© Birkhäuser Verlag, Basel, 2001 Inflamm. res. 50 (2001) 102– 106 1023-3830/01/020102-5 $ 1.50+0.20/0
Inflammation Research
Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet E. Martínez-Domínguez 1, R. de la Puerta 1 and V. Ruiz-Gutiérrez 2 1
2
Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor García González s/n 41012 Sevilla, Spain, Fax: 34-(9)- 5-4233765, e-mail:
[email protected] Instituto de la Grasa, (C.S.I.C.), c/ Padre García Tejero 4, 41012 Sevilla, Spain
Received 19 May 2000; returned for revision 11 October 2000; accepted by G. Geisslinger 12 October 2000
Abstract. Objective and Design: The aim of the present study was to examine the effect of a virgin olive oil enriched diet in acute and chronic inflammation models in rats and to determine the effect of supplementing this oil with a higher content of its polyphenolic fraction. The response was compared to oils rich in monounsaturated fatty acids (high oleic sunflower oil and palm olein) and rich in polyunsaturated fatty acids (fish oil). Diets: Groups of 6–8 male Wistar rats were fed from weaning on six purified diets differing in type of oil: 2% corn oil (basal diet, BD), 15% high oleic sunflower oil (HOSO), 15% virgin olive oil (VOO), 15% virgin olive oil supplemented with 600 p.p.m. polyphenols from this oil (PSVOO), 15% palm olein (POL), and 15% fish oil (FO). Materials and methods: Rats were fed for 8 weeks with BD, HOSO, VOO, PSVOO, POL and FO diets before injecting carrageenan. Rats were fed for 3 weeks with BD, PSVOO and FO diets before induction of adjuvant arthritis. Dietary treatment with or without indomethacin continued during 3 weeks. The data were evaluated using an analysis of variance (ANOVA) followed by the least-significant differences. Results: In carrageenan oedema test, the inflammation indices of animals fed on a diet rich in olive oil (VOO) were lower compared to animals fed with oils high in oleic acid (HOSO, POL) and polyunsaturated fatty acids (FO), and markedly diminished in the group fed on PSVOO. In established adjuvant arthritis, the PSVOO diet was even more effective than FO diet in the prevention of inflammation. Both groups of animals showed an increase in weight during the latter days of the experiment compared to the BD. Indomethacin administered to every diet group, exerted a strong inhibitory effect on the inflammatory process throughout which was augmented by the PSVOO and FO diets. Conclusions: This study demonstrates that virgin olive oil with a higher content of polyphenolic compounds, similar to that of extra virgin olive oil, shows protective effects in both models of inflammation and improves the disease associated Correspondence to: R. de la Puerta
loss of weight. This supplementation also augmented the effects of drug therapy. Key words: Virgin olive oil – Diets – Polyphenols – Inflammation – Rheumatoid arthritis.
Introduction A great deal of epidemiological and experimental data support the existence of relationships between the composition of dietary fatty acids and the development of many inflammatory and inmmunological diseases [1–3]. Alteration on the type of dietary fatty acids may beneficially influence these processes. It is widely accepted that a reduction in saturated fatty acid (SFA) intake is one appropiate prevention strategy; both polyunsaturated (PUFA) and monounsaturated fatty acids (MUFA) have been used to counteract the negative effects of high saturated fatty acid intake [4, 5]. PUFA from marine oils, which are relatively rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are thought to be beneficial in many ways and in particular in the treatment of cardiovascular diseases, rheumatoid arthritis and psoriasis [1, 2, 6]. It has been assumed that the beneficial effects of fish oil are mediated by changes in eicosanoid production, favouring the formation of less biologically active substances; n-3 PUFA have been reported to reduce prostaglandin E2 (PGE2), thromboxane A2 (TXA2), and leukotriene B4 (LTB4) production whereas to correspondingly increase of PGE1 , TXA3 , LTB5 , non vasoconstrictory, non proaggregatory and weakly chemotactic agents respectively [7]. Furthermore, inclusion in the diet of high levels of n-3 PUFA has markedly affected the functions of leukocyte, and cells of the immune system such lymphocytes and macrophages tested in vitro [8]; several studies have indicated that these diets can decrease the inflammation process and are immunosupressive in vivo [3, 4]. MUFA, especially oleic acid from olive oil, have been associated with a lower risk of coronary heart disease
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Protection on inflammation of a virgin olive oil diet
[9–11]. Less attention has been paid to the effects of MUFA on the immune system, though a smaller number of studies have suggested that there may be beneficial effects of olive oil consumption on rheumatoid arthritis [12]. Studies on the Mediterranean diet have considered the possible protective effects of antioxidants, which occur naturally in fruits, vegetables, beverages like red wine, and in olive oil [13]. The phenolic compounds are presented in the range of 50–800 p.p.m. in virgin olive oil and varies according to factors such as climate, cultivar and stage of maduration of the olives, being highest in the first-expressed oil, the so called extra virgin olive oil. The major phenolics include simple phenols based on cinamic acid (e.g. caffeic acid), the secoiridoid glycoside oleuropein and its hydrolisis products hydroxytyrosol, also known as 2 (3,4-dihydroxyphenyl)ethanol, and tyrosol (4-hydroxyphenylethanol) [14]. During the inflammatory process, the release of reactive oxygen metabolites by phagocytic cells contribute to cell death (host cells or invading cells) by attacking macromolecules such as membrane lipids, proteins and DNA [15]. Olive oil phenolics have been shown to reduce in vitro the generation of these reactive species by phagocytic cells and, in addition, to inhibit the activities of the cyclooxygenase and lipoxygenase enzymes [16, 17]. The aim of the present study was to examine the effect of a virgin olive oil enriched diet in an acute and a chronic inflammation model in rats. In addition, the effect of supplementing this oil with a higher content of its polyphenolic fraction was examined. The results were compared with those of oils rich in MUFA, such high-oleic acid sunflower oil and palm olein, and fish oil rich in PUFA.
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Materials and methods Animals Male Wistar rats (Centro de Instrumentación Científica, Granada, Spain) aged 3 weeks weighing about 60 g were placed in cages with wire-net floors in a controlled room (on a 12 h light dark-cyle, temperature 22 ± 2 °C, humidity 70–75%). The rats were randomly divided into six groups of 8 animals, excepting basal diet that contained 6 rats. Each group was fed one of the six experimental diets. Animals had free access to food and water. The food intake and the weight of the animals were monitored regularly.
Diets All diets contained: 51.6% sucrose, 20.7% casein, 15.5% corn starch, 5.2% alphacel, 0.3% DL-methionine, 0.2% choline chloride (Musal & Chemical, Granada. Spain), 3.5% AIN-93 mineral mix, 1% AIN-93 vitamin mix (ICN, USA) [18]. The diets had different lipid compositions: 2% corn oil (basal diet group, BD); 15% high-oleic acid sunflower oil (HOSO group) from Koipe SA, Spain; 15% virgin olive oil (VOO group) from Aceites Toledo, Spain; 15% palm olein (POL group) from Lesier Alimentaire, France; 15% fish oil (FO group) from AFAMSA, Vigo, Spain; and 15% virgin olive oil supplemented with 600 p.p.m. of polyphenols (PSVOO group) obtained by extraction from olives [19]. The original virgin olive oil contains 200 p.p.m. of polyphenols. To minimize oxidation, all diets were prepared twice weekly and stored at 4 °C under an atmosphere of N2 until needed. Changes in composition during storage were not detected. The fatty acid composition of the oils were determined as previously described [20] and are shown in Table 1.
Table 1. Fatty acid composition of dietary oils (g/100g total fatty acids). Fatty acids
Corn oil
HOSO
VOO
POL
FO
14 :0 16 :0 16 :1 (n-7) 17 :0 18 :0 18 :1 (n-9) 18 :1 (n-7) 18 :2 (n-6) 18 :3 (n-3) 18 :3 (n-6) 1 8 :4 (n-3) 20 :0 20 :1 (n-9) 20 :2 (n-6) 20 :3 (n-6) 20 :4 (n-6) 20 :5 (n-3) 22 :4 (n-6) 22 :5 (n-3) 22 :6 (n-3) 24 :0 SFA MUFA PUFA
0.01 10.36 0.12 – 2.36 33.05 1.15 50.89 0.71 0.43 – – 0.22 0.03 – – – 0.11 – – – 12.73 34.64 52.17
– 4.30 0.10 0.10 4.70 80.20 – 9.40 0.10 – – 0.40 0.20 – – – – – – – 0.40 9.90 80.50 9.50
– 11.80 0.90 0.40 2.80 79.2 – 3.50 0.60 – – 0.30 0.20 – – – – – – – 0.40 15.70 80.20 4.10
0.04 34.13 0.24 – 3.85 47.60 – 12.74 0.48 0.35 – – 0.20 0.01 0.01 – – 0.02 – – – 38.02 48.00 13.61
0.66 24.11 11.49 – 4.54 15.59 4.75 2.98 0.60 0.58 3.34 – 2.13 0.15 0.17 0.93 18.61 0.88 2.84 5.64 – 29.31 33.96 36.72
Corn oil included in a 2% in basal diet; HOSO: high-oleic acid sunflower oil; VOO: virgin olive oil; POL: palm olein; FO: fish oil. SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids. Note: PSVOO (supplemeted virgin olive oil) has the same fatty acid composition as VOO.
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Carrageenan-induced oedema Paw oedema was induced by injecting 0.1mL of 1 % w/v sterile carrageenan (Sigma, USA) in saline solution into the hind paw under the plantar aponeurosis of rats [21]. The animals weighing 250 ± 20 g, were fed on the six diets for 4 weeks. At 1, 3 and 5 h after carrageenan, the paw volume was measured with a plethysmometer (LI-7500, Letica) and the inflammation index was calculated as the difference between the final volume of the carrageenan injected paw (Vt) and the initial volume of the same paw before injecting it (Vo). A
CFA-induced arthritis Adjuvant arthritis was induced in rats by injecting 0.2 mL of Freund’s adjuvant-complete (1 mg/mL, Sigma, USA) into the animal hindpaw, by the classic technique of Newbold [22]. Paw volume was measured daily until day 16, and the inflammation index was calculated. The animals had been fed during 3 weeks with basal (BD), supplemented virgin olive oil (PSVOO) and fish oil (FO) diets. The experiment was repeated including the same dietary groups but initiating a daily treatment with the COX inhibitor indomethacin, 1mg/Kg (Sigma, USA) one day before injecting CFA and daily until day 13. During the experiment, rats were kept on their corresponding diets and the gain of weight of each animal was monitored daily.
Statistical analysis Data are expressed as mean ± SEM. Analysis of variance (ANOVA) followed by the least-significant differences were used for statistical evaluation.
Results The subplantar injection of carrageenan produces an increasing local oedema from the 1st to the 5th hour after the injection of carrageenan, as can be observed on the basal diet (Fig. 1). In this experimental model, the inflammation index significantly diminished in rats fed VOO, at 5 h after injection, and it was markedly reduced in the PSVOO group both at 3 and 5 h after injection compared to the BD group. No significant changes in oedema formation were observed in the other diet groups, including the FO group.
Fig. 1. Inflammation index of dietary groups on carrageenan-induced oedema in rats. (BD: basal diet; HOSO: high-oleic acid sunflower oil; VOO: virgin olive oil; PSVOO: supplemented virgin olive oil; POL: palm olein; FO: fish oil. ANOVA test: * p < 0.05, ** p < 0.01 significantly different from BD values.
B
Fig. 2. (A): Inflammation index of rats with Freund’s adjuvant-complete arthritis from dietary groups and (B): idem plus administered 1 mg/Kg indomethacin. (BD: basal diet; BDIndo: basal diet treated with 1 mg/Kg indomethacin; PSVOO: supplemented virgin olive oil. PSVOOIndo: supplemented virgin olive oil treated with 1 mg/Kg indomethacin; FO: fish oil; FOIndo: fish oil treated with 1 mg/Kg indomethacin. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from BD values.
Means of inflammation indices associated with established arthritis by Freund’s complete adjuvant in rats treated with BD, PSVOO and FO, and the same groups treated with 1 mg/Kg indomethacin are presented in Figs. 2A and 2B respectively. Both groups (PSVOO and FO) were able to prevent to some extent the development of the inflammation. PSVOO was even more effective than FO compared to BD in this model of chronic inflammation (Fig. 2A). The daily doses of indomethacin administered to every diet group exerted an inhibitory effect on the inflammatory process along the duration time of the experiment (Fig. 2B), this effect was stronger in rats fed on PSVOO diet. Figure 3 compares changes in weight in arthritic rats. The results show that treatment with indomethacin 1 mg/Kg animal on PSVOO, FO and BD produced a very significant increase in the mean weight of the animals. No such gain on weight could be observed in the groups where indomethacin was not administered, however a substantial increase in weight was detected in the rats of the PSVOO, specially at later days of the experiment compared to the BD group. Discussion We found in the acute carrageenan oedema model, that inflammation indices of animals fed on a diet rich in oleic acid (VOO and PSVOO groups) were lower compared to
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Protection on inflammation of a virgin olive oil diet
Fig. 3. Influence of dietary treatment on the evolution of the weight of the arthritic rats. (BD: basal diet; BDIndo: basal diet treated with 1 mg/Kg indomethacin; PSVOO: supplemented virgin olive oil; PSVOO: supplemented virgin olive oil treated with 1 mg/Kg indomethacin; FO: fish oil ; FOIndo: fish oil treated with 1 mg/Kg indomethacin. ANOVA test: * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from BD values.
those of animals fed on high-oleic acid sunflower oil and palm olein. The difference might be attributable to the presence of specific polyphenols in virgin olive oil since the levels of MUFA such oleic acid are similar in virgin olive and in high-oleic acid sunflower oils. This possibility is consistent with results from the PSVOO group. The development of the oedema was further reduced in rats fed virgin olive oil supplemented with polyphenolic compounds (800 p.p.m.) compared to those fed virgin olive oil (200 p.p.m.) alone. Carrageenan induces an inflammatory response mediated at least in part by arachidonic acid metabolism. Lipoxygenase products have been implicated in leukocyte accumulation during this process in rats [23, 24]. It has been suggested that agents able to inhibit leukotriene production would exert a better control of inflammatory conditions than selective inhibitors of cyclooxygenase activity [25]. In previous work we found that phenolics from virgin olive oil inhibited the leukocyte 5-lipoxygenase enzyme in the micromolar range and additionally these compounds were also powerful scavengers of superoxide anions and hydrogen peroxide and were capable of preventing the generation of reactive oxygen species by intact leukocytes [17]. This study further extends the antioxidant properties demonstrated by other authors for these compounds [26, 27] and could be one mechanism by which these dietary constituents might support their biological activity in this model. No changes were observed in rats fed on fish oil diet (FO) in the carrageenan test. It has been assumed that the beneficial effects of fish oil are mediated by changes in eicosanoid production, favouring the formation of less biologically active substances [7]. However, there are controversial studies about the influence of dietary fish oils on carrageenaninduced edema in foot paw of rats. Depending on the dietary protocol and duration of feeding, the effects appear to be different. Yoshino and Ellis found no influence in rats administered EPA and DHA, despite a significant reduction in the production of pro-inflammatory eicosanoids PGE2 and LTB4 [28]. In contrast, other authors observed beneficial effects on the extent of inflammation in animal fed on these fatty acids or fish oil diet [29–31].
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In adjuvant induced arthritis in rats, we tested the PSVOO diet and compared it to FO diet, previously described as beneficial in rheumatoid arthritis [2, 32, 33]. We observed that the PSVOO diet was even more effective than FO diet. Fish oil supplementation can affect inflammation by changes in the fatty acid composition of cell membranes and modify the eicosanoids involved in inflammatory joint disease [2, 8, 32, 33] and it can alter the cytokine production [34]. For instance, the abnormally high level of cytokines such as interleukin-1 (IL-1) and tumour necrosis factor- alpha (TNF-a) expressed by monocytes, in the synovial fluid of patients with rheumatoid arthritis, can be reduced after fish oil supplementation [35]. The acute-phase protein, C-reactive protein, a biological measure of disease activity is also reduced [36]. Kremer examining the effects of fish oil supplementation on the progression of rheumatoid arthritis, using olive oil as placebo, showed similar results of clinical evaluation and immunological test between both oils. Production of IL-1 by macrophages was decreased in the olive oil group, although not to the same extent as in either of the fish oil group [37, 38]. Previously, Linos had suggested beneficial effects of olive oil in rheumatoid arthritis in the Greek population [39]. We observed that the daily dose of 1 mg/Kg indomethacin exerted an inhibitory effect on the development of arthritis in basal diet group. However dietary supplementation further augmented the effect of indomethacin. The substantial increase in weight detected in animals from the PSVOO group could be related to its potent prevention effect on inflammation, as it is known that inhibition of eicosanoid synthesis in this arthritis experimental model, reduces inflammation index and stabilizes body weight [40]. This study demonstrates that virgin olive oil with a high content of polyphenolic compounds, similar to those of extra virgin olive oil, shows protective effects in both models of inflammation. Human clinical studies could be required to determine whether the effects of a drug-extra virgin olive oil diet combination might be beneficial, as has been proposed for fish oil dietary supplements in humans [33]. Acknowledgements.We thank the “Comisión Interministerial de Ciencia y Tecnología” (CICYT) and the “Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria” (INIA) for partial financial support of this work (Projects ALI96-0456 and CAO99-006 respectively).
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