Diets high in cholesterol and saturated fats could stimulate dyslipidemia. Such diet signaled the body to produce fewer LDL receptors. A decrease in the number of LDL receptors prolong the circulation of LDL particles in the blood stream- an outcome that increase the chances that particles and cholesterol they contain will be incorporated in atherosclerotic plaque. High intake of dietary fructose exerts a number of adverse metabolic effects. The aim of the present study was to investigate whether aqueous extract of cassia fistula stem bark alleviates high-fructose diet-induced insulin resistance in rats. An attempt has been perform to treat this insulin resistance by aqueous extract of stem bark of cassia fistula. The obtained results suggest that oral administration of Cassia fistula has the ability to improve insulin sensitivity and delay the development of insulin resistance in rats, which may be used as an adjuvant therapy to patients with insulin resistance and also evaluate the effect on haematological parameter.
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Key words: Insulin resistance, cassia fistula, haematology, fructose
Cardiovascular disease, a multifactorial diseases are the principal cause of death in countries throughout the world and is posing an increasing threat to our population, infiltrating into younger age groups and poor strata of the society. Despite the progress of its prevention by the advent of newer pharmacological approaches and changes in life style, the incidence of cardiovascular disease continues to be fast increasing (Breslow, 1997; Shattuck, 1997). It is now established that hyperlipidaemia represents a major risk factor for the premature development of atherosclerosis and its cardiovascular complications. (Stokes et al., 1987). In india, as in many developing countries, most hyperlipidaemic individuals use medicinal plants as folk medicine to treat hyperlipideamia and atherosclerosis. Therefore, there is a strong interest locally to search for natural hypolipemic substances derived from medicinal plants used.
Apporimately 2/3rd of cholesterol circulating in the blood is made up in the liver. Hepatocytes synthesize cholesterol and bile acids from acetate and secrete them in bile into the intestine where they are involved in fat absorption. Fat absorbed in the form of TG-rich chylomicrons and free fatty acids are cleaved from TG by lipoproteins lipase (LPL), an enzyme on the surface of the endothelial cells. Chylomicrons remnants are taken up by hepatocytes to complete the exogenous cycle. The endogenous cycle consists of the secretion of TG rich lipoproteins particles (VLDL) that also contain cholesterol, by the liver into the blood, followed by the removal of free fatty acids by LDL in the capillaries. This results in progressive enrichment of the particles with cholesterol with an increase in their density through IDL to LDL. This circulating LDL cholesterol is especially artherogenic. LDL cholesterol particles bind to receptors located in coated pits on the surface of hepatocytes, hence the plasma concentration of LDL cholesterol is determined by a balance between LDL synthesis and hepatic uptake. LDL cholesterol consists of about 2000 molecules of cholesterol, 1000 molecules of phospholipids and one large protein on the surface called apolipoprotein B-100 (apo B-100). High levels of LDL cholesterol are so firmly established as a risk for coronary heart disease that many people refer to it as 'bad cholesterol'.
Research on medicinal plants has been increased all over the world. Various medicinal plants have been used in traditional systems, as they have potential against numerous diseases. Many tribal people in the tropical regions use plants for their medicinal needs. In some cases, a number of tropical species have yielded substances that are pharmacologically important in the manufacture of ethical drugs (Soforowo et al., 1983)
The last 25 years have witnessed a marked increase in total per capita fructose intake as a sweetener in the food industry, primarily in the form of sucrose (a disaccharide consisting of 50% fructose) and high-fructose corn syrup (Bray et al., 2004).
Today, India has a primary position in the global insulin resistance epidemiology map as it is the home of nearly 33 million insulin resistance subjects which is the highest number in the world (Ramachandran et al., 2002). As in many developing countries, most hyperlipidemic individuals use medicinal plants as folk medicine to treat hyperlipidemia and prevent atherosclerosis. Therefore, there is a strong interest, locally, in natural hypolipidemic substances derived from medicinal plants. Cassia fistula L. is a fast-growing, medium-sized, deciduous tree which is now widely cultivated worldwide as an ornamental tree for its beautiful showy yellow flowers. In traditional medicine, it is used in the treatment of hematemesis, pruritus, intestinal disorders, leucoderma, diabetes, antipyretic, analgesic, anorexia, rheumatism, jaundice and laxative (Kirtikar and Basu., 1991, Ansarullah et al., 2009).
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Abbreviations: CFAE, Cassia fistula aqueous extract;
Material and method
Triton WR-1335 (a non-ionic detergent, iso octyl polyoxy ethylene phenol, formaldehyde polymer) was obtained from SRL Lab Chemicals, Delhi. Simvastatin was used marketed brand of Micro Pharmaceuticals. Fructose was obtained from Rankem (Ranbaxy chemical) Mumbai for estimation of glucose, cholesterol and triglyceride using Nicholas enzymatic estimation kits. All other chemicals were of analytical grade and obtained locally.
The stem bark of Cassia fistula L. was collected in the blooming season July 2009, from medicinal garden of Lovely Professional University, Phagwara. The collected part was authenticated by Dr. S. S. Poonia, Department of Botany, Haryana Agriculture University, Hissar (Haryana). The bark material was washed with water, dried in the shade at room temperature after chopped in to small pieces with stainless steel knife. The stem bark material was powdered using mechanical grinder. The powdered bark was defatted with petroleum ether for 24 hrs and further extracted with water to get an aqueous extract. The extract was filtered and the filtrate was placed in the rotatory evaporator under a reduced pressure to remove the extract and until semi-solid substances were obtained. Then, the extract was placed in the desiccator until further use.
Male Albino Wister rats (140-160 g) used for the present study were procured from National Institute of Pharmaceutical Education Research (NIPER, Mohali, India). The animals were acclimatized for 7 days in our animal house (Regd. No. 954 /AC/06/CPCSEA/09/13) before dietary manipulation. They were housed two per cage in an air-conditioned room (22 Â± 20C) with 12 h light/dark cycle and had free access to standard pellet diet and water. All the procedures were performed in accordance with the Institutional Animal Ethics Committee.
Triton induced hyperlipidemia:
The animals were divided into five groups of five rats each. The animals were administered CFAE extract (250 & 500 mg/kg/day, p.o) and Simvastatin (4mg/kg/day, p.o) for seven consecutive days via intragastric tube. On 8th day, the animals were fasted for 18 hrs (had only free access to water) and Triton WR-1339 (200mg/kg) dissolved in 0.9% saline was injected intraperitoneal. Blood samples were collected from retro-orbital puncture under light ether anaesthesia at 6th and 24th hr after triton injection. Samples were centrifuged at 2500 rpm for 10 min to separate the serum, which was further used for the evaluation of total cholesterol, HDL-cholesterol and triglyceride using Nicholas enzymatic kits.
Fructose induced hyperlipidemia, insulin resistance and hyper insulinemia
The animals were divided into four groups of five rats each. Group1: Normal control (NC) - Fed with tap water and saline p.o. Group 2: 10% w/v fructose solution ad libitum in feeding bottle. Group 3: 10% fructose + CFAE (250 mg/ kg/day, p.o.) Group 4: 10% fructose +CFAE (500 mg/ kg/day, p.o.) for 30 days (Shlam et al., 2006). All the animals were fasted for half an hour prior to drug administrations. On day 31st day, the animals were anaesthetized with anaesthetic ether and blood was collected by retro orbital puncture and the serum was separated for estimation of glucose, cholesterol and triglyceride using Nicholas enzymatic estimation kits.
All the animals are fasted for half an hour prior to drug administrations. On day 31, the animals are anaesthetized with anaesthetic ether and blood is collected by retro orbital puncture and the serum is separated for estimation of glucose, cholesterol and triglyceride using Nicholas enzymatic estimation kits. Blood is also collected for haematological study.
On day 31, the animals are anaesthetized with anaesthetic ether and blood is collected by retro orbital puncture in EDTA containing anticoagulant tubes. Blood sample further used for total Cell Blood Count(CBC) . Equipment used for haematological is of semi autoanalyser, Sysmex fully Automatic Cell Counter.
Evaluation of cholesterol in fecal content
In triton induced hyperlipidemia model, collection of fecal matter was performed central and last day of study i.e 3rd and 7th day respectably. Then fecal mater dried naturally and tritrurate in motor pastel. Then weigh amount of fecal content mix with solution of ethanol : water (9:1). Cholesterol content from fecal material comes in the ethnolic layer of solution in seprating funnel. Ethanolic layer seprated study for cholesterol content in auto analyser.
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The induction of insulin resistance in rats fed with fructose-rich chow.
Wistar rats received oral administration of cassia fistula at the different dosage of extract along with fructose except control group every 24 h, daily. Plasma glucose levels were meaThe concentration of plasma glucose was measured by the glucose oxidase method, using Span Diagnostic Kit (Surath, India). Plasma triglyceride level was estimated by GPO-POD enzymatic method using the Nicolas Diagnostic kit (Mumbai, India).
All results were expressed as means Â± SEM for the number, n = 5 of animals in the group as indicated in the figures and table. To determine the statistical significance of clinical and laboratory findings t-test was used. P values of less than 0.05 were regarded as significant.
The rats treated with triton showed many folds change in mean serum lipids profile when compared with control animals at 6th and 24th hrs intervals (Table No:1 & 2). The treated group which received aqueous extract of showed a dose dependent inhibition of cholesterol as compared to those animals which administered with tritone alone (p<0.0).
Triton WR-1339, a non-ionic detergent (oxyethylated tertiary octyl phenol formaldehyde polymer), has been widely used to produce acute hyperlipidaemia in animal models in order to screen natural or chemical drugs (Schurr, Schultz, & Parkinson, 1972) and to study cholesterol and triacylglycerol metabolism (Zeniya & Reuben, 1988). The accumulation of plasma lipids by this detergent appears to be especially due to the inhibition of lipoprotein lipase activity (Hayashi, Niinobe, Matsumoto, & Suga, 1981).
Triton WR 1339 is known to reduce the influx of plasma cholesterol into the liver in rats. It also increases the biliary excretion of lysosomal enzymes and certain lipids (bile acids and phospholipids) and markedly increases serum cholesterol in experimental animal. Triton WR 1339 also accumulates in rat hepatocyte lysosomes, and this accumulation exhibits morphological characteristics that are in many respects similar to those found in lysosomal storage diseases10. Furthermore, it has been shown that Triton WR 1339 increases hydroxymethylglutaryl coenzyme A reductase (HMGCoA reductase) activity in the liver, and an inhibitor of HMG-CoA reductase, simvastatin, has been reported to decrease dolichol and ubiquinol levels in the liver of hypercholesterolemic rats. On the other hand, it has been demonstrated that another HMG-CoA reductase inhibitor (mevinolin) had only limited effects on blood dolichol, although liver dolichol levels increased after this treatment. In humans, HMG-CoA reductase inhibitors have been demonstrated to have no marked effect.
The body weights of four groups of animals during experimental period are represented in Fig. No significant variation in body weights of groups II, III and IV was observed when compared with group-I. The weight was observed from 1st day to onwards till the end of experimental period.
Several studies have demonstrated that chronic fructose feeding leads to insulin resistance, glucose intolerance, hyperinsulinmia and hypertriglyceridemia in a relatively short time in normal rats (Zavaroni et al., 1980).
Insulin resistance as a widespread feature of atherogenic diseases predisposes the affected individuals to various diseases including hypertension, dislipidemia, obesity, cardiovascular diseases and type 2 diabetes mellitus (Zheng et al., 2005; Hotamisligil, 2000). Lowering endogenous insulin levels is the primary key step to successful therapy directed at insulin resistance related diseases (Goldstein, 2002).
The development of insulin resistance in fructose fed rats is well documented in the literature (Yagi et al., 1995; Thorburn et al., 1989) and was also interpretive from our study in our laboratory (Reddy et al.,2008). Results of the present study showed high-fructose feeding in rats for 30 days leads to hyperglycemia, hypertriglyceridemia, and impaired leading to the development of insulin resistance.
Further, as fructose-induced insulin-resistant animal model has been recommended for assessing the therapeutic efficacy of insulin sensitizers and drugs that are likely to have effect on insulin sensitivity, we selected this model to study the efficacy of CFAE in preventing insulin resistance. The hyperglycemia, hypertriglyceridemia
Observed in group-II at 30th day of study. Hypertriglyceridemia after fructose feeding results from the enhanced rate of hepatic VLDL-triglyceride synthesis (Thorburn et al., 1989; Zavaroni et al., 1982) and a decrease in peripheral triglyceride clearance (Mayes, 1993). Our studies have shown that the intravenous injection of triton WR-1339 (200mg/kg body weight) into normolipidemic rats causes nearly a twofold increase of plasma cholesterol. The study shown resemblance with intravenous administration of triton WR-1339 to normolipidemic dogs. (Edelstein C et al., 1985)
Excessive fructose consumption is associated with dyslipidemia, culminating in markedly elevated lipid levels in plasma and increased fat deposition in liver. We show that high fructose feeding resulted in significant alterations in multiple pathways in hepatic metabolism. These include: functions in fatty acid transportation, VLDL-TG assembly, and cholesterol metabolism; enzymes for the accommodation of fructose catabolism in response to increased fructose influx into liver. Our data clearly indicate the beneficial effect of CFAE against fructose-induced hyperglycemia, hyperinsulinemia and hypertriglyceridemia. Although increased consumption of fructose-rich sweeteners in soft drinks is considered a contributing factor for the prevalence of obesity in industrial countries our studies support the idea of limiting excessive fructose addition in beverages to counteract the epidemic of obesity and type 2 diabetes mellitus.
Table 1A: Antihyperlipidemic effect of aqueous extract of Cassia fistula on triton induced rats (after 6 hours)
1% CMC + Triton
250mg/kg + Triton
500mg/kg + Triton
4mg/kg + Triton
The values given are MeanÂ±SEM, n=5. aChanges control vs triton control. bThe values given in the parenthesis are percentage change from triton vs CFAE-I & II, Simvastatin. cchanges control vs triton, CFAE-I& II, Simvastatin.
Table 1B: Antihyperlipidemic effect of aqueous extract of Cassia fistula on triton induced rats (after 24 hours)
1% CMC + Triton
250mg/kg + Triton
500mg/kg + Triton
4mg/kg + Triton
The values given are MeanÂ±SEM, n=5. aChanges control vs triton control. bThe values given in the parenthesis are percentage change from triton vs CFAE-I & II, Simvastatin. cchanges control vs triton, CFAE-I&II, Simvastatin.
Table: 2A Fructose induced hyperlipidemia
1% CMC + fructose
500mg/kg + fructose
The values given are MeanÂ±SEM, n=5. aChanges control vs fructose control. bThe values given in the parenthesis are percentage change from fructose vs CFAE-I & II. cchanges control vs fructose, CFAE-I&II.
Table No: 12 Effect of 30 days administration of Cassia fistula bark aqueous extract on haematological parameters of Wister Albino rats in fructose induced hyperlipidemia.
*P<0.05, **P<0.01, ***P<0.001 vs control and different fraction of aqueous extract. Values are mean Â± SEM, n = 5, t-value= 2.34 (P<0.05), 3.36 (P<0.01), 5.04 (P<0.001)
HGB- haemoglobin, RBC- red blood cells, HCT- hematocrit, MCV- mean cell volume, MCH- mean cell haemoglobin, MCHC- mean cell haemoglobin concentration, RDW- red cell distribution width
WBC- white blood cells, NEU%- neutrophils, LYM%- lymphocytes, MXD%- mixed cells, NEU#- absolute neutrophils, LYM#- absolute lymphocytes, MXD# absolute mixed cells.
PLT- platelets, PDW- platelets distribution width, MPV- mean platelets volume, P-LCR- Platelets least cell ratio.