Jin-Hyang
Suh‡
a,
Cindy
Romain‡
a,
Rocío
González-Barrio
b,
Jean-Paul
Cristol
a,
Pierre-Louis
Teissèdre
c,
Alan
Crozier
b and
Jean-Max
Rouanet
*a
aJoint Research Unit 204 NUTRIPASS, Prevention of Malnutritions & Linked Pathologies, University Montpellier South of France, Jean-Max Rouanet, UMR 204 NUTRIPASS, CC 023, Université Montpellier 2, Place E. Bataillon, 34095, Montpellier Cedex 05, France. E-mail: jm.rouanet@univ-montp2.fr; Fax: (+33) 04 67 14 35 21; Tel: (+33) 04 67 14 35 21
bPlant Products and Human Nutrition Group, Centre for Population and Health Sciences, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
cUMR 1219 Œnologie, Institut de la Vigne et du Vin de Bordeaux-Aquitaine, Université Victor Ségalen Bordeaux 2, 210 chemin de Leysotte, CS 50008, 33882, Villenave d'Ornon Cedex, France
First published on 13th June 2011
The effects of raspberries on early atherosclerosis in Syrian hamsters were investigated using three juices prepared from var. Cardinal, Glen Ample and Tulameen berries. The hamsters received an atherogenic diet for 12 weeks and at the same time a juice at a daily dose corresponding to the consumption of 275 ml by a 70 kg human. A control group received the same diet with water instead juice. The principal polyphenolic compounds in the juices were anthocyanins and ellagitannins, which were present at concentrations of 218–305 μg mL−1 and 45–72 μg mL−1, respectively. The three juices had similar but not identical effects. They all inhibited cardiac and aortic production of superoxide anion and increased hepatic glutathione peroxidase activity although only Tulameen juice brought about a significant increase in superoxide dismutase activity. Glen Ample was the only juice to significantly increase plasma paraoxonase activity. All the juices lowered plasma triglyceride level while consumption of Tulameen and Cardinal, but not Glen Ample, significantly lowered plasma total cholesterol and LDL-cholesterol. Cardinal was the sole juice to significantly increase HDL-cholesterol and likewise it also significantly reduced body weight. These findings suggest that moderate consumption of raspberry juices can help to prevent the development of early atherosclerosis, with the underlying mechanisms related to improved antioxidant status and serum lipid profiles.
The beneficial health effects of fruit consumption have been emphasized by epidemiological studies. Although the evidence is not unequivocal, some studies have shown a relationship between a reduced risk of cardiovascular disease (CVD) and fruit consumption.4,5 The protective effects could be derived from micronutrients and micro-constituents, in particular flavonoids and related phenolic compounds,6 which in general are powerful antioxidants.7
Red raspberries contain substantial amounts of polyphenolic compounds with the principal antioxidants being anthocyanins and the ellagitannins sanguin H-6 and lambertianin C, compounds which in vitro also induce vasorelaxation.8,9 Consumption of raspberry, strawberry and bilberry juices, as well as green and black tea, has been shown to bring about a marked reduction in aortic lipid deposition in hypercholesterolemic golden Syrian hamsters.10 In this paper we report on the impact of raspberry juice consumption on oxidative stress and early markers of atherosclerosis in hamsters.
Compound | Cardinal | Glen Ample | Tulameen |
---|---|---|---|
Cyanidin-3-O-sophoroside | 114 ± 3 | 134 ± 0.7 | 216 ± 3 |
Cyanidin-3-O-(2′′-O-glucosyl)rutinoside/glucoside | 117 ± 2 | 65 ± 1.7 | 65 ± 1.4 |
Pelargonidin-3-O-sophoroside | 6.2 ± 0.2 | 4.8 ± 1.3 | 8.4 ± 0.1 |
Cyanidin-3-O-rutinoside | 27 ± 0.7 | 12 ± 0.1 | 13 ± 6.4 |
Pelargonidin-3-O-(2′′-O-glucosyl)rutinoside | 6.9 ± 0.2 | 2.4 ± 0.0 | 2.9 ± 0.0 |
Total anthocyanins | 271 ± 2 | 218 ± 2 | 305 ± 7 |
Sanguin H-10 | 5.8 ± 0.5 | 5.4 ± 0.1 | 8.5 ± 0.3 |
Sanguin H-6 | 52 ± 1 | 67 ± 2.1 | 36 ± 0.9 |
Total ellagitannins | 58 ± 1 | 72 ± 2 | 45 ± 1 |
Ellagic acid pentoside conjugate-1 | 1.3 ± 0.0 | 1.8 ± 0.6 | 3.2 ± 0.0 |
Ellagic acidpentoside conjugate-2 | 1.3 ± 0.1 | 1.4 ± 0.7 | 1.1 ± 0.0 |
Ellagic acid-like compounds | 0.4 ± 0.0 | 0.5 ± 0.1 | 1.6 ± 0.1 |
Total ellagic acid-like compounds | 2.9 ± 0.1 | 3.7 ± 0.4 | 5.8 ± 0.0 |
Total HPLC phenolics | 332 ± 2 | 294 ± 5 | 356 ± 8 |
Diet | Initial BW (g) | Final BW (g) | Weight gain (g) | Food intake (g/d) | Energy intake (kJ/d) |
---|---|---|---|---|---|
a Values are the means ± SEM (n = 12). For each dietary treatment, mean values in a column with different superscripts (*) are significantly different from the controls, P < 0.05. | |||||
Control | 79.6 ± 2.1 | 90.0 ± 2.3 | 10.3 ± 3.3 | 4.4 ± 0.5 | 96.8 ± 11.0 |
Cardinal | 78.2 ± 1.8 | 80.3 ± 5.2* | 1.9 ± 2.8* | 4.0 ± 0.4 | 88.0 ± 8.8 |
Glen Ample | 79.4 ± 2.0 | 85.3 ± 2.0 | 5.7 ± 1.8 | 4.1 ± 0.5 | 90.2 ± 11.0 |
Tulameen | 77.4 ± 1.7 | 84.5 ± 2.9 | 7.1 ± 2.0 | 4.1 ± 0.5 | 90.2 ± 11.0 |
Groups | Glycemia | TC | HDL-C | LDL-C | TG |
---|---|---|---|---|---|
a Data are expressed as the mean values in mmol L−1 ± SEM (n = 12). For each dietary treatment, mean values in a column with different superscripts (*) are significantly different from the controls, P < 0.05. TC: total cholesterol; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol; TG: triglycerides. | |||||
Control | 8.44 ± 2.27 | 7.32 ± 1.42 | 2.77 ± 0.47 | 3.62 ± 0.90 | 2.04 ± 0.62 |
Cardinal | 7.31 ± 2.05 | 6.35 ± 0.76* | 2.89 ± 0.32 | 2.96 ± 0.54* | 1.10 ± 0.38* |
Glen Ample | 6.65 ± 1.37 | 7.73 ± 0.75 | 3.07 ± 0.20* | 4.05 ± 0.79 | 1.35 ± 0.24* |
Tulameen | 8.00 ± 1.27 | 5.56 ± 0.77* | 2.93 ± 0.25 | 2.15 ± 0.55* | 1.05 ± 0.08* |
Diet | GSHPx (U/mg protein) | SOD (U/mg protein) | PON (U/mL) |
---|---|---|---|
a Data expressed as mean values ± SEM (n = 12). For each dietary treatment, mean values in a column with different superscripts (*, **) are significantly different from the controls, P < 0.05. GSHPx: glutathione peroxidase. SOD: superoxide dismutase. PON: paraoxonase. | |||
Control | 949 ± 45* | 158 ± 13* | 60 ± 16* |
Cardinal | 1412 ± 76** | 161 ± 9* | 70 ± 17* |
Glen Ample | 1352 ± 72** | 144 ± 10* | 120 ± 29** |
Tulameen | 1278 ± 68** | 203 ± 12** | 61 ± 13* |
Plasma PON activity was enhanced significantly by consumption of Glen Ample juice but not the other varieties of raspberry (Table 4). In plasma, PON is localized in HDL, and when PON activity is expressed as a ratio to HDL, Glen Ample remains the only effective juice (Fig. 1).
Fig. 1 Ratio paraoxonase activity (PON)/HDL concentration in hamsters fed a high-fat diet (CTR), or a CTR diet plus either Cardinal (CAR), Glen Ample (GLAM) or Tulameen raspberry juice (TUL) for 12 weeks. Values are mean ± SEM (n = 6). For each dietary treatment, bars with different index letters are statistically significantly different (P < 0.05). |
Fig. 2 Cardiac superoxide anion production in hamsters fed a high-fat diet (CTR), or a CTR diet plus either Cardinal (CAR), Glen Ample (GLAM) or Tulameen raspberry juice (TUL) for 12 weeks. Values are expressed as mean ± SEM (n = 6). For each dietary treatment, bars with different index letters are statistically significantly different (P < 0.05). |
Fig. 3 Thoracic aorta superoxide anion production in hamsters fed a high-fatdiet (CTR), or a CTR diet plus either Cardinal (CAR), Glen Ample (GLAM) or Tulameen raspberry juice (TUL) for 12 weeks. Values are expressed as mean ± SEM (n = 6). |
Juice | Anthocyanins (mg mL−1) | Ellagitannins (mg mL−1) | Body weight | TG | TC | LDL-C | HDL-C | Cardiac O2˙− | Thoracic O2˙− | GSHPx | SOD | PON |
---|---|---|---|---|---|---|---|---|---|---|---|---|
a n.s.: effect not significant. TC: total cholesterol; HDL-C: HDL-cholesterol; LDL-C: LDL-cholesterol; TG: triglycerides. GSHPx: glutathione peroxidase. SOD: superoxide dismutase. PON: paraoxonase. | ||||||||||||
Tulameen | 305 | 45 | n.s. | reduced | reduced | reduced | n.s. | reduced | reduced | increased | increased | n.s. |
Cardinal | 271 | 58 | reduced | reduced | reduced | reduced | increased | reduced | reduced | increased | n.s. | n.s. |
Glen Ample | 218 | 72 | n.s | reduced | n.s. | n.s. | n.s. | reduced | reduced | increased | n.s. | increased |
The main phenolic compounds of the three raspberry juices were anthocyanins and ellagitannins, and there were minor but not substantial differences in the levels of these compounds in the individual juices. Although they did not have identical effects, all the juices induced potentially protective effects when consumed by hamsters on a daily basis for 12 weeks (Table 5). There was, for instance, a trend towards reduced body weight with juice consumption but the effect was only statistically significant with the juice prepared from Cardinal berries (Table 2). Elevated LDL, triglycerides and total cholesterol are known to be risk factors for atherosclerosis. All three raspberry juices lowered triglyceride levels. However, Cardinal and Tulameen, but not Glen Ample, reduced both total cholesterol and LDL-cholesterol but did not impact on HDL-cholesterol which was increased significantly by ingestion of Glen Ample juice (Table 5).
All three juices also significantly reduced the production of cardiac and aortic O2˙− levels (Fig. 2 and 3, Table 5), via the decreased activity of NADPH oxidase and therefore the oxidative stress induced by the atherogenic diet. As noted above, the atherogenic diet led to high level of total and non-HDL-C (i.e. ≈ LDL-C) which is known to induce aortic fatty streak deposition.11 Interestingly, the diet-induced hypercholesterolemia was accompanied by an overproduction of O2˙− (data not shown), as previously shown in rat and hamster models of atherosclerosis.12,13 Thus, according to the oxidative hypothesis of atherosclerosis, it could be postulated that NADPH oxidase activity works with high LDL cholesterol to induce foam cells fatty streak14 and subsequent atherosclerosis.15
Consumption of all three raspberry juices resulted in significantly higher GSHPx activity than in control hamsters. In contrast, only intake of Talameen juice brought about a significant increase in SOD activity (Table 5). PON activity is another marker of oxidative stress. HDL has long been known to be antiatherogenic but the exact mechanism of action has yet to be identified although it has been attributed to a role in the reverse transport of cholesterol and to their antioxidant properties. It has been proposed that PON plays a crucial role in the antioxidant activity of HDL.16 It has been reported that PON is implicated in the protection of LDL and HDL from oxidation induced by copper ions and other free radical generators.17 This protection is most probably related to the ability of PON to hydrolyse oxidized phospholipids18 and/or lipid peroxide products.17 PON is therefore believed to be a protective factor against atherosclerosis.19 Some studies have shown that PON can reduce oxidative stress in aortic lesions.20,21 Moreover, a decrease in the specific anti-atherogenic activity of HDL might also contribute. In animal models, an impaired HDL antioxidant defense has been observed in dyslipidemic, obese mice.22In the current study, only Glen Ample was effective in increasing PON activity (Table 5). This is consistent with a lower production of cardiac O2°−in animals fed Glen Ample juice (Fig. 2).
Tulameen juice had the highest anthocyanin content of the three raspberry juices that were investigated, while Glen Ample juice contained the highest levels of ellagitannins. Regarding the bioactivity of the juices, Tulameen was the most efficient in modulating dyslipidemia (TC and LDL-C) and Glen Ample the least effective. In contrast, Glen Ample was the most efficient in reducing the induction of oxidative stress (O2˙− and PON activity) and Tulameen was the least effective juice. While it is tempting to speculate that the different effects of the juices are related to their differing anthocyanin and ellagitannin profiles, this would be premature. However, the protective effects of raspberries and the relationship between anthocyanins and ellagitannins are topics that merit further investigation. Neither anthocyanins nor ellagitannins per se are likely to enter the bloodstream in sufficient quantity to induce the protective effects observed with the hypercholesterolemic hamsters. In a separate study in which human volunteers consumed raspberries, it has been shown that the major components that enter the bloodstream are derived from the colon. Here ellagitannins are converted to urolithins, which are absorbed as O-glucuronides, and anthocyanins undergo C-ring fission yielding a number of phenolic acids.23 Urolithin aglycones and several phenolic acids have recently been shown in vitro to have anti-inflammatory properties,24 antiglycative effects and to protect neurons from oxidative stress.25
Ingredients | Control |
---|---|
a Mineral mixture contained (mg kg−1 of diet): CaHPO4, 17200; KCl, 4000; NaCl, 4000; MgO, 420; MgSO4, 2000; Fe2O3, 120; FeSO4·7H2O, 200; trace elements, 400 (MnSO4·H2O, 98; CuSO4. 5H2O, 20; ZnSO4·7H2O, 80; CoSO4·7H2O, 0,16; KI, 0.32; sufficient starch to bring to. 40 g (per kg of diet). Vitamin mixture contained (mg kg−1 of diet): retinol, 12; cholecalciferol, 0.125; thiamin, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300; cyanocobalamin, 0.1; menadione, 80; nicotinic acid, 200; choline, 2720; folic acid, 10; p-aminobenzoic acid, 100; biotin, 0.6; sufficient starch to bring to 20 g (per kg of diet). | |
Casein | 200 |
DL-Methionine | 3 |
Corn starch | 393 |
Sucrose | 154 |
Cellulose | 50 |
Lard | 150 |
Mineral mix | 35 |
Vitamin mix | 10 |
Cholesterol | 5 |
The liver was excised, weighed and sectioned for analyses and stored at −80 °C. Liver was homogenized in ice cold 0.1 mol L−1 potassium phosphate buffer (pH 7.4) and the homogenate was spun at 13000 g for 15 min at 4 °C. Glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activities of the supernatant were assayed on an automat Pentra 400 (HORIBA ABX, Montpellier, France) using commercial kits (Ransod kit no. SD 125 and Ransel no. RS505, Randox Laboratories LTD, Crumlin, UK, respectively). The heart and the thoracic aorta were removedand stored at 4 °C in PBS for subsequent analysis.
Following blood collection and tissues removal, the intact aortic cross of 6 hamsters per group were removed then immersed in Krebs buffer, and was cleaned of fat and connective tissue and cut into 2–3 mm wide. Superoxide anion (O2˙−) production was evaluated in thoracic aorta immersed and equilibrated in Krebs buffer containing lucigenin which enhanced luminescence, in the presence of 7.5 μL of NADPH (2 mM). The intensity of luminescence was recorded on a luminometer (Berthold Technology, France) for 30 min. Results were expressed as relative luminescence units (RLU/μg tissue). Cardiac superoxide anion production was also evaluated. Briefly, the left ventricle (150 mg) was homogenized with 10-fold volume of Krebs buffer and centrifuged.29 Supernatant was placed in a well with lucigenin (200 μM) and NADPH (2 mM). The intensity of luminescence was recordedand the results were expressed as relative luminescence units (RLU/mg of protein).
Protein content of tissues was determined by using a commercial protein assay (Sigma, Saint Quentin Fallavier, France) according to the method of Smith et al. and using bovine serum albumin as standard.30
Footnotes |
† The authors have declared no conflict of interest. |
‡ Authors made an equal contribution. |
This journal is © The Royal Society of Chemistry 2011 |