olive oils. Food Res Int. 2013; 54:1860–1867.
  19. 19. De Luna-Bertos E, Ramos-Torrecillas J, García-Martínez O, Díaz-Rodríguez L, Ruiz C, Effect of aspirin on cell growth of human MG-63 osteosarcoma line. ScientificWorldJournal 2012: 834246. pmid:22654634
  20. 20. Díaz-Rodríguez L, García-Martínez O, De Luna-Bertos E, Ramos-Torrecillas J, Ruiz C. Effect of ibuprofen on proliferation, differentiation, antigenic expression, and phagocytic capacity of osteoblasts. J Bone Miner Metab. 2012; 30:554–560. pmid:22543821
  21. 21. Tsai SW, Liaw JW, Kao YC, Huang MY, Lee CY, Rau LR, Huang CY, Wei KC, Ye TC. Internalized gold nanoparticles do not affect the osteogenesis and apoptosis of MG63 osteoblast-like cells: a quantitative, in vitro study. PLoS One. 2013; 8(10):e76545. pmid:24098527
  22. 22. De Luna-Bertos E, Ramos-Torrecillas J, García-Martínez O, Guildford A, Santin M, Ruiz C. Therapeutic doses of nonsteroidal anti-inflammatory drugs inhibit osteosarcoma MG-63 osteoblast-like cells maturation, viability, and biomineralization notential. Sci World J 2013; doi: http://DX.DOI.ORG/10.1155/2013/809891
  23. 23. Hosoya S, Suzuki H, Yamamoto M, Kobayashi K, Abiko Y. Alkaline phosphatase and type I collagen gene expressions were reduced by hydroxyl radical-treated fibronectin substratum. Mol Genet Metab. 1998; 65:31–34. pmid:9787092
  24. 24. Notoya M, Nishimura H, Woo JT, Nagai K, Ishihara Y and Hagiwara H. Curcumin inhibits the proliferation and mineralization of cultured osteoblasts. Eur J Pharmac. 2006; 534: 55–62.
  25. 25. Lee JW, Ahn JY, Hasegawa S, Cha BY, Yonezawa T, Nagai K, Seo HJ, Jeon WB and Woo JT. Inhibitory effect of luteolin on osteoclast differentiation and function. Cytotechnology. 2009; 1: 125–134.
  26. 26. Stalikas CD. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci. 2007; 30:3268–3295. pmid:18069740
  27. 27. Folwarczna J, Zych M, Burczyk M, Trzeciak H and Trzeciak HI. Effects of natural phenolic acids on the skeletal system of ovariectomized rats. Planta Med. 2009;75:1567–1572. pmid:19598079
  28. 28. Lai YL and Yamaguchi M., Phytocomponent p-hydroxycinnamic acid inhibits osteoclast-like cell formation in mouse bone marrow cultures. Int J Mol Med Sci. 2007;19:123–128.
  29. 29. Chen JR, Lazarenko OP, Wu X, Kang J, Blackburn ML, Shankar K, Badger TM and Ronis M.J., Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/β-catenin canonical Wnt signalling. J Bone Min Res. 2011; 25:2399–23411.
  30. 30. Eriksen EF, Colvard DS, Berg NJ, Graham ML, Mann KG, Spelsberg TC and Riggs BL, Evidence of estrogen receptors in normal human osteoblast-like cells. Science 1998; 241:84–86.
  31. 31. Sugimoto E,Yamaguchi M. Stimulatory effect of Daidzein in osteoblastic MC3T3-E1 cells. Biochem Pharmacol. 2000; 59:471–475. pmid:10660113
  32. 32. Chang CJ, Chiu JH, Tseng LM, Chang CH, Chien TM, Wu CW et al. Modulation of HER2 expression by ferulic acid on human breast cancer MCF7 cells. Eur J Clin Invest. 2006; 36:588–596. pmid:16893382
  33. 33. Stromeier S, Petereit F, Nahrstedt A. Phenolic esters from the rhizomes of Cimicifuga racemosa do not cause proliferation effects in MCF-7 cells. Planta Med. 2005; 71:495–500. pmid:15971118
  34. 34. Zych M, Folwarczna J and Trzeciak H.I., Natural phenolic acids may increase serum estradiol level in ovariectomized rats. Acta Biochim Pol 2009; 56:503–507. pmid:19753332
  35. 35. Puel C, Mathey J, Galias A, Kati-Coulibaly S, Mardon J, Obled C et al. Dose-response study of effect of oleuropein, an olive oil polyphenol, in an ovariectomy/inflammation experimental model of bone loss in the rat. Clin Nutr. 2006; 25:859–868. pmid:16740345
  36. 36. Puel C, Quintin A, Agalias A, Mathey J, Obled C, Mazur A, Davicco MJ, Lebecque P, Skaltsounis AL, and Coxam V, Olive oil and its main phenolic micronutrient (oleuropein) prevent inflammation-induced bone loss in the ovariectomised rat. Br J Nutr. 2004; 92:119–27. pmid:15230995
  37. 37. Habauzit V, Horcajada MN. Phenolic phytochemicals and bone. Phytochemistry Reviews 2008; 7: 313–344.
  38. 38. Yang XJ, Wong MS, Wang NL, Chan SC, Yao XS. Lignans from the stems of Sambucus williamsii and their effects on osteoblastic UMR106 cells, J Asian Nat Prod Res. 2007; 9: 583–591. pmid:17701563
  39. 39. Kim TH, Jung JW, Ha BG, Hong JM, Park EK, Kim HJ and Kim SY, The effects of luteolin on osteoclast differentiation function in vitro and ovariectomy-induced bone loss. J Nutr Biochem. 2011; 22:8–15. pmid:20233653
  40. 40. Choi EM, Modulatory effect of luteolin on osteoblastic function and inflammatory mediators in osteoblastic MC3T3-E1 cells, Cell Biol Int. 2007; 31: 870–877. pmid:17368935
  41. 41. Yamaguchi M, and Jie Z, Effect of polyphenols on calcium content and alkaline phosphatase activity in rat femoral tissues in vitro. Biol Pharm Bull. 2001; 24:1437–1439. pmid:11767119
  42. 42. Artajo LS, Romero MP, Motilva MJ, Transfer of phenolic compounds during olive oil extraction in relation to ripening stage of the fruit. J Sci Food Agr. 2006; 86:518–527.
  43. 43. Vissers MN, Zock PL, Roodenburg A.J.C, Leenen R, and Katan MB. Olive oil phenols are absorbed in humans. J Nutr. 2002;132:409–417. pmid:11880564