Hypolipidemic Effect of Tomato Juice in Hamsters in High Cholesterol Diet-Induced Hyperlipidemia

Tomato is a globally famous food and contains several phytonutrients including lycopene, β-carotene, anthocyanin, and flavonoids. The increased temperature used to produce tomato juice, ketchup, tomato paste and canned tomato enhances the bioactive composition. We aimed to verify the beneficial effects of processed tomato juice from Kagome Ltd. (KOT) on hypolipidemic action in hamsters with hyperlipidemia induced by a 0.2% cholesterol and 10% lard diet (i.e., high-cholesterol diet (HCD)). Male Golden Syrian hamsters were randomly divided into two groups for treatment: normal (n = 8), standard diet (control); and experimental (n = 32), HCD. The 32 hamsters were further divided into four groups (n = 8 per group) to receive vehicle or KOT by oral gavage at 2787, 5573, or 13,934 mg/kg/day for six weeks, designated the HCD-1X, -2X and -5X groups, respectively. The efficacy and safety of KOT supplementation was evaluated by lipid profiles of serum, liver and feces and by clinical biochemistry and histopathology. HCD significantly increased serum levels of total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), LDL-C/HDL-C ratio, hepatic and fetal TC and TG levels, and degree of fatty liver as compared with controls. KOT supplementation dose-dependently decreased serum TC, TG, LDL-C levels, LDL-C/HDL-C ratio, hepatic TC and TG levels, and fecal TG level. Our study provides experiment-based evidence to support that KOT may be useful in treating or preventing the onset of hyperlipidemia.


Introduction
Tomato is low in fat and calories, cholesterol free, and a good source of fiber and protein. It is also rich in vitamins A and C, β-carotene, potassium, and lycopene [1]. Tomato is now used in enormous quantities in the fresh state and heads the list of all vegetables as a canned product. Most tomato is consumed as a processed product, such as pastes, concentrates, ketchup, salsa and juice. Processed tomato products are important sources of minerals and vitamins in diets [2]. Tomato and processed tomato contain many health-benefit components, such as lycopene, anthocyanin, ascorbic acid, total phenolics, glycoalkaloids, and tomatine and low levels of carotenoids [3][4][5][6][7][8]. Lycopene bioavailability can be affected by food processing. The bioavailability in food is higher for cis-isomers than all-trans-isomers. Lycopene bioavailability is higher in processed tomato products than in unprocessed fresh tomatoes [9][10][11].
Tomato juice is made by heating, crushing and simmering tomatoes. To maintain nutrition, tomatoes are usually boiled first to inactivate enzymes that decrease vitamin C and other nutrients when tomatoes are crushed. Some foods lose their nutrient content when they are cooked or juiced, but the heating process actually boosts certain of tomatoes' properties. Lycopene is more available in tomato juice than in fresh tomatoes because of the heat and oil used to produce juice. Canned and bottled tomato juice is often fortified, thereby increasing the levels of vitamins [12,13]. In a previous study, the US Department of Agriculture recorded that 1/2 cup of tomato juice provides 10% and 35% of the recommended daily amount of vitamins C and A, respectively [14].
Tomato juice is known to have lipid-lowering effects and antioxidant activities [11]. In previous study, a tomato processed product improved blood lipid profiles in postmenopausal hyperlipidemic rats [10]. A high dietary intake of tomato products has atheroprotective effects by significantly reducing liver and serum cholesterol levels [15,16]. Tomato from the processing of tomato products contains many bioactive components, including those that act as antioxidants, such as the vitamins C and E and carotenoids. Lycopene is one of the main carotenoids in tomatoes. Previous study demonstrated that lycopene shows greater stability at low than high temperature and benefits from the processed tomato products. The lycopene bioactivity can be more accurately predicted in processed tomato than fresh tomato because lycopene is more soluble in lipids than water and has greater interaction with cellulose. Therefore, grinding tomato and cooking with oil could increase the bioavailability [17][18][19]. Another study showed that a higher lycopene concentration could protect against cardiovascular disease [20]; tomato-processed foods contain lycopene that can help reduce serum triglyceride (TG) levels with human high fat-induced [21][22][23]. One recent study reported that 13-oxo-9,11-octadecadienoic acid in tomato extract acts as a peroxisome proliferator-activated receptor α (PPARα) agonist and ameliorates obesity-induced dyslipidemia and hepatic steatosis [24].
Many studies used the hamster model to evaluate the hypolipidemic effect because it has many similarities with human fat-induced atherosclerotic disease. Similar to humans, hamsters are endowed with cholesterol ester transfer protein and all of the enzymatic pathways in lipoproteins and bile metabolism; atherosclerotic plaques develop in response to a fat diet in lesion-prone areas similar to humans [25][26][27]. Therefore, we used hamsters to evaluate the preventive effectiveness of supplementation with tomato juice from Kagome Ltd. (KOT) that is produced by a process for increasing lycopene and dietary fiber on hyperlipidemia regulation. We also examined the biochemical parameters and liver tissues by histopathology.

Materials, Animals, and Experiment Design
KOT was obtained from Taiwan Kagome Co. (Tainan, Taiwan). In this study, the dose of KOT designed for humans was 22.595 g per day (lyophilized powder), which would be equivalent to a daily recommended dose of KOT at 280 mL/serving/day. To ensure precise and accurate dosing of test animals, KOT was lyophilized by freeze-drying to obtain powder extract. The nutrition facts, dietary fiber and lycopene of KOT were provided by Kagome Co. and are shown in Table 1. The hamster dose (2787 mg/kg) we used was converted from a human-equivalent dose (HED) based on body surface area by the following formula from the US Food and Drug Administration: assuming a human weight of 60 kg, the HED for 22.595 (g)/60 (kg) = 377ˆ7.4 = 2787 mg/kg; the conversion coefficient 7.4 was used to account for differences in body surface area between hamsters and human as we recently described [28]. Specific pathogen-free (SPF) male Golden Syrian hamsters (12 weeks old) were purchased from the National Laboratory Animal Center (NLAC), Taipei City, Taiwan. Animals were housed in the animal facility at National Taiwan Sport University at temperature (22˘1˝C) and 50% to 60% relative humidity, with a 12 h light-dark cycle (light on 7:00 a.m.). Distilled water and standard laboratory chow diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) were provided ad libitum. Before the experiments, the hamsters were acclimatized for 1 week to the environment and diet. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) approved all animal experimental protocols, and the study conformed to the guidelines of the protocol IACUC-10306 approved by the IACUC ethics committee.
The experimental design is in Figure 1. A total of 40 hamsters were randomly divided into 5 groups for treatment (n = 8/each group): (1) control, standard chow diet with vehicle (water); (2) HCD, standard chow (No. 5001) with 0.2% cholesterol and 10% lard diet with vehicle treatment; (3) KOT-1X, HCD with KOT supplementation at 2787 mg/kg; (4) KOT-2X, HCD with KOT supplementation at 5573 mg/kg; (5) KOT-5X, HCD with KOT supplementation at 13,934 mg/kg. The vehicle treatment was the volume of solution to body weight (BW). The food intake and water consumption were monitored daily, and BW was recorded weekly.  Specific pathogen-free (SPF) male Golden Syrian hamsters (12 weeks old) were purchased from the National Laboratory Animal Center (NLAC), Taipei City, Taiwan. Animals were housed in the animal facility at National Taiwan Sport University at temperature (22 ± 1 °C) and 50% to 60% relative humidity, with a 12 h light-dark cycle (light on 7:00 a.m.). Distilled water and standard laboratory chow diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) were provided ad libitum. Before the experiments, the hamsters were acclimatized for 1 week to the environment and diet. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) approved all animal experimental protocols, and the study conformed to the guidelines of the protocol IACUC-10306 approved by the IACUC ethics committee.
The experimental design is in Figure 1. A total of 40 hamsters were randomly divided into 5 groups for treatment (n = 8/each group): (1) control, standard chow diet with vehicle (water); (2) HCD, standard chow (No. 5001) with 0.2% cholesterol and 10% lard diet with vehicle treatment; (3) KOT-1X, HCD with KOT supplementation at 2787 mg/kg; (4) KOT-2X, HCD with KOT supplementation at 5573 mg/kg; (5) KOT-5X, HCD with KOT supplementation at 13,934 mg/kg. The vehicle treatment was the volume of solution to body weight (BW). The food intake and water consumption were monitored daily, and BW was recorded weekly.

Clinical Biochemical Profiles
At the end of the experiment, after 12 h of food deprivation all hamsters were anaesthetized with 5% isoflurane at the rate of 0.5 L/min and euthanized by exsanguination after 12 h of food deprivation. Blood samples were collected from abdominal aortas. Serum was collected by centrifugation at 1500ˆg for 15 min and the clinical biochemical variables including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein (TP), blood urea nitrogen (BUN), creatinine, and glucose were measured by using the Beckman DxC 800 analyzer (Beckman Coulter, Brea, CA, USA). Hamsters were sacrificed after 6 weeks of KOT supplementation; liver, kidney, heart and epididymal fat pad (EFP) were removed and tissue weight was recorded for evaluating body composition. All tissue samples were snap-frozen and stored at´80˝C until further analysis.

Liver and Fecal Lipid Analysis
We used a metabolic cage (Muromachi Kikai, Tokyo) to collect hamster feces for analysis of fecal TG and total cholesterol (TC) levels. Fecal lipids were extracted by using chloroform-methanol (2:1, v/v) with a Bullet Blender (Next Advance, Cambridge, MA, USA). The suspension was filtered through Whatman No. 5 filter paper (Whatman, Maidstone, UK), and the solvent was aspirated, and evaporated. The residue was resuspended in 1 mL of DMSO solution. Fecal TG and TC levels were measured colorimetrically as described previously. Hepatic TG and TC were extracted by chloroform-isopropanol-NP40 (7:11:0.1, v/v) with a Bullet Blender. After centrifugation (12,000ˆg; 10 min), TG and TC levels were quantified by using a commercial enzymatic kit for TG (No. 10010303) and a kit for TC (No. 10007640) from Cayman Chemical (Ann Arbor, MI, USA).

Histological Staining of Tissues
Liver tissues were carefully removed, minced and fixed in 10% formalin. All samples were embedded in paraffin and cut into 4-µm thick slices for morphological and pathological evaluations. Tissue was stained with hematoxylin and eosin (H&E) and examined under a light microscope equipped with a CCD camera (BX-51, Olympus, Tokyo, Japan) by a veterinary pathologist.

Statistical Analysis
All data are expressed as mean˘SD (standard deviation). Statistical differences were analyzed by one-way analysis of variance (ANOVA) and the Cochran-Armitage test for trend analysis of dose-effect of KOT supplementation with use of SAS 9.0 (SAS Inst., Cary, NC, USA). p < 0.05 was considered statistically significant.

Hamster BW and Daily Intake
The growth curves for hamsters are in Figure 2. At the start of the experiment, the BW of the five groups did not significantly differ ( Table 2). During the experimental period, BW was stable and steadily increased in each group. At the end of the experiment, the BW did not differ among the groups. Therefore, the HCD did not affect BW. With KOT supplementation, the BW curve was still stable and steadily increased, with no significant differences among groups. The daily intake is shown in Table 2. The diet intake did not differ among the groups, but water intake significantly decreased in HCD-induced hyperlipidemia groups (HCD, KOT-1X, -2X and -5X) as compared with controls. This result was same as for our previous study; hamsters fed an HCD to induce hyperlipidemia showed decreased daily water intake [30]. in HCD-induced hyperlipidemia groups (HCD, KOT-1X, -2X and -5X) as compared with controls. This result was same as for our previous study; hamsters fed an HCD to induce hyperlipidemia showed decreased daily water intake [30].

Effect of Six-Week KOT Supplementation on Hepatic TG and TC Levels in Hyperlipidemic Hamsters
At the end of the experiment, liver TG content was 80.6˘6, 112˘10, 96˘7, 78˘7 and 74˘13 (mg/g liver) for control, HCD, KOT-1X, -2X and -5X groups, respectively ( Figure 4A), and was higher, by 1.40-fold (p < 0.001), with HCD alone than for controls. Liver TG content was lower, by 14.8% (p = 0.0007), 30.2% (p < 0.0001) and 33.8% (p < 0.0001) for KOT-1X, -2X and -5X groups, respectively, than with HCD alone. On trend analysis, liver TG content was dose-dependently decreased with KOT supplementation under HCD-induced hyperlipidemia (p < 0.0001). Furthermore, liver TC content was 3.51˘0.49, 11.46˘0.41, 9.22˘1.15, 6.64˘0.96 and 7.78˘1.07 (mg/g liver) for control, HCD, KOT-1X, -2X and -5X groups, respectively ( Figure 4B), and was higher, by 3.27-fold (p < 0.001), with HCD alone than for controls. Liver TC content was lower, by 19.6% (p < 0.0001), 42.0% (p < 0.0001) and 32.1% (p < 0.0001) for KOT-1X, -2X and -5X groups, respectively, than with HCD alone. On trend analysis, liver TC content was dose-dependently decreased with KOT supplementation (p = 0.0001). Accumulation of liver fat is often associated with abnormal accumulation of TGs in liver [41]. Therefore, KOT supplementation could significantly mitigate the increased liver TC and TG content induced by the HCD hyperlipidemia model. One recent study demonstrated that 9-oxo-10(E),12(Z),15(Z)-octadecatrienoic acid in tomato extract promotes fatty acid metabolism via PPARα activation in liver cells and has potential for use in the management of dyslipidemia [42]. In addition, 13-oxo-9(Z),11(E),15(Z)-octadecatrienoic acid in the extract of tomato induced PPARγ expression in adipose tissue and resulted in the regulation of adipogenesis [43]. From these previous studies, we suggest that KOT may activate PPARα and PPARγ by two different pathways to reduce TG level and increase insulin sensitization; there were activation of PPARα reduces triglyceride level and is involved in regulation of energy homeostasis and activation of PPARγ causes insulin sensitization and enhances glucose and enhances fatty acids metabolism.

Effect of Six-Week Supplementation with KOT on Fecal TG and TC Levels in Hyperlipidemic Hamsters
After the six-week KOT supplementation, we collected all hamsters' feces for analysis of fecal TG and TC levels. At the end of the experiment, the fecal TG content was 17.7 ± 3.4, 22.5 ± 6.2, 20.0 ± 4.2, 19.2 ± 5.0 and 17.8 ± 3.2 (mg/g feces) in control, HCD, KOT-1X, KOT-2X and KOT-5X, respectively ( Figure 5A). The fecal TG content of the HCD group was significantly higher, by 1.27-fold (p = 0.0415), as compared with the control. The fecal TG content was decreased by 20.9% (p = 0.044) with KOT-5X than with HCD alone. On trend analysis, KOT supplementation dose-dependently decreased fecal

Effect of Six-Week Supplementation with KOT on Fecal TG and TC Levels in Hyperlipidemic Hamsters
After the six-week KOT supplementation, we collected all hamsters' feces for analysis of fecal TG and TC levels. At the end of the experiment, the fecal TG content was 17.7˘3.4, 22.5˘6.2, 20.0˘4.2, 19.2˘5.0 and 17.8˘3.2 (mg/g feces) in control, HCD, KOT-1X, KOT-2X and KOT-5X, respectively ( Figure 5A). The fecal TG content of the HCD group was significantly higher, by 1.27-fold (p = 0.0415), as compared with the control. The fecal TG content was decreased by 20.9% (p = 0.044) with KOT-5X than with HCD alone. On trend analysis, KOT supplementation dose-dependently decreased fecal TG content under HCD-induced hyperlipidemia (p = 0.0406). Fecal TC content was 2.28˘0.51, 2.92˘0.46, 4.12˘0.69, 3.70˘0.49 and 3.58˘0.59 (mg/g feces) for control, HCD, KOT-1X, -2X and -5X groups, respectively ( Figure 5B), and was higher, by 1.28-fold (p = 0.0273), with HCD alone than for controls. Furthermore, fecal TC content was higher, by 1.41-fold (p = 0.0001), 1.27-fold (p = 0.0078) and 1.23-fold (p = 0.0224), for KOT-1X, -2X and -5X groups, respectively, than with HCD alone. Therefore, our HCD could increase both fecal TG and TC content. KOT treatment could reduce excessive fecal TG levels and increase fecal TC level excretion. A previous study demonstrated that tomatine decreased serum LDL-C level via formation of a tomatine-cholesterol complex, which was subsequently excreted in feces. Alternatively, the TG and TC content may be reduced by a diet rich in fiber, which may reduce the risk of cardiovascular disease by several mechanisms. Many studies showed significantly reduced cholesterol level associated with dietary fiber intake and cholesterol exerted by feces [44]. We previously showed that viscous flaxseed dietary fibers may be useful for lowering blood cholesterol level than fibers in the solid state [45]. KOT, in accordance with the intervention, allows for decreasing intestinal absorption of dietary lipids and also affects cholesterol homeostasis and lipid transport in the gut, which could explain the decreased hepatic and fecal TG levels with KOT supplementation.

Effect of KOT Supplementation on Biochemical Analyses at the End of the Experiment
In our study, we observed the beneficial effects of KOT on indicators of lipid-lowering capacity. We further investigated whether six-week KOT treatment had any adverse effect on other biochemical markers in hamsters. We examined the tissue-and health status-related biochemical parameters and liver tissues by histopathology (Table 4 and Figure 6). KOT supplementation for six weeks had no adverse effects. Levels of biochemical indices, including albumin, total protein (TP), blood urea nitrogen (BUN), creatinine and glucose, did not differ among groups (p > 0.05, Table 4). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity was higher, by 1.29-fold (p = 0.0183) and 1.15-fold (p = 0.0224), respectively, with HCD alone than for controls. In addition, KOT supplementation under HCD-induced hyperlipidemia could significantly decrease the serum AST, ALT and LDH activity, respectively, as compared with HCD alone. On trend analysis, serum AST and ALT levels were dose-dependently decreased (p = 0.0031, p = 0.0006) under HCD-induced hyperlipidemia with KOT supplementation. Therefore, the effect of KOT on decreasing AST and ALT activity was associated with decreased adipose tissue accumulation. The most important adverse side effects of statins (cholesterol-lowering drugs) are increased concentration of liver enzymes and muscle problems [46]. As compared to statins, KOT did not cause liver damage. Therefore, KOT supplementation could provide alternative nutrient supplementation to ameliorate the side effects of statins and has a potential effect on lowering hyperlipidemia.

Effect of KOT Supplementation on Histology at the End of the Experiment
In a previous study, the high-fat diet-induced pathological morphology of livers significantly differed among rodent species; the fat was microvesicular in hamsters and mixed (macro-and microvesicular) in mice [47]. Liver slices from our hamsters fed a normal chow diet showed a clear hepatic cord and sinusoid ( Figure 6). Significant fatty steatosis was detected in all animals of the HCD, KOT-1X, -2X and -5X groups, with hepatocytes comprising microvesicles filled with small lipid droplets, which is similar to the previous pathological observation. The degree of fatty steatosis was significantly lower in the KOT-5X than other HCD-induced hyperlipidemic groups.

Conclusions
In our study, KOT had lipid-lowering actions by decreasing serum TG and TC levels, liver TG and TC levels, fecal TG levels and serum LDL-C and LDL-C/HDL-C levels in hyperlipidemic hamsters. Six-week KOT supplementation significantly improved the hyperlipidemia syndrome in hamsters. Hamsters showed decreased cholesterol levels in serum, and the KOT effect was exerted

Effect of KOT Supplementation on Histology at the End of the Experiment
In a previous study, the high-fat diet-induced pathological morphology of livers significantly differed among rodent species; the fat was microvesicular in hamsters and mixed (macro-and microvesicular) in mice [47]. Liver slices from our hamsters fed a normal chow diet showed a clear hepatic cord and sinusoid ( Figure 6). Significant fatty steatosis was detected in all animals of the HCD, KOT-1X, -2X and -5X groups, with hepatocytes comprising microvesicles filled with small lipid droplets, which is similar to the previous pathological observation. The degree of fatty steatosis was significantly lower in the KOT-5X than other HCD-induced hyperlipidemic groups.

Conclusions
In our study, KOT had lipid-lowering actions by decreasing serum TG and TC levels, liver TG and TC levels, fecal TG levels and serum LDL-C and LDL-C/HDL-C levels in hyperlipidemic hamsters. Six-week KOT supplementation significantly improved the hyperlipidemia syndrome in hamsters. Hamsters showed decreased cholesterol levels in serum, and the KOT effect was exerted via increased fecal lipid excretion. In biochemical study, we found no gross abnormalities attributed to KOT treatment. Thus KOT may be beneficial to human health by reducing the risk of developing cardiovascular disease. Previous studies have demonstrated that the tomato glycoalkaloid tomatine lowered serum LDL-C and cholesterol levels in hamsters and mice [38,48]. The possible mechanism for reducing serum cholesterol level by KOT is inhibition of acyl-CoA:cholesterol acyl-transferase (ACAT)-1 and (ACAT)-2; ACAT-1 is located in the Kupffer cells of the liver, kidneys, and adrenal cortical cells, an important component of cellular cholesterol homeostasis [49]. We provide experiment-based evidence to support that KOT may have potential as a therapy for reducing blood lipid levels and lowering hyperlipidemic effects.