Background
Obesity is one of the most prevalent heath conditions which may foster various diseases such as dyslipidemia, insulin resistance, hypertension and increased risks of cardiovascular mortality [
1]. Being overweight is not the only problem in obesity but the accumulation of excess dietary calories into visceral fat and the release of high concentrations of free fatty acids into various organs eventually lead towards metabolic syndrome. According to the world health organization it has been defined as a “medical condition in which excess body fat is accumulated to the extent that can effect negatively on health”. It is a chronic, multifactorial and complex disease resulting from a long-term imbalance between the energy intake and expenditure, however, genetic, physiological, behavioral and environmental factors are also involved [
2]. There is presently a global epidemic of obesity found in all age groups and in both developed and developing countries. The worldwide prevalence of obesity is more than doubled between 1980 and 2014. Currently, more people die due to being overweight than being underweight in the world (WHO 2015). Hence, obesity is an increasing concern of society which reduces overall quality of life and leads to premature death. A large body of evidences also indicate that the global epidemic of obesity is fuelled by the societal factors that promote sedentary lifestyle and the consumption of high-fat, energy-dense diets for global epidemic of obesity [
3].
Furthermore, it is also found that obesity predisposes the condition of oxidative stress which further leads to the various complications like endothelial dysfunction, nonalcoholic fatty liver disease, microvascular complications and nephropathy [
4,
5]. Multiple mechanisms may be involved for oxidative stress condition due to obesity. Some of them are oxidation of fatty acid by mitochondria and peroxisome, lipid rich content diet and overconsumption of oxygen etc. Generally the obese people possesses low antioxidant defense than the normal weight people due to diminished production of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). With the increase of central obesity, the antioxidant defense further decreases in an inversely proportional fashion. Obesity is also characterized by enhanced levels of reactive oxygen species (ROS) or reactive nitrogen species (RNS). Studies showed that the hepatic inflammation caused by obesity may also promote tumor formation in dietary induced obese mice [
6].
Syzygium cumini is an evergreen tree habitat in Bangladesh, India, Eastern Africa, South America and Madagascar. Traditionally the
Syzygium cumini fruits, leaves, seeds, and bark are used in ayurvedic medicine [
7].
Syzygium cumini is used for the cure of various diseases including cough, diabetes, dysentery, inflammation and ringworm [
7]. Bark decoctions are taken for asthma and bronchitis, used as mouth wash for the astringent effect on mouth ulcerations, and spongy gums and for stomatitis [
8]. Gallic acid, ellagic acid, ellagitannins, isoquercetin, quercetin, caffeic acid, ferulic acid, guaiacol, resorcinaldimethyl ether, lignaglucoside, veratrole, β–sitosterol and palmitic acid are isolated from the seed of
Eugenia jambolana [
7].
Syzygium cumini extract showed anti-inflammatory activity in animal model [
9,
10].
Syzygium cumini extract also reduced the production of prostaglandin E
2, serotonin, and histamine in vivo [
11]. Previous report also suggests that
Syzygium cumini fruit extract possesses hypoglycemic action in diabetic rats [
12]. The seed powder of
Syzygium cumini is also reported to have hypoglycemic action in streptozotocin induced diabetic rats [
13].
Syzygium cumini extract also protected the oxidative stress by improving antioxidant enzymes in diabetic rats [
14]. Earlier report also suggests that
Syzygium cumini extract may prevent hepatic damage in carbon tetrachloride induced rats [
15]. Flavonoid-rich extract of
Eugenia jambolana seed reduced total cholesterol, LDL-cholesterol, and triacylglycerol while raised HDL-cholesterol levels in diabetic mice [
13]. A recent investigation reported that
Syzygium cumini leaves extract improves peripheral insulin sensitivity, stimulates β-cell insulin release, lowered body weight gain, body mass index, and white adipose tissue mass in monosodium glutamate induced obese rats [
16]. Gallic acid and ellagic acid were found to improve metabolic dysfunction and dyslipidemia in high fat diet fed rats [
17,
18]. However, no study has reported the anti-obesity, anti-inflammatory and anti-fibrotic role of
Syzygium cumini seeds extract in high fat diet induced obese animal. Thus the current investigation was undertaken to evaluate the effect of
Syzygium cumini seed powder supplementation in hepatic fibrosis and inflammation in diet induced obese rats.
Materials
Chemicals
The beef tallow was used as a source of high fat in the diet which was obtained from the local beef market and processed well by heating to solidify it for using in the high carbohydrate high fat (HCHF) diet formulation. Thiobarbituric acid (TBA) was purchased from Sigma Chemical Company (USA). Reduced glutathione (GSH) was purchased from J.I. Baker (USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride liquid, Cholesterol (total) liquid, LDL and HDL assay kits were obtained from DCI diagnostics (Budapest, Hungary). All other chemicals and reagents used were of analytical grade.
Plant material
Syzygium cumini seeds were collected from the local market of Dhaka, Bangladesh. Syzygium cumini seed was authenticated by Dr. Bokhtiar Uddin, Associate Professor and botanist, Chittagong University, Chittagong, Bangladesh and a catalog accession number was assigned (SBU 119). Then the seeds were undergone processes including removal of the extra pulp and drying. The dry seeds were then grinded to fine powder and mixed well with powdered chow food for using as a supplement.
Syzygium cumini seeds powder (20 g) was also used to prepare crude extract using ethanol as solvents in a Soxhlet extractor (temperature 45 °C). This ethanol extract was then used for phenolic content analysis.
HPLC detection and quantification of polyphenolic compounds
Detection and quantification of selected phenolic compounds in the ethanol extract were determined by HPLC-DAD analysis followed by as previously described method [
19]. It was carried out on a Dionex UltiMate 3000 system equipped with quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). Separation was performed using Acclaim® C
18 (5 μm) Dionex column (4.6 × 250 mm) at 30 °C with a flow rate of 1 ml/min and an injection volume of 20 μl.
Composition of foods used in this study
Two types of foods were used in this study. One of them was the normal laboratory chow food composed of mainly wheat, wheat bran, rice polishing and fish meal (Table
1). Normal chow diet contained calories as percentage, e. g. 14% proteins, 57% carbohydrates, 13.5% fat. The other type of diet was high carbohydrate high fat diet (HCHF), mainly composed of chow food, sugar, beef tallow and condensed milk. HCHF diet contained calories as percentage, e. g. 14% proteins, 37% carbohydrates, 48% fat.
Table 1
Composition of normal and high carbohydrate high fat diet used in this study (for 100 g)
Wheat | 40% | Powdered normal rat feed | 15.5% |
Wheat bran | 20% | Sugar | 17.5% |
Rice Polishing | 5.5% | Beef tallow (fat) | 20.0% |
Fish meal | 10.0% | Condensed milk | 39.5% |
Oil cake | 6.0% | Vit-B complex | 0.1% |
Gram | 0.39% | Salt | 0.5% |
Pulses | 0.39% | Water | 100 ml |
Milk | 0.38% | | |
Soybean Oil | 1.5% | | |
Molasses | 0.095% | | |
Salt | 0.095% | | |
Embavit (vitamin) | 0.1% | | |
Animals and treatment
All experimental protocols were approved by the Ethical Committee of North South University for animal care and experimentation. Twenty eight Wistar male rats (Ten to twelve weeks old, 185–200 g) were obtained from Animal production unit of Animal House at Department of Pharmaceutical Sciences, North South University and kept in individual cages at temperature controlled room with a 12 h dark/light cycles environment having free access to standard laboratory feed and water. To study the effects of high carbohydrate high fat diet and its attenuation by supplementation of jam seed, rats were randomly divided into four experimental groups (
n = 7 each), control (group I), control +
Syzygium cumini seed (group II), HCHF (Group III) and HCHF+
Syzygium cumini (Group IV). Animals of group-I were given the normal laboratory food and water every day for the whole of the study period (8 weeks). Group II received similar treatment as of group I; however, this group was supplemented with
Syzygium cumini seed powder every day for 8 weeks. Animals of group III received only HCHF treatment, however animals of group IV received both HCHF treatment for 8 weeks and
Syzygium cumini seed powder mixed in food supplementation every days (2.5% of food,
w/w). HCHF diet was prepared in our laboratory in pellet forms (Table
1). To assess the glycemic activity before and after the HCHF feeding, OGTT was performed for all four groups before and after finishing of treatment of HCHF. Measurements of body weight and food and water intakes were taken daily.
Oral glucose tolerance test
At the end of the feeding protocol, rats were kept starved overnight (12 h) and an oral glucose tolerance test was performed. Normal water was supplied during the food deprivation period. Basal blood glucose concentrations were measured in blood taken from the tail vein using (Bionim Corporation, Bedford, MA, USA). The rats were administered 2 g/kg body weight of glucose as a 40% aqueous solution via oral gavage. Tail vein blood samples were taken at 30, 60, 90 and 120 min following glucose administration.
Animal sacrifice and sample collection
At the end of 8 weeks, all animals were weighed and sacrificed under high dose pentobarbitone sodium (90 mg/kg) anesthesia. Immediately after the sacrifice, blood sample was drawn from abdominal aorta from each rats and placed in citrate buffer containing tubes. Collected blood samples were centrifuged at 8000 rpm and separated the plasma and stored in refrigerator at −20 °C for further analysis. All internal organs such as heart, kidney, spleen and liver were also harvested. Immediately after collection of the organs, they were weighed and stored in neutral buffered formalin (pH 7.4) for histological analysis and in refrigerator at −20 °C for further analysis.
Plasma biochemistry
Blood was centrifuged at 8000 rpm for 15 min within 30 min of collection into citrate buffer containing tubes. Plasma was separated and transferred to Eppendorff tubes for storage at −20 °C before analysis. Plasma concentrations of total cholesterol, triglycerides, LDL, HDL and activities of plasma alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were determined using kits supplied by Diatec diagnostic kits (Hungary) according to manufacturer-provided standards and protocols. Plasma insulin was also estimated using insulin kit obtained from Diatec diagnostic kits (Hungary) according to the manufacturer’s protocol.
Preparation of tissue sample for the assessment of oxidative stress markers
For determination of oxidative stress markers, liver tissue was homogenized in 10 volumes of Phosphate buffer containing (pH 7.4) and centrifuged at 8000 rpm for 15/30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below.
Estimation of lipid peroxidation
Lipid peroxidation in liver was estimated calorimetrically measuring thiobarbituric acid reactive substances (TBARS) followed by previously described method [
20]. Lipid peroxidation in the sample was estimated by using 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5), which was further treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 15% TCA). This solution was then taken in sealed eppendorf tube and placed in hot water bath for 15 min and cooled in room temperature. The absorbance of clear supernatant was measured against reference blank at 535 nm. MDA concentration was measured using a MDA standard curve straight-line equation. MDA concentration was expressed as nmol/mL or nmol/g tissues.
Assay of nitric oxide (NO)
Nitric oxide (NO) was determined according to the method described by Tracey et al. as nitrate [
21] using Griess reagents. In this study, Griess-Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1%
w/
v) instead of 1-napthylamine (5%). Tissue homogenates (2 mL) and phosphate buffer saline (0.5 mL) were incubated with the reaction mixture (3 mL) at 25 °C for 150 min. A pink colored chromophore was formed. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions. NO level was measure by using standard curve and expressed as nmol/ml or nmol/g of tissue.
Advanced oxidation protein products (APOP) assay
Determination of APOP level was performed by modification of the method of Witko-Sarsat et al. [
22] and Tiwari et al. [
23]. Two mL of plasma was diluted 1: 5 in PBS. Potassium iodide (0.1 mL of 1.16 M) was then added to each tube, followed by 0.2 mL acetic acid after 2 min. The absorbance of the reaction mixture was immediately read at 340 nm against a blank containing 2 mL of PBS, 0.1 mL of KI, and 0.2 mL of acetic acid. The chloramine-T absorbance at 340 nm was found linear within the range of 0 to 100 nmol/mL, AOPP concentrations were expressed as nmol·mL − 1 chloramine-T equivalents.
Catalase assay (CAT)
CAT activities were determined using previously described method by Chance and Maehly [
24]. The reaction solution of CAT activities contained: 2.5 ml of 50 mmol phosphate buffer (pH 5.0), 0.4 ml of 5.9 mmol H
2O
2 and 0.1 ml tissue homogenates. Changes in absorbance of the reaction solution at 240 nm were determined after one minute. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.
Estimation of superoxide dismutase (SOD) activity
SOD was assayed in plasma and tissue homogenates by using previously described method [
25]. Three ml reaction mixture consisted of aliquot of tissue homogenates and PBS to make up the volume to 2.94 ml. The reaction was started by addition of 0.06 ml of 15 mM epinephrine. Change in absorbance was recorded at 480 nm for one min at 15 s interval. Control consisting of all the ingredients, except tissue homogenates, was run simultaneously. One unit of enzyme activity has been defined to cause 50% inhibition of auto-oxidation of epinephrine present in the assay system.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. [
26]. Tissue homogenate (1.0 ml) was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4 °C for 1 h and then centrifuged at 1200×g for 20 min at 4 °C. The total volume of 3.0 ml assay mixture was composed of 0.1 ml tissue homogenate, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM). The yellow color of the mixture was developed which was read immediately at 412 nm on a Smart SpecTM plus Spectrophotometer and expressed as ng/mg protein.
Estimation of myloperoxidase (MPO) activity
MPO activity was determined by a dianisidine-H
2O
2 method [
27], modified for 96-well plates. Briefly, plasma sample (10 μg protein) was added in triplicate to 0.53 mM
o-dianisidine dihydrochloride (Sigma) and 0.15 mM H
2O
2 in 50 mM potassium phosphate buffer (pH 6.0). The change in absorbance was measured at 460 nm. Results were expressed as units of MPO/mg protein.
Histopathalogical determination
For microscopic evaluation liver tissues were fixed in neutral buffered formalin and embedded in paraffin, sectioned at 5 μm and subsequently stained with hematoxylin/eosin to see the architecture of hepatic tissue and inflammatory cell infiltration. Sirius red staining for fibrosis and Prussian blue staining for iron deposition were also done in liver sections. Milligan trichrome staining was done for estimation of collagen deposition. Sections were then studied and photographed under light microscope (Zeiss Axioscope) at 40X magnifications.
Statistical analysis
All values are expressed as mean ± standard error of mean (SEM). The results were evaluated by the One-way ANOVA followed by Newman- Keuls post hoc test using Graph Pad Prism Software (USA). Statistical significance was considered at p < 0.05 in all cases.
Discussion
Obesity is considered as a major health risk and is associated with various health disorders such as insulin resistance, hyperlipidemia, non-alcoholic fatty liver diseases hypertension and cardiovascular dysfunction [
28]. Recent shift of dietary behavior from low carbohydrate, high fiber diet to high fat high carbohydrate diet amongst people living in both developed and developing countries is one of the causes of obesity progression [
29]. Moreover, sedentary life style also limits the energy expenditure in both young and aged individuals [
30]. Obesity treatment is a time consuming and relatively complicated process and there is no easy solution. Moreover, very few drugs are available in the market that are approved by FDA and that should be taken with precaution due to undesirable side effects. The alternative medicine and functional food rich in high polyphenolic component and antioxidants showed promise to reduce body weight gain and related health complications [
18,
31]. In this study, we have developed an obese rat model using high fat high carbohydrate diet. These rats mimic human obesity and metabolic syndrome and showed increased body weight gain, fat deposition in peritoneal region, developed glucose intolerance and dyslipidemia. Moreover, high fat diet feeding in rats also increased oxidative stress in liver.
High fat diet feeding is associated with the development of central obesity, insulin resistance, high circulating plasma insulin concentration and non-alcoholic fatty liver [
32]. Polyphenolic compound rich food supplement offers a great benefit in obesity related complications [
32,
33]. Our investigation found that, high fat diet feeding in rats showed significant glucose intolerance and high level of plasma insulin concentration. Previous report suggests that flavonoid rich extract from
Syzygium cumini seed has hypoglycemic activity in streptozotocine induced diabetic rats [
13]. Our investigation also suggest that Gallic acid and ellagic acid rich
Syzygium cumini seed powder normalized the impaired glucose tolerance and circulating insulin concentration in high fat diet induced rats. One possible mechanism of
Syzygium cumini seed powder to be effective on postprandial blood glucose is due to inhibition of carbohydrate metabolizing-amylase and -glucosidase enzymes or improved insulin resistance in the peripheral tissues such as muscle or adipose tissues [
34‐
36].
Insulin resistance and increased plasma insulin concentration were also observed in obese individual. In this investigation, high fat diet feeding in rats was showed an increased plasma insulin concentration which was further normalized by
Syzygium cumini seed powder. This effect could be attributed to the capacity of
Syzygium cumini extract to dual up regulation of both the peroxisome proliferators-activated receptors (PPARα and PPARγ) which were previously reported in liver of streptozotocin induced diabetic rats [
13,
35]. Moreover, recent investigations have suggested a role for adipose tissue in the development of insulin resistance. In fact, free fatty acids and various adipokines released from adipose tissue have been involved in the development of insulin resistance [
28]. Thus, increased insulin sensitivity to adipose tissues would be another mechanism of improving the insulin resistance in obesity. Adiponectin is one of the adipose-specific adipokine and possesses insulin-sensitizing effects and treatment with adiponectin increases insulin sensitivity in animal models [
37,
38]. However, adiponectin concentrations are found low in obese individuals [
39]. Thus, body fat mass and insulin resistance are inversely correlated with adiponectin levels. In this study, we have not measured adeponectin level in plasma of high fat diet fed rats. Despite,
Syzygium cumini seed powder treatment resulted in lowering of fat deposit and improvement of insulin sensitivity in high fat diet fed rats. Previous studies also suggest that restoration of adeponectin level by polyphenol rich extract of
Terminalia paniculata bark prevented fat deposition in high fat diet-fed rat [
40]
. Further, lowering of peritoneal fat or total fat deposit would be beneficial in obese individual by lowering the adipose tissues derived inflammatory mediators and cytokines production [
41].
Oxidative stress is considered as the most crucial events while developing complications in diet induced obesity in rats. Oxidative stress in rats due to high fat diet feeding also increases the glucose intolerance and insulin resistance [
42,
43]. Our investigation also revealed that high fat diet increased plasma and tissues level of oxidative stress markers. The results obtained in this study demonstrate that obesity increases lipid peroxidation in hepatic tissues as expressed by increased tissue levels of MDA. High fat diet feeding in rats also increased APOP, nitric oxide level whereas decreased the antioxidant enzyme activities such as SOD and catalase. Milagro et al. showed that obesity is an independent risk factor for increasing lipid peroxidation and decreased activity of cytoprotective enzymes [
44]. Moreover, High fat diet feeding in rats decreased the glutathione concentration. Similar findings were also reported in previous studies [
42,
45].
Syzygium cumini seed powder prevented the rise of plasma oxidative stress markers and restored the antioxidant enzyme activities. Antioxidants such as gallic acid and ellagic acid present in the
Syzygium cumini seed powder may be responsible for the observed protective mechanism and antioxidant action [
18,
46].
Cellular antioxidant enzymes constitute a supportive defense against reactive oxygen species. In the present study, hepatic antioxidant activities of SOD, CAT, GPx and GSH contents were significantly decreased in HCHF diet fed rats as compared to normal diet rats. Another interesting point is that
Syzygium cumini seed powder normalized the activities of SOD, CAT, GPx and GSH content in hepatic tissue. These results demonstrate that eight weeks HF diet feeding induces oxidative stress to impair the liver tissue. Moreover, hyperlipidemia is considered a major clinical symptom associated with high fat diet feeding in rats. In our study, high fat diet feeding in rats also increased total cholesterol, triglycerides and HDL cholesterol in our study. Chronic dyslipidemia has been characterized as a major risk factor for cardiovascular risk and well as nonalcoholic fatty liver diseases [
47]. Previous studies further suggest that high fat diet-induced obesity and abnormal lipid metabolism trigger inflammation, congestion, and nonalcoholic fatty liver disease (NAFLD) leading to hepatic failure marked as boost in AST, ALT, and ALP activity in the serum [
48,
49]. Our results showed that consumption of high-fat diet induces fatty liver or hepatic steatosis in rats.
Syzygium cumini seed powder prevented the rise of plasma total cholesterol and triglyceride levels in our study.
High fat diet feeding in rats further develops inflammation, steatosis and increased fibrosis in liver. Our histological analysis confirmed the inflammatory cells infiltration in liver and lipid accumulation in hepatocytes. Hepatocyte damage and infiltrating inflammatory cells may release inflammatory cytokines and activates hepatic Kuffer cells and hepatic stellate cells (HSCs). Another finding of this study is the increased fibrous tissue accumulation in portal vein and bile duct areas. Activated HSCs are considered the main source of hepatic collagens in fibrosis [
50]. Furthermore, an association between IR and mild hepatic iron accumulation has been found particularly in patients with NAFLD. Iron deposits are found in hepatocytes, and/or Kupffer/sinusoidal cells, promoting cell damage. Our investigation also showed increased iron accumulation in liver sections of high fat diet fed rats.
Syzygium cumini seed powder supplementation ameliorated the inflammatory cells infiltration, fibrosis and iron over load in high fat diet fed rats.
Polyphenol rich extract and pure antioxidants supplementation showed beneficial effect in high fat diet fed obese animals in experimental condition [
51,
52]. Some of the phenolic antioxidants such as gallic acid and ellagic acid may prevent pre-adipocyte differentiation to adipocyte [
53,
54]. The phenolic antioxidants gallic acid and ellagic acid present in
Syzygium cumini seed powder could be responsible for the less adipose tissue deposition in high fat diet fed rats. Moreover, gallic acid may also prevent hyperlipidemia and insulin resistance in experimental animals [
17,
46]. Previous experiment also suggests that ellagic acid may also regulate fat metabolism in liver and prevent obesity and hyperlipidemia in obese rats [
18].
Acknowledgement
This research was partially supported by Research Grant (Special allocation grant), 2015-1016 to Dr. Hasan Mahmud Reza and Dr. Md Ashraful Alam, from Ministry of Science and Technology, Bangladesh. Research was conducted in the Department of Pharmaceutical Sciences, North South University, Bangladesh. The authors also gratefully acknowledge the logistic support provided by the Department of Pharmaceutical Sciences, North South University Bangladesh.