Comparative evaluation of the metabolic effects of hydroxytyrosol and its lipophilic derivatives (hydroxytyrosyl acetate and ethyl hydroxytyrosyl ether) in hypercholesterolemic rats

1 Comparative evaluation of the metabolic effects of hydroxytyrosol and its 1 lipophilic derivatives (hydroxytyrosyl acetate and ethyl hydroxytyrosyl 2 ether) in hypercholesterolemic rats 3 4 María Tabernero § , Beatriz Sarriá ∞ , Carlota Largo § , Sara Martínez-López ∞ , Andrés Madrona ‡ , 5 José Luis Espartero ‡ , Laura Bravo ∞ , Raquel Mateos ∞ *. 6 7 § Hospital La Paz Health Research Institute (IdiPaz); Madrid, Spain. ∞ Instituto de Ciencia y 8 Tecnología de Alimentos y Nutrición (ICTAN-CSIC), Madrid, Spain. ‡ Dpto. Química Orgánica y 9 Farmacéutica. Facultad de Farmacia, Universidad de Sevilla, Seville, Spain. 10 11 12 Running title: Metabolic effects of hydroxytyrosol and its lipophilic derivatives 13 * Corresponding author: Raquel Mateos. Department of Metabolism and Nutrition, 14 Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC). José Antonio 15 Novais 10, Ciudad Universitaria, 28040, Madrid, Spain. Phone: +34.91.544.56.07; Fax: 16 +34.91.549.36.27; e-mail: [email protected] 17 18 Page 1 of 28 Food & Function 2 19 Abstract 20 Hydroxytyrosol (HT), a virgin olive oil phenolic phytochemical with proven health benefits, 21 has been used to generate new lipophilic antioxidants to preserve fats and oils against 22 autoxidation. The aim of this work is to comparatively evaluate the physiological effects of 23 HT and the lipophilic derivatives, hydroxytyrosyl acetate (HT-Ac) and ethyl hydroxytyrosyl 24 ether (HT-Et) in a high-cholesterol fed animals. Male Wistar rats (n=8) were fed a standard 25 diet (C group), a cholesterol-rich diet (Chol group) or cholesterol-rich diets supplemented 26 with the phenolic compounds (HT group, HT-Ac group and HT-Et group) for 8 weeks. Body 27 and tissue weights, lipid profile, redox status, and biochemical, hormonal, and inflammatory 28 biomarkers were evaluated. Plasma levels of total, LDL-cholesterol, glucose, insulin and 29 leptin, as well as malondialdehyde in serum increased in Chol compared to C (p<0.05). Rats 30 fed the test diets improved their glucose, insulin, leptin and MDA levels and antioxidant 31 capacity status, being HT-Ac the most effective compound. The studied phenolic compounds 32 also modulated TNF-α and IL-1β plasma levels compared to Chol. HT-Ac and HT-Et 33 improved adipose tissue distribution and adipokine production, decreasing MCP-1 and IL-1β 34 levels. Our results confirm the metabolic effects of HT, which are maintained and even 35 improved by the hydrophobic derivatives, particularly in HT-Ac. 36 37 Keywords: Ethyl hydroxytyrosyl ether; hydroxytyrosol; hydroxytyrosyl acetate; 38 hypercholesterolemic rats; metabolic stress. 39 40 Page 2 of 28Food & Function 3 1. Introduction 41 The most representative phenolic compound in virgin olive oil, hydroxytyrosol 42 (HT), plays and important role in the prevention of degenerative diseases 1-2 . Explicitly, it 43 is involved in the reduction of LDL oxidation, a well-known cardiovascular risk factor 3 , 44 and it is transiently associates with LDL lipoproteins 4 . Moreover, HT is involved in the 45 inhibition of lipid and protein oxidation in human plasma 5 , having anti-inflammatory 6 46 and antiplatelet aggregation activities 7 . Additionally, there is evidence that olive oil 47 phenolic compounds decrease plasma glucose levels in rats 8-9 . HT can be efficiently 48 recovered from olive by-products, which has prompted the study of the biological and 49 health effects of HT from alperujo 8, 10 or olive leaves. 9, 11 50 On the other hand, the food industry demands new lipophilic antioxidants to 51 preserve fats and oils against autoxidation. Two series of hydrophobic derivatives of HT, 52 hydroxytyrosyl esters 12 and hydroxytyrosyl ethers 13 , have been synthesized. 53 Hydroxytyrosyl acetate (HT-Ac) merits special interest among hydroxytyrosyl esters, 54 since it is an antioxidant naturally present in virgin olive oil 14 that is transported across 55 the small intestinal epithelial cell barrier more efficiently than HT, 15 showing a higher 56 hepatic bioavailability than HT 16 . Moreover, HT-Ac has shown protective effects against 57 oxidative DNA damage in blood cells 17 , iron-induced oxidative stress in human cervical 58 cells, 18 and oxidative stress in HepG2 cells 19 . Hydroxytyrosyl ethyl ether (HT-Et), with an 59 alkyl chain with the same length as the acyl chain in HT-Ac, was included in this study to 60 assess the influence of the different functional groups (etherification versus esterification) 61 on the biological activity of the two phenolic compounds. HT-Et is absorbed to a higher 62 extent in Caco-2 cells than its precursor HT, 20 with absorption rate similar to that of HT-63 Ac, 15 being also broadly taken-up by HepG2 cells 21 . In addition, HT-Et protects hepatic 64 human HepG2 cells against oxidative stress 22 and inhibits platelet activation after oral 65 Page 3 of 28 Food & Function 4 administration in rats 23 .These antecedents justify using this compound although it is not 66 naturally present in virgin olive oil. 67 Both HT-Ac and HT-Et maintain the orthodiphenolic group intact and show 68 higher antioxidant capacity than HT 24-25 , preserving their potential application as 69 antioxidants to stabilize foodstuffs or as functional food ingredients. 70 With these antecedents, the aim of the present study was to comparatively 71 analyse the ability of HT and its lipophilic derivatives, HT-Ac and HT-Et, counteracting 72 the metabolic deregulation derived from consuming a high-cholesterol diet, focusing on 73 the effects on plasma lipids, glucose levels, hormone response, oxidative stress and pro-74 inflammatory status. In addition, body weight gain, adipose tissue distribution and 75 secretion of adipokines from visceral fat were evaluated. 76 77 2. Experimental 78 2.1. Materials and Chemical 79 Cholesterol, cholic acid, palmitic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-80 carboxilic acid (Trolox), 1,1,3,3-tetraethoxypropane, fluorescein, 2,2’-azobis(2-81 amidinopropane) dihydrochloride (AAPH), trizmabase, dithiothreitol, 1,1,3,3-82 tetraethoxypropane and 2,4-dinitrophenylhydrazine were purchased from Sigma-Aldrich 83 Chemical (Madrid, Spain). Sodium hydroxide, sulphuric acid, sodium hydrogen phosphate 84 and potassium dihydrogen phosphate were from Panreac (Madrid, Spain). The Bradford 85 reagent was from BioRad (BioRad Laboratories S.A., Madrid, Spain). Free fatty acids (FFA) 86 colorimetric kit was purchased from Roche (Roche Applied Science, Madrid Spain). Rat 87 adipocyte (RADPCYT-82K, MilliplexMap Rat Adipocyte Panel) and adipokine (RADPK-88 81K, MilliplexMap Kit Rat Serum Adipokine Panel) Milliplex kits were acquired from 89 Page 4 of 28Food & Function 5 Millipore (Millipore, Billerica, MA, US, USA). Other reagents were of analytical or 90 chromatographic quality. 91 HT was isolated with 98% purity from olive oil waste water following a patented 92 procedure 26 and further purified by column chromatography. 93 HT-Ac was obtained from HT in ethyl acetate after incubation with p-94 toluenesulfonic acid and purification by column chromatography following a patented 95 procedure 12 . 96 HT-Et was obtained from HT by chemical synthesis as described elsewhere 13 . 97 98 2.2. Diet, animals and experimental design 99 Forty male Wistar rats (200–225 g body weight) were purchased from an accredited 100 supplier (Charles River Laboratorios España, S.A. Barcelona, Spain) and housed in 101 metabolic cages. They had free access to food and water, and were maintained under a 102 normal light–dark cycle in the Experimental Surgery Service of La Paz University Hospital 103 (registration number: 280790001941). After one week of acclimation, animals were 104 randomly distributed into 5 different experimental groups (8 rats per group). One group 105 received the standard, maintenance rodent diet (A04-SAFE, Augy, France), which is the 106 control (C group), and four groups were fed the standard diet supplemented with 2% 107 cholesterol and 0.4% cholic acid in order to generate the hypercholesterolemic model as 108 described elsewhere 27 . In three of these groups the hypercholesterolemic diet was 109 supplemented with 0.04% of HT (HT group), HT-Ac (HT-Ac group) and HT-Et (HT-Et 110 group), respectively, to assess the potential beneficial effects of these phenolic compounds in 111 the hypercholesterolemic rat model. Nutritional and energetic content of each diet is 112 summarized in Table 1. During the whole intervention, animal health status and dietary 113 tolerance were monitored by veterinary individual observation in addition to weekly control 114 Page 5 of 28 Food & Function 6 of animals’ weight (Table 2). After 8 weeks animals were subjected to complete 115 exsanguination under general anesthesia (isoflurane 2%). Plasma and serum fractions were 116 separated by centrifugation (10 minutes at 2500 g) using pretreated EDTA or Silica Act Cot 117 Activator blood collection tubes (BD Vacutainer, Plymouth, UK) respectively, and stored at -118 20ºC until further analysis. Liver, kidney, heart and adipose tissue from retroperitoneal and 119 epididymal areas were collected, weighed and washed in ice-cold phosphate buffered saline. 120 Samples were immediately frozen in liquid nitrogen and stored at -80ºC until analysis. The 121 study protocol was approved by the Institutional Animal Ethics Committee of La Paz 122 University Hospital (Madrid, Spain) and procedures were performed in accordance with 123 Spanish law for the protection of experimental animals and other research purposes (RD 124 53/2013, BOE nº 34 Sec I pg 11370 8 th February 2013). 125 126 2.3. Blood biochemical analyses 127 Triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, glucose and 128 creatinine in plasma were determined using an automated analyser (Beckman Coulter-Former 129 Olympus Diagnostics AU 5420, Nyon, Switzerland). Plasma free fatty acids (FFA) 130 concentration was determined with a colorimetric commercial assay kit (Free Fatty Acids, 131 Half Micro Test, Roche Applied Science) using palmitic acid as standard. 132 133 2.4. Determination of oxygen radical scavenging capacity (ORAC) in serum. 134 Serum antioxidant activity was analysed using the hydrophilic oxygen radical 135 scavenging capacity (ORAC) assay according to the method developed by Huang, Ou, 136 Hampsch-Woodill, Flanagan& Prior 28 . The fluorescence at 485 and 528nm excitation and 137 emission wavelengths, respectively, was determined in a 96 well microplate reader (Bio-Tek, 138 Winooski, VT, USA). ORAC values were expressed as micromoles of Trolox / mL serum. 139 Page 6 of 28Food & Function 7 140 2.5. Determination of Malondialdehyde (MDA) in serum and liver. 141 MDA was determined as its hydrazone by high-performance liquid chromatography 142 using 2,4-dinitrophenylhydrazine for derivatization 29 . Livers (0.5g) were homogenized (1:5 143 w/v) in ice-cold 0.25M Trizma base buffer pH 7.4 containing 5mM dithiothreitol using an 144 Ultra Turrax (IKA® Works Inc., WilmingtonNC) at 18000 rpm. After centrifugation 145 (11000rpm 30min, 4ºC), supernatants were collected for MDA quantification. Serum samples 146 were analysed directly. Standard MDA was prepared by acidic hydrolysis of 1,1,3,3-147 tetraethoxypropane in 1% sulphuric acid. Concentrations were expressed as nanomoles of 148 MDA per milligram of protein in liver tissue and per millilitre in serum. Protein content in 149 liver homogenates was estimated by the Bradford method using a Bio-Rad protein assay kit. 150 151 2.6. Determination of cytokines and hormones in plasma and adipose tissue by 152 immunoassay. 153 A sample of white adipose tissue from the retroperitoneal area (0.2g) was collected 154 and homogenized (1:3.5 w/v) in PBS (pH 7.4) with Triton X-100 and protease inhibitor by 155 low temperature sonication (17 microns, 3 consecutive pulses of 15 seconds at 30 seconds 156 intervals). After centrifugation (5000rpm, 10min, 4ºC), the lipid layer was removed and 157 supernatants were collected for adipokine determination. The concentration of leptin, 158 interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), monocyte 159 chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in the 160 retroperitoneal adipose tissue were determined using a Rat Adipocyte Multiplex Kits 161 (RADPCYT-82K Milliplex Map Kit, Rat Adipocyte Panel, Millipore, Billerica, MA, US). 162 IL-1β, IL-6, TNF-α, insulin and leptin concentrations were determined in plasma samples 163 obtained at the end of the intervention using a Rat Adipokine multiplex kits (RADPK-81K, 164 Page 7 of 28 Food & Function 8 Milliplex Map Kit, Rat Serum Adipokine Panel, Millipore, Billerica, MA, US). Multianalyte 165 profiling panels were used according to manufacturer’s instructions and analysed on a 166 LuminexLX200 Analyzer. Data were analysed using the 3.1 xPONENT software (Millipore); 167 high and low concentration quality controls were used with all the biomarkers. Protein 168 content in adipose tissue homogenates was estimated by the Bradford method using a Bio-169 Rad protein assay kit. 170 171 2.7. Statistical analysis. 172 All data were expressed as the mean ± standard deviation (SD). Non parametric 173 significant test (Kruskal-Wallis one-way analysis of variance and Mann-Whitney U test) was 174 performed to compare values. Differences were considered statistically significant when p < 175 0.05 and in Mann-Whitney U test the significance was adjusted with a Bonferroni test. Data 176 were analysed using the statistical package SPSSv.19.0 software (SPSS, Institute Inc, Cary, 177 NC). 178 179 3. Results 180 3.1.Food intake, body weight gain and tissues weights 181 All diets provided equivalent amounts of carbohydrates and proteins. However, 182 cholesterol-rich diets led to an excess of lipids resulting in an 8.5 Kcal/100g higher caloric 183 intake. During the 8-week study period, animals in the five experimental groups had similar 184 daily food intakes (approximately 20g/d). 185 Therefore, the groups consuming the cholesterol-rich diets (Chol, HT, HT-Ac and 186 HT-Et) presented a daily caloric excess of approximately 2 Kcal/day. However, this did not 187 induce significant differences in the body weight gain compared with the control animals, as 188 can be seen in Table 2. 189 Page 8 of 28Food & Function 9 Considering an average daily food intake of 20g and an average body weight of 190 300g, the daily phenolic compound intake was 25mg/kg body weight/day. This is a relatively 191 low dose, comparable with doses used in some other studies 8-11 and within a range proven to 192 elicit no toxic effects in rats. Accordingly, no pathologic alterations or behaviours were 193 observed in the animals during the study. Organs, including kidney or heart among others, 194 presented normal appearance by gross observation. However, livers in animals fed the 195 cholesterol-rich diets showed a whitish appearance compared with the C group, suggestive of 196 fat accumulation, being significantly heavier than those of the C group (Table 2). Adipose 197 tissue depots were obtained from retroperitoneal and epididymal areas, and when their 198 weights were normalized for the final animal body weight, no differences were observed 199 among different treatments. However, the ratio obtained with the adipose tissue from 200 retroperitoneal area/epididymal area showed significant differences, being higher in the Chol 201 and HT groups than in the C group. 202 203 3.2. Plasma cholesterol profile 204 As Figure 1 shows, animals fed the cholesterol-rich diets had a marked 205 hyperlipidaemia, with high LDL-cholesterol and total cholesterol concentrations, although 206 HDL-cholesterol levels were not affected as compared to C group. Although not reaching the 207 values of control animals, supplementation of the cholesterol-rich diet with 0.04% HT-Ac 208 significantly decreased the total cholesterol and LDL-cholesterol levels as compared with the 209 Chol group (p<0.05), which was not attained in the HT and HT-Et groups. 210 211 3.3. Biochemical parameters and hormones involved in energy homeostasis 212 The high-cholesterol diet used in the present study induced a significant increase in 213 glucose levels in the Chol group compared to control animals (p<0.05). All the tested 214 Page 9 of 28 Food & Function 16 modify gene expression in different tissues and cell lines, the chemical structure of the 364 phytochemical plays an important role not only in their direct scavenging of free radicals, but 365 also in the target protein and therefore in the molecular mechanism involved in its biological 366 action. Thus, considering the extensive hydrolysis that HT-Ac undergoes during intestinal 367 absorption yielding HT 15 , both compounds could follow similar mechanisms to regulate the 368 unbalance induced by the high-cholesterol rich diet. Our results suggest changes in the 369 expression of redox and inflammatory related genes, based on studies recently published by 370 other research groups; however, further work is required to elucidate the molecular 371 mechanisms underlying the regulatory metabolic effect of HT and its derivatives. 372 A limitation of this study is that a control group fed a standard diet supplemented 373 with HT was not included to assess the effect of this compound within a balanced diet in 374 healthy animals. 375 In summary, HT and its lipophilic derivatives, HT-Ac and HT-Et, were able to 376 reduce the metabolic unbalance induced by a high-cholesterol diet in rats, being HT-Ac, the 377 lipophilic HT derivative naturally present in virgin olive oil, the most effective phenolic 378 compound. Since HT-Ac can be easily obtained from natural HT 12 , this compound might be 379 proposed as an interesting bioactive ingredient in the production of functional foods, having 380 the added value of contributing to induce beneficial metabolic properties. 381 382 Page 16 of 28Food & Function 17 List of abbreviations: C group: Control group ; Chol group: cholesterol-rich diet group; 383 AAPH: 2,2’-azobis (2-amidinopropane) dihydrochloride; COX 2: cyclooxigenase-2; FFA: 384 Free fatty acids; HT: hydroxytyrosol; HT-Ac: hydroxytyrosyl acetate; HT-Et: ethyl 385 hydroxytyrosyl ether; IL-1b: interleukin-1β; IL-6: interleukin-6; LRP1: LDL receptor-386 related protein-1; MCP-1: monocyte chemoattractant protein-1; ORAC: oxygen radical 387 scavenging capacity; PAI-1: plasminogen activator inhibitor-1; SD: standard deviation; 388 TNF-α αα α: tumour necrosis factor-α ; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-389 carboxilic acid 390 391 Acknowledgements 392 Study funded by projects RTA2007-00036 (INIA), Excellence Project AGR-5098 393 (Junta de Andalucia) and Consolider-Ingenio (CSD2007-00063). The authors thank the 394 technical support and expertise in blood analysis provided by CORE lab (University Hospital 395 La Paz).SM-L is a JAE predoctoral fellow funded by CSIC and the European Social Fund. 396 397 398 Conflict of interest statement 399 The authors have declared no conflict of interest. 400 401 402 403 404 405 406 Page 17 of 28 Food & Function 18 References 407 1. M. I. Fernandez-Mar, R. Mateos, M. C. Garcia-Parrilla, B. Puertas, and E. Cantos-408 Villar, Food Chem., 2012, 130, 797-813. 409 2. A. Dey and J. Lakshmanan, Food Funct., 2013, 4, 1148-1184. 410 3. K. De la Torre-Carbot, J. L. Chávez-Servín, O. Jaúregui, A. I. Castellote, R. M. 411 Lamuela-Raventós, T. Nurmi, H. E. Poulsen, A. V. Gaddi, J. Kaikkonen, H. F. 412 Zunft, H. Kiesewetter, M. Fitó, M. I. Covas, and M. C. López-Sabater. J. Nutr., 413 2010, 140, 501-508. 414 4. M. Gonzalez-Santiago, J. Fonolla, and E. Lopez-Huertas. Pharm. Res., 2010, 61, 415 364-370. 416 5. J. Ruano, J. López-Miranda, F. 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Bars 515 without a common letter differ, p< 0.05. 516 517 518 Page 22 of 28Food & Function 23 Figure 1. Page 23 of 28 Food & Function 24 Table 1. Dietary composition of the experimental diets. Component (g/100g dry weight) C Chol HT HT - Ac HT - Et Carbohydrates 65.93 64.20 64.17 64.17 64.17 Protein 17.58 17.12 17.11 17.11 17.11 Lipids (others than cholesterol) 3.30 3.21 3.21 3.21 3.21 Cholesterol 0.00 2.19 2.19 2.19 2.19 Cholic acid 0.00 0.44 0.44 0.44 0.44 HT and derivatives 0.00 0.00 0.04 0.04 0.04 Caloric content (Kcal/100g) 219.2 227.7 227.7 227.7 227.7 Page 24 of 28Food & Function 25 Table 2. Body and tissue weights of rats in the control group (C), the group consuming the non-supplemented cholesterol-rich diet (Chol) or the 1 cholesterol-rich diet supplemented with hydroxytyrosol (HT), hydroxytyrosyl acetate (HT-Ac) and ethyl hydroxytyrosyl ether (HT-Et). Data 2 represents the mean of 8 determinations ± SD*. 3 * Mean values in a row with unlike superscripts are significantly different according to Kruskal-Wallis one-way analysis of variance and Mann-Whitney 4 U test (p< 0.05). 5 6 7 Standard diet Hypercholesterolemic diet C Chol HT HT-Ac HT-Et Body weight (g) Inicial 242 + 6 244 + 8 244 + 7 239 + 11 243 + 4 Final 336 + 18 326 + 30 331 + 16 317 + 10 329 + 14 Body weight gain (g/56d) 94 + 15 82 + 32 87 + 12 78 + 11 86 + 12 Tissue weights (g) Epididymal fat/ body weight 0.016 + 0.003 0.016 + 0.002 0.015 + 0.001 0.017 + 0.003 0.015 + 0.004 Retroperitoneal fat/ body weight 0.018 + 0.004 0.015 + 0.002 0.014 + 0.002 0.017 + 0.001 0.016 + 0.005 Ratio Retroperitoneal/epididymal 0.88 + 0.09 a 1.07 + 0.08 b 1.07 + 0.08 b 1.00 + 0.05 ab 0.93 + 0.07 ab Liver / body weight 0.024+ 0.002 a 0.038 + 0.003 b 0.029 + 0.018 a b 0.036 + 0.002 b 0.037 + 0.002 b Kidney / body weight 0.0053 + 0.0022 0.0057+ 0.0005 0.0058+ 0.0008 0.0061+ 0.0005 0.0059 + 0.0006 Heart / body weight 0.0026 + 0.0003 0.0028 + 0.0005 0.0025 + 0.0001 0.0029 + 0.0005 0.0028 + 0.0003 Page 25 of 28 Food & Function