Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells

www.elsevier.com/locate/issn/10434666 Cytokine 36 (2006) 211–217

Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells Ana Marquez-Martin a, Rocio De La Puerta b,*, Angeles Fernandez-Arche b, Valentina Ruiz-Gutierrez a, Parveen Yaqoob c a Instituto de la Grasa (C.S.I.C.) Avda, Padre Garcı´a Tejero n4, 41012 Seville, Spain Department of Pharmacology, Faculty of Pharmacy, University of Seville, c/ Profesor Garcı´a Gonza´lez n2, 41012 Seville, Spain The Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK b

c

Received 11 October 2006; received in revised form 28 November 2006; accepted 16 December 2006

Abstract Olive pomace oil, also known as ‘‘orujo’’ olive oil, is a blend of refined-pomace oil and virgin olive oil, fit for human consumption. Maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes, found in the non-glyceride fraction of orujo oil, which have previously been reported to have anti-inflammatory properties. In the present work, we investigated the effect of these minor components on pro-inflammatory cytokine production by human peripheral blood mononuclear cells in six different samples. Uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1b and IL-6 production in a dose-dependent manner. All three compounds significantly reduced TNF-a production at 100 lM; however, at 10 lM, uvaol and oleanolic acid enhanced the generation of TNF-a. In contrast, maslinic acid did not significantly alter the concentration of those cytokines, with the exception of a slight inhibitory effect at 100 lM. All four triterpenes inhibited production of I-309, at 50 lM and 100 lM. However, uvaol enhanced I-309 production at 10 lM. The triterpenic dialcohols had a similar effect on MIG production. In conclusion, this study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and anti-inflammatory properties depending on chemical structure and dose, and may be useful in modulating the immune response.  2006 Elsevier Ltd. All rights reserved. Keywords: Olive pomace oil; Pentacyclic triterpene; Peripheral blood mononuclear cells; Cytokines; Inflammation

1. Introduction The Mediterranean diet is associated with a lowered risk of cardiovascular disease [1,2]. One of the most well-known and important characteristics of this diet is the presence of olive oil as the principal source of energy from fat [3] In recent years, the number of reports describing the beneficial properties of olive oil has dramatically increased and data suggests that some of the minor components in olive oil may have more significant health benefits than previously recognised [4,5]. Studies have demonstrated antiatheroscle-

*

Corresponding author. E-mail address: [email protected] (R. De La Puerta).

1043-4666/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2006.12.007

rotic and/or anti-inflammatory effects of both oleic acid [6,7] and the phenolic components [8,9] of olive oil. Moreover, minor components present in the unsaponifiable matter of olive oil and olive pomace oil, have shown interesting activities. Olive pomace oil, also called ‘‘orujo’’ olive oil, is a subproduct of olive oil fit for human consumption traditionally produced in Spain, although the properties and potential effects on human health are not characterised yet. This oil, extracted using a new improved procedure (patent number 200400755) is a rich source of biologically active components. Oleanolic acid, maslinic acid, erythrodiol, and uvaol are all pentacyclic triterpenes present in the unsaponifiable fraction of ‘‘orujo’’ oil; these compounds have a hydroxyl function in the C-3 position of their chemical structure

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and a carboxylic or a methanolic function in the C-17 position (Fig. 1). Oleanolic acid and maslinic acid are found in significant quantities (0.1% and 0.4%, respectively), in edible ‘‘orujo’’ olive oil, in contrast with olive oil, where they are found only in minor amounts [10,11]. Triterpenoids have many biological activities, such as hepatoprotective, antitumor, anti-inflammatory, antiulcer, and nitric oxide (NO) inhibitory effects, among others [12–15]. We have previously reported some findings relating to the triterpene components from the unsaponifiable matter of olive oil, including, a significant reduction of oedema in a tetradecanoylphorbol acetate (TPA) induced inflammation model and a significant decrease in myeloperoxidase activity after treatment with erythrodiol [16]. Both erythrodiol and oleanolic acid were found to have endothelium-dependent vasorelaxant capacity in rat aorta [17] and it has also been suggested that the potent antioxidant effect exerted by erythrodiol might protect the polyunsaturated fatty acids present in rat-liver microsomal membranes from oxidation [18]. More recently, we have described prevention of oxidative stress and inhibition of pro-inflammatory cytokine generation by maslinic acid in peritoneal murine macrophages [19]. Cytokines are soluble glycoproteins, released by living cells of the host, which play a crucial role in initiating, maintaining and regulating immunologic, homeostatic

and inflammatory processes [20]. Cytokines are produced by a number of cells, including those present in the peripheral blood mononuclear fraction [21,22]. The functions of these proteins are diverse and include roles in normal T-cell mediated immunity, the inflammatory response, cancer, autoimmunity, and allergy [23]. There are different families of cytokines, including lymphokines, interferons, or colony stimulating factors [24]; we have focused our interest on tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-6 (IL-6), considered as central mediators in inflammation [25]. Chemokines are a family of chemoattractant proteins which mediate several cellular functions and are central to inflammatory processes [26,27]. This study focuses on I-309/CCL1 (I-309) and monokine induced by IFN-c (MIG/CXCL9). I-309 is secreted by both monocytes and lymphocytes and is a potent chemoattractant for the same cell types [28]. Th2 lymphocytes preferentially express the receptor for I-309, CCR8, giving this chemokine a role in allergy [28]. Mig, on the other hand, is a prototypical Th1 chemokine. It is induced by IFNc, dramatically enhanced by TNF-a, and plays a role in rheumatoid arthritis [29]. To date, there are no reports of the effects of these pentacyclic triterpene on cells of the immune system, particularly with respect to cytokine and chemokine production. Therefore, in this study, the effects of uvaol (UV), erythrodiol (ER), oleanolic acid (OA), and

COOH COOH

HO

HO HO

H

Maslinic acid

Oleanolic acid

CH2OH

CH2OH

HO

HO

Erythrodiol

Uvaol

Fig. 1. Chemical structures of pentacyclic triterpenes from orujo olive oil.

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maslinic acid (MA) on the production of selected pro-inflammatory cytokines and chemokines by human peripheral blood mononuclear cells were investigated. 2. Materials and methods 2.1. Materials Erythrodiol, uvaol, and oleanolic acid were obtained from Extrasynthese (Genay, France); maslinic acid was obtained from pomace, the solid waste of olive oil, by the patented method from Garcı´a-Granados [30]; RPMI medium, glutamine, antibiotics (penicillin and streptomycin), and phytohaemagglutinin (PHA) were obtained from SIGMA (Poole, UK); Lympholyte was obtained from Cedarlane (Hornby, Ontario, Canada); cytotoxicity LDH kits were obtained from Oxford Biomedical Research (Oxford, MI, USA) and cytokine ELISA kits were obtained from R&D Systems Europe (Abingdon, UK). 2.2. Cytotoxicity of pentacyclic triterpenes towards peripheral blood mononuclear cells (PBMC)

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PBMC (2 · 106) were resuspended in medium supplemented with 2.5% autologus plasma and 10 lg/ml phytohaemagglutinin (PHA) and cultured in presence of ER, UV, MA, or OA, at concentration of 10–100 lM (in 1% EtOH) for 24 h at 37 C and 5% CO2 atmosphere in 24-well plates; the final culture volume was 2 ml. At the end of the incubation, the plates were centrifuged for 10 min at 480g and RT, and the culture medium was collected and frozen in aliquots. TNF-a, IL-1b, IL-6, I-309, and MIG were analysed using ELISA kits; according to the manufacturer’s instruction. Results are expressed as pg/ml of cytokine or chemokine. 2.4. Statistical analysis Data were analysed using one-way ANOVA, followed by Student’s t test as post-hoc analysis versus control group. p < 0.05 (a); p < 0.01 (b) were taken as significant. 3. Results 3.1. Cytotoxicity of orujo olive oil derivates to PBMC

Blood samples were obtained from six healthy, overnight-fasted volunteers and collected in heparinized tubes. The blood was layered onto Lympholyte separation medium and centrifuged for 15 min at 800g and room temperature (RT). The cells were collected from the interface and washed once with RPMI medium containing 0.75 mM glutamine/L and antibiotics (penicillin and streptomycin) (culture medium). After resuspension on 2.5 ml culture medium, the cells were layered onto 2.5 ml Lympholyte. They were centrifuged (15 min, 800g, RT) washed with culture medium and resuspended in 0.5 ml medium. Finally the cells were counted in a Z1 Coulter Counter (Beckman Coulter, High Wycombe, UK). Cytotoxicity of the compounds was determined by measuring cellular release of lactate dehydrogenase (LDH) using a commercial kit. The cells were cultured in the presence of uvaol (UV), erythrodiol (ER), oleanolic acid (OA), or maslinic acid (MA), at a concentration of 10, 25, 50, or 100 lM in 1% EtOH for 24 h at 37 C and 5% CO2 atmosphere in 96-well microtitre plate at a density of 106 cells/ml in a total volume of 200 ll. Four wells were setup to contain 200 ll medium only (blanks), four were set up as controls (cell suspension + medium + vehicle) and four contained cell suspension plus lysis reagent, representing maximal release of LDH. At the end of the incubation period 100 ll of culture medium from each well was transferred to a second microtitre plate. The concentration of LDH in the medium was measured according to the manufacturer’s instruction and results were expressed as percentage of cytotoxicity. 2.3. Cytokine production Blood samples were obtained from healthy, overnightfasted volunteers and PBMC were prepared as above.

There were no cytotoxic effects of the studied compounds on lactate dehydrogenase (LDH) release by peripheral blood mononuclear cells at any of the tested doses (10–100 lM) (data not shown). 3.2. Effects of orujo olive oil triterpenoids on the production of pro-inflammatory cytokines and chemokines by PBMC PHA-stimulated PBMC cultures (2 · 106) produced on average 354 pg/ml (218, 487) of IL-1b. The triterpenoids, ER, and OA inhibited IL-1b production in a dose-dependent manner compared to PHA control cells where it was added only the vehicle, EtOH (Fig. 2). In contrast, UV and MA did not significantly affect the release of this cytokine, although there was a strong trend towards a dose-dependent inhibition (Fig. 2). ER was the most potent compound, significantly decreasing IL-1b production at all doses. OA significantly (p < 0.05) down-regulated IL-1b liberation at the highest doses assayed (50–100 lM). Mean IL-6 concentration in the PHA-stimulated PBMC supernatants was 15199 pg/ml (10364, 18227) and its production was inhibited by all four compounds, although to different extents (Fig. 3). As with IL-1b, ER had the most potent inhibitory effect on IL-6 production, inhibiting at all doses (Fig. 3). The inhibitory effect of UV on IL-6 production was more potent than on IL-1b production, since its effects were statistically significant at all but the lowest dose (Figs. 2 and 3). The inhibition of IL-6 production by OA and MA was statistically significant only at 100 lM (p < 0.05). Stimulation of PBMC with PHA produced a mean TNF-a concentration of 241 pg/ml (216, 275). At the

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A. Marquez-Martin et al. / Cytokine 36 (2006) 211–217 TNF-α

IL-1β 150

a a

50

% Release

% Release

100

a a a a

0

100 a a

50

0

UV

ER

OA

MA

UV

50μM

25μM

100μM

Fig. 2. Effect of UV, ER, OA, and MA on IL-1b production by PBMC. (2 · 106 cell/ml) PBMC were incubated with PHA (10 lg/ml ) in the presence of several concentrations of the compounds for 24 h. Each value represents means ± SEM for six experiments. Results are expressed as percentage of cytokine liberation versus control (control release 354 ± 68.6 pg/ml). Statistically significant differences are denoted as ‘‘a’’ p < 0.05 vs. control (Anova + Student’s t test).

MA

10μM

50μM

25μM

100μM

Fig. 4. Effect of UV, ER, OA, and MA on TNF-a production by PBMC (2 · 106 cell/ml). PBMC were incubated with PHA (10 lg/ml ) in the presence of several concentrations of the compounds for 24 h. Each value represents means ± SEM for six experiments. Results are expressed as percentage of cytokine liberation versus control (control release 241.3 ± 17.5 pg/ml). Statistically significant differences are denoted as ‘‘a’’ p < 0.05 vs. control (Anova + Student’s t test).

effect on TNF-a production, although there was a trend towards a decrease at the highest concentration (Fig. 4). Stimulation with PHA resulted in a mean I-309 concentration of 86 pg/ml (75, 100). The only significant effect of the triterpenes on I-309 production was an increase in response to 10 lM UV (Fig. 5). There was a trend towards

IL-6

100

% Release

OA

control (cells+PHA+EtOH)

control (cells+PHA+EtOH) 10μM

ER

a a a

a a a

50

a

a

I-309

a

200

0

UV

ER

OA

MA

control (cells+PHA+EtOH) 10μM

50μM

25μM

100μM

Fig. 3. Effect of UV, ER, OA, and MA on IL-6 production by PBMC (2 · 106 cell/ml). PBMC were incubated with PHA (10 lg/ml ) in the presence of several concentrations of the compounds for 24 h. Each value represents means ± SEM for six experiments. Results are expressed as percentage of cytokine liberation versus control (control release 15199.9 ± 1745.3 pg/ml). Statistically significant differences are denoted as ‘‘a’’ p < 0.05 vs. control (Anova + Student’s t test).

highest dose of 100 lM, UV and OA significantly inhibited TNF-a production (Fig. 4). However, at lower doses these compounds appeared to have a slight stimulatory effect. Although the increase was not statistically significant, this is suggestive of a biphasic response to these compounds (Fig. 4). ER and MA, on the other hand, had no significant

%Release

a

100

0

UV

ER

OA

MA

control (cells+PHA+EtOH) 10μM

50μM

25μM

100μM

Fig. 5. Effect of UV, ER, OA, and MA on I-309 production by PBMC (2 · 106 cell/ml). PBMC were incubated with PHA (10 lg/ml ) in the presence of several concentrations of the compounds for 24 h. Each value represents means ± SEM for six experiments. Results are expressed as percentage of cytokine liberation versus control (control release 86.0 ± 6.7 pg/ml). Statistically significant differences are denoted as ‘‘a’’ p < 0.05 vs. control (Anova + Student’s t test).

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MIG 200

b b

% Release

a

100

0

UV

ER

OA

MA

control (cells+PHA+EtOH) 10μM

50μM

25μM

100μM

Fig. 6. Effect of UV, ER, OA, and MA on MIG production by PBMC (2 · 106 cell/ml). PBMC were incubated with PHA (10 lg/ml ) in the presence of several concentrations of the compounds for 24 h. Each value represents means ± SEM for six experiments. Results are expressed as percentage of cytokine liberation versus control (control release 117.3 ± 12.6 pg/ml). Statistically significant differences are denoted as ‘‘a’’ p < 0.05; ‘‘b’’ p < 0.01 vs. control (Anova + Student’s t test).

an inhibitory effect at the higher concentrations for all four compounds, although this was not statistically significant (Fig. 5). Again, this is suggestive of a biphasic effect for UV. Mean production of MIG by PHA-stimulated cells production was 117 pg/ml (89, 151). All four triterpenes increased MIG generation at the lowest concentration, with UV and ER having the most potent effects (Fig. 6). None of the compounds decreased MIG production at higher concentrations, although, as with I-309, the response of this chemokine to triterpenes appeared to be biphasic (Fig. 6). 4. Discussion Pentacyclic triterpenes are natural compounds which are widely distributed in plants. These natural products have been demonstrated to possess anti-inflammatory properties [12,14]. Among others, maslinic acid, erythrodiol, and oleanolic acid, all triterpenoids present in orujo olive oil, have been reported to possess antioxidant properties, since they prevent lipid peroxidation [18,31] and suppress superoxide anion generation [32]. The triterpenes have a history of medicinal use in many Asian countries [33]. However, the effects of these orujo olive oil pentacyclic triterpenes have not been studied in human immune cells. The purpose of this study was to examine the influence of these compounds on cytokine and chemokine secretion by human PBMC following mitogen stimulation with PHA. Previous studies have demonstrated a favourable cytokine response under these conditions [34].

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IL-6, TNF-a, and IL-1b are pro-inflammatory cytokines which are part of a larger network of inflammatory regulation [35], and their overproduction is believed to play a key role in a number of inflammatory diseases [21,36–38]. In addition, the effects of these compounds on production of two chemokines, the monokine induced by IFN-c (MIG) and I-309, were examined. These chemokines are expressed by cells during contact with pathogenic agents and are specialised for the recruitment of effector cells [39]. I-309 is a C–C chemokine which acts as chemoattractant for monocytes and lymphocytes, and exerts it effects through the CCR8 receptor [40–42]. MIG is a C–X–C chemokine that mainly acts as a chemotactic factor for stimulated cells, and exerts it effects through the CXCR3 receptor [43–45] which is expressed on activated T cells [46] and plays an important role in the inflammatory process [47]. It has also been demonstrated that this chemokine is strongly expressed in primary malignant melanomas, playing an important role in the selective recruitment of tumor-infiltrating lymphocytes [48]. Besides chemoattraction of lymphocytes, an antiangiogenic property has been described [49,50]. In the present study, all four triterpenes inhibited production of IL-1b and IL-6 to some degree, with ER emerging as the most potent anti-inflammatory compound. The behaviour of UV and OA was suggestive of a biphasic response in terms of TNF-a production, with an increase at 10 lM and a decrease at the higher concentrations. MA only produced a significant effect at the highest dose (100 lM) for IL-6 production. This latest result is in agreement with those we measured in stimulated murine macrophages [19]. This inhibitory cytokine effect could help to explain the anti-inflammatory effect observed for these compounds. A number of dietary antioxidants are suggested to exert anti-inflammatory properties by inhibiting cytokine production through down-regulation of the activation of key nuclear transcription factors such as NF-jB [51,52] which is required for the induction of some cytokine genes [53–55]. The inhibitory effects on cytokine expression by these triterpenes could be mediated, at least in part, through an NF-jB pathway inhibition as has been demonstrated for other pentacyclic oleanane-triterpenoids as betulinic acid [56]or boswellic acid derivatives [57]. However, the biphasic nature of the TNF-a response and the production of MIG and I-309 to some of the compounds suggests that at low concentrations these compounds activate other, as yet unknown, pathways to up-regulate chemotactic and inflammatory responses. The consequences of this up-regulation should be taken into consideration; it could be considered that up-regulation of chemoattractant activity would be desirable in situations where improved wound healing etc., are required. It is notable that MIG is known to be dramatically increased in response to TNF-a and the effects of the compounds on these inflammatory factors are somewhat similar. According to their chemical structure, these pentacyclic triterpenoids can be classified as alcoholic or acid

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derivatives depending on the function present at the C17 position (Fig. 1). Although the relationship between chemical structure and suppression of cytokine generation by these compounds is unclear at present, it might be noteworthy that the chemical structure of MA differs from the others by the presence of an additional hydroxyl group at the C-2 position. (Fig. 1), which likely confers it antioxidant properties [19,31]. It might be predicted, based on this structural characteristic, that the effects of MA would be potent. However, MA actually appears to be the weakest of the four compounds in terms of its influence on the production of inflammatory markers, suggesting that the mechanisms by which these compounds act may be independent of their antioxidant activity. Although experiments have shown immunomodulatory activity for OA and ursolic acid (UA), for example their ability to inhibit the hypersensitivity reaction [55]; to our knowledge, this is the first time that triterpenoid compounds have been shown to modulate inflammatory cytokine and chemokine production by human cells depending on the dose. OA, MA, and ER are found in minor amounts in virgin olive oil, but in edible ‘‘orujo’’ oil they reach concentrations of between 300–500 ppm. Juan et al., [58] have recently estimated the colonic bioavailability of pentacyclic triterpenes from virgin olive oil, which contains approximately 172 mg/kg maslinic acid and 231 mg/kg oleanolic acid [11]. A daily consumption of 33 g of virgin olive oil per day would therefore provide 6 mg maslinic acid and 8 mg oleanolic acid whereas a daily consumption of the same amount of commercialised ‘‘orujo’’ oil, which contains approximately 721 mg/kg maslinic acid and 5592 mg/kg oleanolic acid [11], would provide 24 mg maslinic acid and 184 mg oleanolic acid. Juan et al. estimate that approximately 70% of this is absorbed [58]. In the absence of full bioavailability studies for these compounds, it is difficult to predict the likely plasma concentration, but if calculations were based on the bioavailability of hydroxytyrosol from virgin olive oil [59], then plasma concentrations of 1–5.5 lM would be achievable following consumption of orujo oil. However, this is based on a number of assumptions and could well be higher-certainly within the 10–20 lM range, as tested in the current study. Furthermore, the higher doses employed in the current study may be relevant to the medicinal use of the triterpenoids in some Asian countries [33]. In view of these results, further studies are required to confirm the immunomodulatory behaviour of these triterpenoid compounds, and particularly, characterise the mechanisms underlying the biphasic nature of some aspects of the inflammatory response. In conclusion, it has been demonstrated that uvaol, erythrodiol, maslinic acid, and oleanolic acid exhibit both proand anti-inflammatory properties depending on chemical structure and dose and may be useful in modulating the immune response.

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