الفهرس 
-Definitions of obesityOnly 14 pages are availabe for public view
 |  | from | 279 |  |  |
| | | from | 279 | | |
Abstract
Obesity is a worldwide epidemiologic syndrome characterized by fat mass accumulation, mainly visceral fat that has an adverse effect on health, leading to reduced life expectancy and/or increased health problems such as type 2 diabetes, cardiovascular diseases and several types of cancer. Many strategies are used to treat obesity, the inhibitory effects of phytochemicals on pancreatic lipase have been extensively recommended as a treatment for obesity, as well as modulating gut microbiota diversity. Multiple phytochemicals combinations due to synergistic activity, affect multiple molecular targets and is recommended for obesity management.
The present study was conducted to investigate the effect of oral administration of cherry and/or pomegranate juices on metabolic disturbances, oxidative stress and inflammatory reactions associated with obesity in rats.
A total number of sixty adult male Wistar Albino rats, weighing 180 ± 5 g were used in this study. Rats were randomly divided into two main sections, group one; 10 rats were fed with balanced diet according to AIN-93 M and received water ad libitum. While section two; 50 rats were fed on a high fat high sucrose (HFHS) diet (for induction of obesity) and received water ad libitum. Induction of obesity was confirmed by calculation of lee index as cubic route body weight (g) divided by the length (cm); where > 0.30 considered obese and BMI; calculated as body weight (g) divided by the square of the anal-nasal length (cm); where > 0.68 considered obese. After the induction of obesity (extended for 6 weeks), rats in section 2 continued to receive (HFHS) and further divided into five subgroups; 2, 3, 4, 5&6 with 10 rats each.
The experimental groups were as follow:
group 1 (- ve control group): Healthy rats fed on the balanced diet and administrated 1 ml distilled water (p.o.) (daily).
group 2 (+ ve control group): Obese rats fed on the HFHS diet and administrated 1 ml distilled water (p.o.) (daily).
group 3 (Cherry group): Obese rats fed on the HFHS diet and administrated 1 ml cherry juice (p.o.) (daily).
group 4 (Pomegranate group): Obese rats fed on the HFHS diet and administrated 1 ml pomegranate juice (p.o.) (daily).
group 5 (Mix group): Obese rats fed on the HFHS diet and administrated 1 ml Mix juice (p.o.) (daily).
group 6 (Chromium group): Obese rats fed on the HFHS diet and administrated 1 ml chromium (8 μg Cr/day) (p.o.) (daily).
The animals were weighed weekly to monitor body weight changes as well as feed efficiency ratio (FER) and feed conversion ratio (FCR) were calculated. In addition to, final lee index and BMI were measured. After six weeks, rats were fasted for 12 hours, the animals were sacrificed under ether anesthesia and blood samples were collected from hepatic portal vein. Serum was separated and part of serum was immediately used for determination of glucose level by colorimetric methods. The remaining samples were kept at -20°C until other biochemical analysis. Liver tissue was stored at -20°C for estimation of biochemical parameters. While those for molecular assays were stored at -80 °C until use for gene expression measurement using the real time PCR technique. Fresh fecal samples were used for apparent digestibility measurements and GC-mass analysis of SCFAs. While some fecal samples are stored at -80 °C for Firmicuties and Bacteriodetes analysis by using PCR technique. White adipose tissue (visceral) was preserved in 10% formalin solution for microscopic examination.
The following analysis were performed:
• Phytochemicals analysis of fruits juices included: Quantitative determination of total polyphenols, flavonoids and anthocyanins as well as qualitative screening of phytochemical components of cherry and pomegranate juices by gas chromatography mass spectrometry (GC-MS) analysis.
• Anthropometrical and biological measurements included: body weight change (g), length in cm (from nose to anus) and calculation of final body mass index (BMI) in g/cm2 and final lee index in g/cm as well as food intake, feed efficiency ratio and feed conversion ratio.
• Fecal analysis including:
-Apparent digestibility measurements of fecal dry weight, pH and total lipids.
-Gas chromatography mass spectrometry (GC-MS) analysis of short chain fatty acids in rat fecal samples.
-Microbial analysis (Bacteroidetes and Firmicutes) of rat fecal samples by using PCR technique.
• Serum analysis including:
-Determination of serum adipokines (visfatin and leptin) and inflammatory biomarkers (IL-6, calprotectin and afamin) levels.
-Determination of the activities of serum pancreatic lipase and α-amylase enzymes.
-Determination of serum glucose homeostasis by measuring serum glucose and insulin levels as well as the calculation of homeostatic model assessment of insulin resistance (HOMA-IR).
-Lipid profile analysis as determination of serum triacylglycerol’s (TAGs), total cholesterol (TC) and high-density lipoprotein cholesterol level (HDL-c). In addition to calculation of very low-density lipoprotein cholesterol (VLDL-c) and low-density lipoprotein cholesterol (LDL-c) concentration, atherogenic coefficient and atherogenic risk factor were calculated.
• Liver tissue measurements including:
-Genetic expression analysis of carbohydrate and lipid metabolic regulatory transcriptional factors: peroxisome proliferator activated receptor gamma (PPAR-γ), sterol regulatory element binding protein (SREBP) and their house keeping gene; β-actin in liver tissue by using real time PCR technique.
-Evaluation of the oxidative stress status in liver by measuring malondialdehyde (MDA) and reduced glutathione (GSH) concentrations.
• Microscopic examination of white adipose tissue (WAT) and measuring adipocyte cell size.
The results of present study can be summarized as follow:
1) Phytochemicals analysis of fruit juices:
Phytochemicals and antioxidants analysis data showed that 1gram of cherry juice contains 47.35mg mg gallic acid and 41.73 mg catechin while 1 gram of pomegranate juice contains 56.43 mg gallic acid and 34.44 mg catechin which are equivalent to total phenols and flavonoids respectively. In addition, total anthocyanins percentage in cherry and pomegranate juice was 73.65 and 31.14 %, respectively. While gas chromatography-mass spectrometry (GC-MS) qualitative screening of phytochemicals in cherry juice sample revealed the presence of catechin, epicatechin, rutin, resveratrol, cyanidine-3 glucoside, apigenin, genistein, quercetin, kaempferol, peonidin, melatonin, caffeic acid, ferulic acid and gallic acid and pomegranate juice sample revealed the presence of punicalagin, pyrogallol, quercetin, catechin, epicatechin, chlorogenic acid, chatechol, P-OH-benzoic acid, ellagic acid, P-coumaric acid, ferulic acid, gallic acid, caffeic acid and ellagitannins.
2) Anthropometrical and biological measurements:
Obesity caused significant increase in the body weight, lee index, and BMI. Meanwhile, administration of juices as well as chromium decreased body weight gain, lee index, and BMI with the highest effect was shown in mix juice and chromium administrated group. In addition to, food intake and feed efficiency ratio was significantly increased in obese group, feed conversion ratio was significantly decreased. On the other hand, juices and chromium administered groups showed significant decreases in food intake and feed conversion ratio while, a significant increase in feed efficiency ratio. Moreover, relative weight of liver was significantly increased in obese control group. While, there were no significant change between treated groups and obese control.
3) Fecal analysis:
3.1. Apparent digestibility measurements of fecal dry weight, pH and total lipids:
High fat high sucrose diet affected fecal data. Moreover, juices administration showed an increased fecal weight and fecal lipid excretion that were associated with low fecal pH due to inhibitory effect of its components on pancreatic lipase. The fecal weight and fat content were found to be the highest in the pomegranate group, followed by mix group, cherry group then, chromium group.
3.2. Fecal intestinal microbiota (Firmicutes and Bacteriodetes) abundance and fecal SCFAs concentration:
Obese control group showed an increased Firmicutes to Bacteroidetes ratio, while administration of juices modulated this ratio thus modulated the gut microbiota of obese rats. Moreover, cherry administration has the better effect on modulating bacterial diversity while, chromium showed little or no effect on Firmicutes to Bacteroidetes ratio. Moreover, the acetic acid, butyric acid, and total SCFAs levels in juices administered groups were found to be significantly increased relative to the obese control group levels, while the levels of these fatty acids in the chromium group did not differ significantly from those in the obese control group. Also, it’s clear that, changes in the gut microbiota modulate SCFAs production which may be responsible for the lowest fecal pH. Modulating SCFAs level decreased systemic and local inflammation caused by obesity.
4) Serum analysis:
4.1. Serum interleukin-6, calprotectin, afamin, visfatin and leptin analysis:
It was noticed that elevation in interleukin-6, calprotectin as well as afamin and visfatin serum levels in obese rats indicating chronic inflammatory condition. In addition to, leptin resistance observed by elevated serum leptin level which was in line with increased BMI and increased visceral adipose tissue mass. Whereas, anti-inflammatory effect observed by significant reduction in interleukin-6, calprotectin, afamin serum levels as well as serum visfatin and leptin levels. The most significant decrease was shown in Mix group. The anti-inflammatory and anti-obesity effects of juices were due to their bioactive components especially anthocyanins. It caused a reduction in adipocyte size and decreased inflammatory cytokines and adipokines.
4.2. Serum pancreatic lipase and α-amylase enzyme activities:
The present study indicated the powerful role of bioactive components of juices in inhibiting pancreatic lipase and α-amylase thus decreased serum activities of both enzymes. The decreased lipase activity in juices administered groups was in line with increased fecal fat excretion as revealed before. Results showed juices effect was better than chromium effect.
4.3. Serum glucose homeostasis measurements:
Obesity caused a disruption in glucose homeostasis shown as hyperinsulinemia, hyperglycemia and elevated insulin resistance value while juices and chromium administration reversed these changes. However, results showed that the effect of chromium on treatment of insulin resistance was more powerful than juices. While, Mix group followed by pomegranate group showed a marked decrease in serum glucose, serum insulin and HOMA-IR, values which enhanced glucose utilization and insulin sensitivity in obese rats.
4.4. Serum lipid profile analysis:
It is clear that, obesity was associated with dyslipidemia; elevation in serum levels of TAGs, TC, LDL-c and VLDL-c, atherogenic coefficient and atherogenic risk factor, with significant decrease in HDL-c level. Meanwhile, juices and chromium administration significantly reduced atherogenic indices and reversed the disruption in lipid profile showing a hypolipidemic effect. Anthocyanins regulated lipid metabolism by suppressing fatty acid synthesis related genes and reducing visceral fat deposition as well as inhibiting lipid absorption.
5) Liver tissue measurements:
5.1. Antioxidant status and lipid peroxidation measurements:
Obesity was associated with increased ROS production and oxidative stress as demonstrated by increased MDA and decreased GSH levels. The current study, indicated that juices administration prevented lipid peroxidation caused by obesity; juices administration caused significant decrease in MDA level, boosted antioxidant defense by increasing GSH level, thus protecting liver and other tissues against oxidative stress and ameliorated metabolic dysfunctions caused by high level of lipid peroxides.
5.2. Gene expression of hepatic sterol regulatory element binding protein (SREBP) and peroxisome proliferator activated receptor (PPAR-γ) measurements:
As observed by the present results that obesity caused up-regulation of SREBP and down-regulation of PPAR-γ. while Anthocyanins decreased hepatic de novo lipogenesis by downregulating SREBP gene expression that regulates fatty acid and cholesterol synthesis and upregulating PPAR-γ gene expression that regulates insulin sensitivity and fatty acid oxidation and act in preventing lipid accumulation. The mix and chromium group were the most significant as compared to other juices groups.
6-Microscopic examination and measuring adipocyte cell size:
Obesity was associated with hypertrophy of the adipocytes, microscopic examination and measuring adipocyte size showed the largest expansion of adipocyte in positive control group. On the other hand, normal adipocytes distribution with regular sizes of cells of the rats was shown in the healthy control group. While mean adipocyte diameter was small in juices and chromium administered groups, the least expansion and small adipocyte diameter were observed in pomegranate and chromium groups. Visceral fat accumulation observed in obese group was a predictor of insulin resistance and dyslipidemia, polyphenols decreased hepatic and adipose tissue fat accumulation preventing visceral adipocyte hypertrophy.
In conclusion, administration of fresh cherry and pomegranate juices as well as their Mix. juice ameliorated the metabolic disturbances associated with obesity induced by HFHS diet by modulating lipogenic genes, gut dysbiosis and inhibiting lipid absorption. However, chromium effect was better than juices in some parameters, but juices affected many pathways that resolved obesity related complications without side effects.