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1502 Journal of Applied Sciences Research, 8(3): 1502-1509, 2012 ISSN 1819-544X This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLES Hypolipidemic Effect of Caffeine Beverages in Fatty Liver Injured Rats Abd El-Ghany, M.A., Rasha, M. Nagib and Hagar, M. El-Saiyed Department of Home Economics, Faculty of Specific Education, Mansoura University, Egypt. ABSTRACT The present study was designed to investigate the effects of caffeine beverages on the nutritional status and some biochemical analyses of serum and liver in hypercholesterolemic rats. Thirty-five healthy Sprague– Dawley albino rats weighing 135±5g were classified into seven groups. One was fed on standard diet and kept as control (-ve) group. The other six group injected intraperitoneal with CCl4 and fed on basal diet with lard in substitution of oil to induce fatty liver injury. The fatty liver injury rats were classified into non-treated group and caffeine, cocoa, Nescafe, coffee and black tea treated groups. Rats were sacrificed after eight weeks. Compared with non-treated group, results revealed the following: Values of final weight, weight gain, weight gain percent, food intake and food efficiency ratio significantly decreased in caffeine treated group but significantly increased in cocoa, Nescafe, coffee and black tea treated groups. Moreover, serum alanine and aspartate amino transferase, creatinine, total bilirubin, urea, alkaline phosphatase and cholesterol and albumin to globulin (A/G) ratio and atherogenic indexes significantly decreased but serum high-density lipoprotein cholesterol significantly increased in all treated groups. Serum very low-density lipoprotein cholesterol (VLDLc) and uric acid were significantly increased but serum triglyceride (TG), total lipids and phospholipids significantly decreased in caffeine and black tea treated groups. However, serum T.G, phospholipids, total lipids and low density lipoprotein cholesterol (LDLc) were significantly decreased in cocoa and coffee treated groups. Serum total protein, globulin, T.G and serum LDLc significantly decreased but serum uric acid significantly increased in Nescafe treated group. Serum T.G, total lipids,phospholipids and LDLc were significantly decreased in coffee treated group while serum globulin and LDLc significantly increased in black tea treated group. Key wards: (Caffeine, Cocoa .Nescafe, Coffee, Black tea &liver disease). Intoduction The liver disorders are a world problem. Despite its frequent occurrence, high morbidity and high mortality, its medical management is currently in adequate; no therapy has successfully prevented the progression of hepatic diseases, even though newly developed drugs have been used to treat chronic liver disorders (Bruck et al., 1996). Hepatic steatosis resulted from an increased dietary intake of fat, in addition to excess caloric intake. Oxidative stress induced by lipid metabolism may be implicated in liver fat accumulation, or steatosis, including fibrosis and necrosis leading to cirrhosis (Fisher et al., 2002 and Meijer et al., 2010). Caffeine is one of the most commonly ingested, pharmacologically active substances. It is present in coffee, tea, soda, cocoa, solid milk chocolate, and many medications. Caffeine is rapidly absorbed from the digestive tract and distributes throughout all tissues (Matissek 1997). Moreover, various mechanisms of action of caffeine like inhibition of phosphodiesterase, mobilization of calcium, binding to benzodiazepine receptors and blocking of adenosine receptors. Caffeine has significant antioxidant ability in protecting membranes against oxidative damage and has ability to quench major reactive oxygen species (Devasagayam et al., 1996). Caffeine content in coffee varies widely depending on the type of coffee bean and the method of preparation used. Certain types of tea may contain somewhat more caffeine than other teas. Besides strength of the brew, growing conditions, processing techniques and other variables also affect caffeine content (Hicks et al., 1996). Chocolate derived from cocoa beans contains a small amount of caffeine. A typical 28-gram serving of a milk chocolate bar has about as much caffeine as a cup of decaffeinated coffee, although some dark chocolate currently in production contains much as 160 mg per 100g (Smit et al., 2004). In a healthy liver, caffeine is mostly broken down by the hepatic microsomal enzymatic system. The resulting metabolites are mostly paraxanthines, theobromine and theophylline and a small amount of unchanged caffeine is excreted by urine. Roast coffee, high in lipophilic antioxidants and chlorogenic acid lactones prevent free radicals from causing cell damage. Theobromine, a methylxanthine alkaloid is widely enjoyed in coffee and cocoa beverages and as an ingredient in many Corresponding Author: Abd El-Ghany, M.A., Department of Home Economics, Faculty of Specific Education, Mansoura University, Egypt. 1503 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 prescriptions and over the counter medications such as stimulants, analgesics and diuretics (Eteng et al., 1997 and Chu et al., 2009). The present study aimed to investigate the effect of caffeine containing beverages on injured liver induced by combination of carbon tetrachloride with high fat diet in experimental rats. Materials and Methods Materials: Caffeine powder and carbon tetrachloride (CCL4) were obtained from El-Gomhoria Company for chemicals, Egypt. The rats injected intraperitoneal with 1 ml/kg body weight as a 50% vegetal oil solution once a week for two months to induce liver injury. Cocoa, pure Nescafe, coffee and black tea were purchased from the local market, EL Mansoura city, Egypt. BioMeriuex Kits were purchased from Alkan Co. for Chemicals and Biodiagnostics (Dokki, Egypt). The basal diet was prepared according to NRC (1995). Thirty-five healthy Sprague–Dawley albino rats weighing 135±5g were purchased from the Agricultural Research Center, Giza, Egypt. Rats were kept under observation for 5 days before experiment and fed on the standard diet and water ad libitum. Methods: 1- Preparation Of Caffeine Products Extracts: Caffeine, cocoa, Nescafe, coffee and black tea extracts were prepared by dissolving 1gm of caffeine in 100 ml of boiling distilled water (Noori et al., 2009). Each rat administered 1 ml of these extracts daily using a stomach tube. 2- Biological Method: The animals were kept under observation for five days and fed on standard diet before the start of the experiment for adaptation. Food and water was provided ad-libtum. Five rats served as normal control and fed on basal diet. The other rats injected intraperitoneal with CCl4 and fed on the basal diet but the oil of the basal diet and 25% of the starch were replaced by lard to induce fatty liver injury (Moritz and Pankow 1989 and Abd El-Ghany and Nanees 2010). The fatty liver injured rats were classified into non-treated group and treated groups with caffeine, cocoa, Nescafe, coffee and black tea extract. Food intake was recorded daily and body weight of rats was measured once weekly. After two months, rats were sacrificed after overnight fasting. Blood samples were immediately collected from each rat and centrifuged to obtain serum for estimation of some biochemical parameters. Liver for every rat was collected and immersed in 10 % neutral buffered formalin as fixative and then sent to Pathological Department of Veterinary Medicine, Cairo University for histopathological examination (Bancroft et al., 1996). Serum alanine and aspartate amino transferase (ALT& AST) and alkaline phosphatase (ALP) enzymes activities were performed according to Reitman Frankel (1957) and Kind and King (1954), respectively. Serum total protein and albumin (A) were determined according to Henry (2001) and Eastham, (1976), respectively. Moreover, serum uric acid, creatinine, total bilirubin and urea were determined according to Barham and Trinder (1972), Bonsens and Taussky (1984), Jendrassik (1938) and Patton and Crouch (1977), respectively. In addition, serum cholesterol (CHO), triglyceride (TG) and high-density lipoprotein cholesterol (HDLc) were measured according to Richmond (1973), Buccolo and David (1973), Grodon and Amer (1977), respectively. Food efficiency ratio (FER) was calculated according to Chapman et al., (1950). Serum globulin (G), phospholipids and A/G ratio were determined according to Coles (1974), Ketes (1972) and Friedwald et al., (1972), respectively. Low-density lipoprotein cholesterol (LDLc) and very low-density lipoprotein cholesterol (VLDLc) were calculated according to Lee and Nieman (1996) while athrogenic indexes (cholesterol /HDLcor LDLc/HDLc) were calculated according to Castelli and Levitar (1977). 3- Statistical Analysis: All the obtained data were statistically analyzed by SPSS computer software. The calculated occurred by analysis of variance ANOVA and follow up test LSD by SPSS ver.11 (Artimage and Berry 1987). 1504 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 Results: Non-treated or caffeine, cocoa, Nescafe , coffee and black tea treated groups showed a significant decrease in final weight, weight gain, weight gain percent , food intake and FER at p<0.05, 0.01 &0.001 compared with normal control group. The caffeine treated group showed a significant decrease in final weight, weight gain, weight gain percent, food intake and FER compared with non-treated group. Cocoa, Nescafe , coffee and black tea treated groups showed a significant increase in final weight, weight gain, weight gain percent and FER compared with nontreated group as shown in table (1). Table 1: Body weight gain, food intake and FER of the experimental rat groups. Variables Initial weight(g) Final weight(g) Weight Weight Food Groups gain(g) gain% intake(g) 195.85+ 60.28+ 44.48+ 19.58+ Normal control 135.57+ a a a a 1.52 2.25 2.09 1.15 a 1.71 Non- Treated 136.32+ 156.60+ 20.27+ 14.88+ 15.65+ 1.76 a 1.69 c** 2.44d*** 1.91 d*** 1.17 b** Caffeine 135.85+ 137.05+ 11.20+ 8.92+ 13.70+ 1.95 a 1.91d*** 2.95e*** 2.46 e*** 1.19 c** Cocoa 134.54+ 166.82+ 32.28+ 24.03+ 16.68+ 3.19 a 1.91 b* 2.12c*** 2.12 c*** 1.19 b* Nescafe 135.60+ 165.21+ 29.61+ 21.85+ 16.52+ 1.72 a 1.47 b* 1.90c*** 1.60 c*** 1.14 b* Coffee 133.28+ 174.28+ 41.01+ 30.77+ 17.42+ 1.79 a 1.79 b* 1.91 b** 1.73 b** 1.17 b* Black tea 133.85+ 168.14+ 34.28+ 25.62+ 16.81+ 1.95 a 1.95 b* 1.79c*** 1.55 c*** 1.19 b* * ** *** Significant with control group p< 0.05 P< 0.01 P< 0.001 Mean values in each column having different subscript (a, b, c, d, ) are significantly different at P<0.05 FER 0.051+ 0.003 a 0.021+ 0.002 cd*** 0.013+ 0.001 e*** 0.032+ 0.001 b** 0.029+ 0.002 b** 0.039+ 0.001 b** 0.033+ 0.002 b** Non- treated group, caffeine, and coffee treated groups showed a significant increase in serum AST, ALT and ALP at p< 0.01&0.001 while cocoa and Nescafe treated groups showed a significant increase in serum AST and ALP at p< 0.01&0.001. Moreover, black tea treated group showed a significant increase in serum AST and ALT at p< 0.01 compared with normal control group. All treated groups showed a significant decrease in serum AST, ALT and ALP compared with non-treated group as shown in table (2). Table 2: AST, ALT and ALP enzymes of the experimental rat groups. Variables AST ALT Groups (U/L) U/L Normal control 44.91+ 38.30+ 5.23 d 2.44 c d Non- Treated 185.81+ 125.37+ 11.06 a*** 11.33 a*** Caffeine 80.35+ 51.28+ 8.08 b** 5.24 b** Cocoa 75.81+ 45.67+ 7.31 c** 5.18 c Nescafe 84.82+ 41.31+ 8.24 b** 3.39 c Coffee 97.76+ 50.60+ 9.18 b** 3.65 b** Black tea 94.90+ 52.18+ 9.45 b** 5.57 b** * ** *** Significant with control group p< 0.05 P< 0.01 P< 0.001 Mean values in each column having different subscript (a, b, c, d, ) are significantly different at P<0.05 ALP U/L 105.34+ 6.97 e 370.74+ 11.67 a*** 223.65+ 13.63 c*** 185.40+ 16.34 d** 251.60+ 19.66 b*** 232.52+ 14.78 bc*** 117.10+ 9.07 e Non- treated group showed a significant decrease in serum albumin and significant increase in A/G ratio at p< 0.05 while Nescafe treated group showed a significant decrease in serum total protein, albumin and globulin at p< 0.05. However, black tea treated group showed a significant increase in serum globulin and significant decrease in A/G ratio at p< 0.01 compared to normal control group. Cocoa treated group showed a significant increase in serum albumin but Nescafe treated group showed a significant decrease in serum total protein and globulin while black tea treated group showed a significant increase in serum globulin compared with non-treated group. All treated groups showed a significant decrease in A/G ratio compared with non-treated group as shown in table (3). 1505 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 Table 3: Total protein, albumin, globulin and albumin to globulin ratio (A/G) of the experimental rat groups. Variables T. Protein Albumin Globulin Groups (g/dl) (g/dl) (g/dl) Normal control 7.90+ 4.15+ 3.75+ a a 1.46 0.72 0.73 b Non- Treated 7.48+ 3.53+ 3.94+ 1.52a 0.24 b* 0.74 b Caffeine 7.44+ 3.72+ 3.72+ 1.29 a 0.22 ab 0.30 b Cocoa 7.84+ 4.09+ 3.75+ 1.55 a 0.37 a 0.48 b Nescafe 5.54+ 3.54+ 2.00+ 1.65 b* 0.83 b* 0.60 c* Coffee 7.22+ 3.70+ 3.51+ 0.69 a 0.36 ab 0.56 b Black tea 8.35+ 3.61+ 4.74+ 1.51 a 0.64 ab 0.96 a** * ** *** Significant with control group p< 0.05 P< 0.01 P< 0.001 Mean values in each column having different subscript (a, b, c, d, ) are significantly different at P<0.05 A/G ratio 1.16+ 0.33b 2.18+ 0.86 a* 1.14+ 0.56 b 1.23+ 0.49 b 1.71+ 0.74 b 1.07+ 0.19 b 0.82+ 0.46 c** In comparing to normal control group, the non-treated group showed a significant increase in serum creatinine, total bilirubin and urea at p< 0.01&0.001 but showed a significant decrease in serum uric acid at p< 0.05 while caffeine treated group showed a significant increase in serum total bilirubin and urea at p< 0.01. Cocoa treated group showed a significant decrease in serum uric acid p< 0.05 but Nescafe treated group showed a significant increase in serum total bilirubin and urea at p< 0.01. Coffee treated group showed a significant decrease in serum uric acid at p< 0.05 and a significant increase in total bilirubin at p< 0.05 but black tea treated group showed a significant increase in serum uric acid, total bilirubin and urea at p< 0.01. All treated groups showed a significant decrease in serum creatinine, total bilirubin and urea while caffeine, Nescafe and black tea treated groups showed a significant increase in serum uric acid compared with nontreated group as shown in table (4). Table 4: Creatinine, uric acid, total bilirubin and urea of the experimental rat groups. Variable Creatinine Uric Acid T.Bilirubin Groups (mg/dl) (mg/dl) (mg/dl) Normal control 0.80+ 1.75+ 0.29+ c b 0.11 0.95 0.08 d Non- Treated 3.66+ 2.73+ 1.57+ 2.01 a** 0.28 c* 0.15 a*** Caffeine 0.84+ 1.70+ 0.59+ 0.16 c 0.12 b 0.15 c** Cocoa 0.88+ 1.11+ 0.26+ 0.13 c 0.55 c* 0.08 d Nescafe 1.00+ 1.68+ 0.40+ 0.21 bc 0.68 b 0.12 c** Coffee 0.84+ 1.35+ 0.36+ 0.13 c 0.46 c* 0.05 c* Black tea 0.81+ 1.35+ 0.73+ 0.13 c 0.26 a** 0.17 b** Significant with control group *p< 0.05 ** P< 0.01 ***P< 0.001. Mean values in each column having different subscript (a, b, c, d, ) are significantly different at P<0.05 Urea (mg/dl) 26.10+ 4.77 c 94.90+ 1.94 a*** 43.05+ 7.11 b** 25.94+ 4.43 c 47.40+ 6.84 b** 29.88+ 3.04 c 48.10+ 1.73 b** In comparing to normal control group, the non-treated group showed a significant increase in serum triglyceride , total lipids and phospholipids at p< 0.001 while caffeine treated group showed a significant increase in serum T.G and VLDLc at p< 0.05&0.001 and a significant decrease in serum total lipids and phospholipids at p< 0.001. Cocoa treated group showed a significant decrease in serum total lipids at p< 0.05 Nescafe treated group showed a significant decrease in serum T.G at p< 0.01 and a significant increase in serum total lipids and phospholipids at p< 0.001 but coffee treated group showed a significant decrease in T.G, total lipids and phospholipids at p< 0.05&0.01. Black tea treated group showed a significant increase in serum T.G and VLDLc at p< 0.001&0.01and a significant decrease in phospholipids at p< 0.05. Caffeine and black tea treated groups showed a significant increase in serum VLDLc and a significant decrease in serum T.G, total lipids and phospholipids but Nescafe treated group showed a significant decrease in serum T.G. Cocoa and coffee treated groups showed a significant decrease in T.G, total lipids and phospholipids compared with non-treated group as shown in table (5). 1506 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 Table 5: Serum T.G, VLDLc, total lipids and phospholipids of the experimental rat groups. Variables T.G VLDLc Total lipids Groups (mg/dl) (mg/dl) (mg/dl) Normal control 29.5+ 5.90+ 527.20+ 3.03 d 2.60 c 96.57 b Non- Treated 128.05+ 7.35+ 785.30+ 5.89 a*** 4.37 c 113.27a*** Caffeine 36.75+ 26.10+ 438.10+ 2.10c* 7.89a*** 97.19c*** Cocoa 31.00+ 6.20+ 490.65+ 3.40 cd 2.28 c 95.67 c** Nescafe 16.00+ 3.20+ 769.00+ 3.61 e** 1.92 c 117.31a*** Coffee 22.00+ 4.40+ 398.10+ 4.40 e** 2.88 c 133.67d** Black tea 92.50+ 18.50+ 546.00+ 5.59 b*** 1.11 b** 98.53 b Significant with control group *p< 0.05 ** P< 0.01 ***P< 0.001. Mean values in each column having different subscript (a, b, c, d) are significantly different at P<0.05 Phospholipids (mg/dl) 413.70+ 21.58 b 642.85+ 27.82a*** 191.35+ 54.09d*** 379.25+ 20.18 bc 668.00+ 24.11a*** 301.40+ 34.79 c* 326.30+ 8.02 c* In comparing to normal control group, the non-treated group and caffeine treated group showed a significant increase in serum CHO, LDLc and atherogenic indexes (CHO/HDLc& LDLc/HDLc) at p< 0.05,0.01&0.001 and a significant decrease in serum HDLc at p< 0.05&0.01. Nescafe treated group showed a significant decrease in serum HDLc at p< 0.05 and a significant increase in serum LDLc and LDLc/HDLc at p< 0.05 while black tea treated group showed a significant increase in serum CHO, LDLc and atherogenic indexes (CHO/HDLc& LDLc/HDLc) at p< 0.05, 0.01&0.001. All treated groups showed a significant decrease in serum CHO and atherogenic indexes (CHO/HDLc& LDLc/HDLc) and a significant increase in serum HDLc compared with non-treated group. Cocoa, Nescafe and coffee treated groups showed a significant decrease in serum LDLc but black tea treated group showed a significant increase in serum LDLc compared with non-treated group as shown in table (6). Table 6: Serum CHO, HDLc, LDLc and atherogenic indexes (CHO/HDLc& LDLc/HDLc) of the experimental rat groups. Variables Groups CHO (mg/dl) HDLc (mg/dl) LDLc (mg/dl) CHO/HDLc LDLc/HDLc Normal control 84.00+ 40.69+ 17.50+ 2.07+ 0.43+ 9.59 c 1.48 a 1.09 d 0.29 c 0.25 d Non- Treated 185.70+ 20.21+ 38.90+ 9.24+ 1.96+ 4.14a*** 1.86 c** 21.64b** 0.78 a*** 1.44 a*** Caffeine 116.25+ 32.52+ 37.95+ 3.57+ 1.16+ 11.01 b* 1.96 b* 5.15 b** 0.20 b* 0.17 b** Cocoa 80.40+ 38.33+ 15.85+ 2.09+ 0.41+ 11.26 c 1.57 a 11.23 d 0.28 c 0.28 d Nescafe 85.00+ 33.76+ 28.00+ 2.52+ 0.82+ 14.59 c 1.75 b* 3.06 c* 0.41 bc 0.54 c* 36.71+ 16.50+ 2.04+ 0.44+ Coffee 74.70+ 2.12 a 1.08 d 0.43 c 0.14 d 14.23 c Black tea 128.20+ 36.68+ 54.10+ 3.49+ 1.47+ 4.81 b* 1.48 a 4.03a*** 0.11 b* 0.07 b** Significant with control group *p< 0. 05 ** P< 0.01 ***P< 0.001. Mean values in each column having different subscript (a, b, c, d, ) are significantly different at P<0.05 Histopathological Examination Of Liver: Microscopically, liver of rat from the normal group revealed the normal histological structure of hepatic lobule (Pict.1). However, Examines sections from the non-treated group revealed kupffer cells activation, congestion of hepatic sinusoids and presence of small vacuoles in the cytoplasm of hepatocytes (Pict.2). Liver of rat from caffeine group showed congestion of central vein (Pict.3). However, liver of rat from cocoa group showed no changes except kupffer cells activation (Pict.4). Examined liver from Nescafe group showed slight congestion of hepatic sinusoids (Pict.5) while liver of rat from coffee group showed slight Kupffer cells activation (Pict.6). Examined liver of rat from black tea group revealed no changes except slight dilation of hepatic sinusoids (Pict 7). 1507 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 1 2 3 4 5 6 7 Discussion: Many authors could explain the obtained nutritional results. Bukowiecki et al., (1983) and Nakabayashi et al., (2008) recorded that the addition of caffeine could promote increased intake of diet beverages, and could thereby exacerbate the increases in energy intake and body weight gain associated with increased consumption of non-caloric sweeteners. On the other hand, there is evidence that caffeine can suppress food intake, body weight gain and adiposity in rodents. Many studies have focused on the biochemical effects of caffeinated beverage. Coffee consumption may mediate levels of gut peptides (glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1), hormones intimately involved in the regulation of satiety and insulin secretion. Finally, coffee may have prebiotic-like properties, altering gut flora and ultimately digestion. Chronic administration of green tea or caffeine decreases in fat stores and increases in muscle mass of mice (Michna et al., 2003 and Tunnicliffe and Shearer 2008). long-term feeding of orange peel extract, black tea extract and caffeine is beneficial for the suppression of high-fat diet-induced obesity, and that their effects may be attributed to the inhibition of adipose tissue formation and reduction of adipose tissue mass (Huang et al., (2009). It is well documented that green tea and coffee are natural products and considered as powerful antioxidant, chemoprotective, antiinflammatory and antitumorigenic agent. Administration of coffee and green tea not only reversed the pathological effects of CCl4 but also counteracted on deleterious effects of CCl4 - induced liver injury by protecting liver cells and triggering immune system (Yang et al., 1999 and Noori et al., 2009). Black tea (Camellia sinensis) extracts could protect liver injury from chronic ethanol toxicity with or without high-fat diet supplement (Wang et al.,2000).Cocoa powder caused significant reductions in the total cholesterol, lowdensity lipoprotein cholesterol, triglycerides, red blood cells and significant increases in white blood cells and platelets of rats (Abrokwah et al., 2009). Cocoa polyphenols (flavanols) have been reported to have a wide range of biological properties including modulating eicosanoid synthesis, increasing nitric oxide synthesis, lowering the rate of LDL-cholesterol oxidation, inhibiting platelets activation and stimulating the production of anti- inflammatory cytokines (Waterhouse et al., 1996 and Wollgast and Anklain 2000). Several researchers have suggested that lower plasma LDL and higher HDL cholesterol in dialyzed coffee drinkers may occur because of a direct influence of coffee ingredients on serum lipid profile. Coffee drinkers 1508 J. Appl. Sci. Res., 8(3): 1502-1509, 2012 showed a more favorable body composition lower fat body mass and better protein nutrition as compared with non drinkers of coffee (Grzegorzewska et al., 2009). However, the diterpenes cafestol and kahweol, present in coffee, have a cholesterol-raising effect because of their action as ligands for farnesoid and pregnane receptors in the liver (Ricketts et al., 2007). Coffee prepared by Italian methods does not alter the cholesterolaemic and lipoproteinaemic profile in young men, reinforcing the hypothesis that the brewing method is a determinant factor in the association between coffee consumption and blood cholesterol increase Caffeine reduces the transintestinal absorption of hydrophobic substances such as cholesterol and fatty acids in rats (Amicis et al., 1996). The obtained nutritional and biochemical results were going in parallel with histopathological results that were confirmed by many researchers. 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