Abstract
Escins, a triterpene glycoside mixture obtained from the ethanol extract of Aesculus hippocastanum L. (Hippocastanaceae) seed, was evaluated for its in vivo effects on the plasma levels of some hormones (leptin, insulin, FT3, FT4) and biochemical parameters (glucose, triglyceride, total cholesterol, HDL-C, LDL-C concentrations) in mice fed with a high fat diet for 5 weeks. A high fat diet induced a remarkable increment in the plasma leptin (p <0.01), total cholesterol (p <0.01) and LDL-C (p <0.001) concentrations compared to control group animals. Combined administration of a high-fat diet with escins decreased leptin (31.6%) (p<0.05) and FT4 (36.0%) (p<0.05) levels, increased HDL-C concentration (17.0%), while remained ineffective on LDL-C concentration in mice. Results have shown that escins may have beneficial effects in the understanding of obesity.
Introduction
Maintenance of stable body weight is attained via a biological process known as energy homeostasis, which depends on the balance of caloric intake and energy expenditure. Furthermore, when changes in the composition of the diet take place, the body must adapt macronutrient oxidation to intake. This adaptation relies on the ability to partition dietary nutrients between oxidation and storage (CitationRavussin & Swinburn, 1993). However, a high dietary fat intake is not always followed by an increase in lipid utilization, eventually diet-induced obesity may develop (CitationIossa et al., 2002). Obesity is a metabolic disorder in which the natural energy reserve, stored in the fatty tissue of mammals, is increased to a point where it is associated with many important complications such as diabetes and cardiovascular disorders (coronary heart disease, stroke, dyslipidemia, venous insufficiency, deep vein thrombosis), diseases of the gallbladder, liver and the musculoskeletal system, reproductive dysfunction, poor wound healing, and more (CitationAkiyama et al., 1996; CitationAbu-Abid et al., 2002; CitationPi-Sunyer, 2002; CitationHaslam & James, 2005).
Fatty diet, as well as imbalance between the food intake and energy expenditure are the factors affecting the prevalence of obesity (CitationAltunkaynak, 2005; CitationMilagro et al., 2006). Leptin, insulin and T3 are among the important hormones to provide this balance (CitationOtukonyong et al., 2005). Leptin, an adipose tissue hormone, regulates body fat mass and body weight by decreasing the appetite and increasing energy expenditure (CitationZabrocka et al., 2006).
Aesculus hippocastanum L. (Hippocastanaceae), horse chestnut, is a common tree growing widespread in Turkey (CitationBaytop, 1999). Seed extract of the plant has been used medically for the treatment of rheumatism, chronic venous insufficiency, traumatic edema, hemorrhoids, pain in the chest and abdomen (CitationSato et al., 2005; CitationHu et al., 2008). The main active constituent of the seeds are triterpenoid saponins, called escins, and also tannins, coumarin derivatives, flavonoids, mainly glycosides of quercetin and kaempferol (CitationSato et al., 2005; CitationKapusta et al. 2007; CitationHu et al., 2008). The triterpene saponin mixture (escins) was also shown to exert significant anti-inflammatory, antiedema, capillary protective effects and particularly has been used to alleviate hemorrhoids, varicose veins and rheumatic pains in medical practice (CitationHu et al., 2008).
In addition to the above-mentioned well-known pharmacological effects, CitationKimura et al. (2006) have recently reported that escins isolated from Japanese horse chestnut (Aesculus turbinate Blume) after treatment with wooden ashes to attenuate bitterness possess significant in vivo hypoglycemic activity as well as inhibitory effect on pancreatic lipase activity. They identified four types of deacetylescins Ia, IIa, Ib, and IIb as well as two types of desacylescins I and II in escin mixture. These components exerted significant inhibitory effects on elevated blood glucose levels when administered in a single oral dose of 100 mg/kg on the oral glucose tolerance test. The order of hypoglycemic potency was as follows: escins > deacetylescins > desacylescins. On the other hand, escins were again found as the most potent in inhibiting the pancreatic enzyme activity, and followed by desacylescins and then deacetylescins. Saponin fraction of seeds significantly lowered the increased body weight, the mass of peritoneal adipose tissues and plasma triacylglycerol levels (CitationKimura et al., 2008). Furthermore, a higher rate of undigested fats in feces was observed possibly due to the inhibition of fat digestion (CitationKimura et al., 2008). Consequently, they suggested escins from Japanese horse chestnut as a novel antiobesity agent.
Several studies have also reported that some triterpenoid saponins from Panax ginseng C.A. Mey. and Panax japonicus C.A. Mey. (Araliaceae) rhizomes or Platycodon grandiflora (Jacquin) A. DC. (Campanulaceae) roots showed strong inhibitory effects on pancreatic lipase in vitro and suppressed the increase of body weight induced by a high fat diet in vivo (CitationHan et al., 2002, Citation2005; Xu et al., 2005). Pancreatic lipase inhibitors have recently drawn attention to treat obesity and orlistat has been clinically used for this purpose (CitationDimitrov et al., 2005), however gastrointestinal side effects have frequently been reported during long-term administration (CitationHu et al., 2008).
In spite of the studies exerting the antiobesity effect of escins from Japanese horse chestnut, analogous activity of escins from European horse chestnut (Aesculus hippocastanum) seeds has not been investigated in detail. Moreover, the effect of escins on the hormones regulating the homeostasis between food intake, reserving energy and energy expenditure and affecting obesity has not been reported yet. Previously, CitationYoshikawa et al. (1994, Citation1996) studied the hypoglycemic activity of several triterpene glycosides from Aesculus hippocastanum seeds using the oral glucose tolerance test in rats, and reported that escins-IIa and IIb exhibited the highest activity, while desacylescins-I and II were inactive. In the present work, we aimed to study the effect of escins obtained from Aesculus hippocastanum seeds on such regulatory hormones as leptin, insulin and thyroid as well as some biochemical parameters (glucose, total cholesterol, HDL, LDL, TG) in order to reveal and elucidate the effect of escins from the scientific viewpoint.
Material and methods
Plant material
The plant material (seed) was collected from the garden of the Faculty of Pharmacy, Ankara University in July, 2006 and was identified by one of the authors (E.K.A.). A voucher specimen is deposited in the Herbarium of the Faculty of Pharmacy, Gazi University (GUE-2591).
Preparation of plant extract
Powdered seeds (2 kg) were extracted with ethanol (EtOH) (2 L) by intermittent stirrer at room temperature for 2 days and the ethanol extract was filtered and evaporated to dryness under reduced pressure below 40°C. This process was repeated several times to remove the extractable components. The combined extract was then dissolved in 400 mL methanol (MeOH). This extract was transferred drop-by-drop to a flask of ice-cold diethyl ether stirring well by using a stirring bar. The white precipitate was then filtered by using a vacuum system through Whatman filter paper. This precipitate was dried in a vacuum oven at 30-35°C (yield: 46.15 g).
Animals and test samples
Male Swiss albino mice (25-35 g) were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health, Ankara, Turkey. The animals had two days for acclimatization to animal room conditions and were maintained on standard pellet diet and water ad libitum. A minimum of ten animals were used in each group. Throughout the experiments, the animals were processed according to the suggested ethical guidelines for the care of laboratory animals (Ref: 07-212 GUAEC).
Test samples were suspended in distilled water and 0.5% sodium carboxymethyl cellulose (CMC), and were administered orally by gastric gavage in 100 mg/kg doses daily for 5 weeks. This dose was determined in a previous preliminary study.
Experimental protocol
Three different control groups were employed in the study. 1) The negative control group animals were maintained on standard pellet diet and water ad libitum without administering any plant extract. 2) The high fat diet group (positive control) animals received a special diet containing 40% beef tallow for 5 weeks. 3) The vehicle control (CMC) group animals received 0.5% CMC suspension in distilled water and were maintained on a high fat diet. On the other hand, for test groups, one group of animals received escin and was maintained on a standard pellet diet with water ad libitum, while the other test group was administered escin alone with a high fat diet. Blood samples were taken from the heart into tubes with heparin and plasma was obtained by centrifugation at 3000 rpm (+4°C) for 10 min.
Biochemical analysis
Plasma leptin (Cat. EZRL-83K) and insulin (Cat. EZRMI-13K) concentrations were determined by ELISA using a rat kit (Linco Research, St. Charles, Missouri USA). The manufacturer’s suggested protocol was followed for the determination of free T3 (FT3) (code no: DSL-10-41100) and free T4 (FT4) (code no: DSL-10-40100i) concentrations by specific ELISA-tests (DSL Diagnostic Systems Laboratories, Texas USA). Plasma glucose, triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol values were measured with commercially available assay kits (Human Diagnostica, Wiesbaden Germany).
Statistical analysis of data
Data obtained from animal experiments were expressed as standard error mean (± SEM). Statistical differences between the treatments and control group were evaluated by ANOVA and Student’s-Newman-Keuls post hoc tests. A difference in the mean values of p <0.05 was considered to be significant.
Results and discussion
In the present study there is a significant increase in the body weight () and plasma leptin level (172.4%) (p <0.01) () in the group fed a high-fat diet in comparison to the control group animals. Administration of escins to mice fed a normal diet induced 37.8% (p >0.05) inhibition in leptin concentration, while in the high fat diet group this inhibitory rate was 31.6% (p <0.05). The results of this study have been found to be consistent with those reporting that a high fat diet leads to lipid accumulation in the visceral tissues and increases body weight (CitationMilagro et al., 2006; CitationYang et al., 2006), as well as between the body fat mass and plasma leptin level (CitationChilliard et al., 2001). Energy homeostasis is maintained by complex and dynamic procedures interacting with each other. Among these, leptin is an adipocyte-derived hormone that acts as a major regulator for food intake and energy homeostasis and exerts its metabolic effects via the central nervous system and the specific receptors in peripheral tissues (CitationHouseknecht et al., 1998); this also stimulates signals from the adipose tissue for energy expenditure (CitationTrayhurn et al., 1999). The residual leptin in the plasma inhibits the release of neuropeptide Y (NPY) in the arcuate nucleus, known as the appetite control center of the body, via the hypothalamic receptors by passing the blood brain barrier. Thus, it causes reduction in appetite, sympathetic nervous system activation, and increase in the metabolic speed and energy expenditure (CitationSchwartz & Seeles, 1997).
Table 1. Change in body weight (g) during the 5-week experimental period.
Table 2. Effects of escin on plasma leptin, insulin, T3, T4 levels.
As shown in , a high fat diet causes 28.3% (p > 0.05) increase in blood glucose level of experimental animals, while escins reduced it in both untreated and high fat diet-treated animals between 19.8% (p < 0.05) and 15.1% (p > 0.05), respectively, representing a hypoglycemic effect. On the other hand, escin increased insulin level both in untreated and high fat diet groups (). CitationAl-Achi (2005) has also reported that some saponin-containing plants increase the production of insulin secretion from the pancreas and glucose utilization by the tissues. The increase in plasma insulin level also causes an increase in plasma leptin and ob gene mRNA levels by inducing lipogenesis in the liver and adipose tissues (CitationMaffei et al., 1995; CitationLopez et al., 2007). Insulin hormone increases glucose entrance especially into the cardiac muscle, skeletal muscle and adipose tissues and the sensitivity of these tissues to insulin (CitationNoyan, 1993). In addition, it also causes other nutrients such as free fatty acids and amino acids uptake into the cell (CitationLopez et al., 2007). Therefore glucose is oxidized in the glycolysis and raises the ATP/ADP ratio in the cytoplasm. The high adenosine triphosphate /adenosine diphosphate (ATP /ADP) ratio triggers exocytosis of insulin-containing granules. Fatty acids also enhance insulin secretion by increasing fatty acids β-oxidation leading to a high ATP/ADP ratio or by activating some protein kinases (CitationLopez et al., 2007). In fact, hyperinsulinemia seen in long-term consumption of high fat diets has been reported to occur as a result of pancreatic hyperplasia, fatty acids uptake and enhanced pancreatic β-cell glucose metabolism (CitationLopez et al., 2007). Parallel to the increase in the plasma leptin level in the study, variations in the insulin and glucose levels of the group consuming a high fat diet were found to be not significant compared to the control groups. In fact, this result is found to be consistent with the study reporting that the variations in insulin (), glucose and triglyceride levels () were insignificant despite high plasma leptin level in rats fed a high-fat diet for 8 weeks (CitationGuglielmacci et al., 2005). Similarly, CitationDuarte et al. (2006) have stated that pancreatic size decreases but the number of beta cells increase in rats fed a high fat diet for a period of 15 weeks, while non-significant variations in the insulin and glucose levels were found.
Table 3. Effects of escin on plasma glucose, total-, HDL-, and LDL-cholesterol and triglyceride.
Previous studies have reported that triterpenoid saponins from Platycodon roots (CitationHan et al., 2002), Panax ginseng saponins (CitationKaru et al., 2007) and tea saponins (CitationHan & Kimura, 2001) decrease hepatic triglyceride and total cholesterol levels in mice consuming a high fat diet by inhibiting pancreatic lipase. Pancreatic lipase is known to be a critical enzyme in the development of obesity. Recently, escins have also been reported to show inhibitory effects on the elevation of blood glucose levels and inhibitory effects on pancreatic lipase activity in mice (CitationHu et al., 2008). In the present study, a decrease in the body weight and plasma leptin levels both in the escin group compared to the untreated control and high fat diet plus escin groups compared to high fat diet control group animals have been found (). However, these values were not statistically significant when compared with the results of the control group. In fact, CitationHu et al. (2008) have suggested that 2% total escin obtained from Aesculus turbinata supplemented to the high fat diet for an 11-week period decreased body weight and the parametrical adipose tissue weight, and reduced dietary fat absorption in the intestine by inhibiting pancreatic lipase and increasing gastrointestinal motility.
Total cholesterol level of untreated animals was not affected by escin, while it significantly prevented severe rise in rats fed a high fat diet (). Escin provides significant increase in HDL cholesterol both in untreated and high fat diet animals. However, it was ineffective against the elevation of LDL-cholesterol and triglycerides.
The thyroid gland principally synthesizes T4, whereas the metabolically active T3 is mainly generated in extra-thyroidal tissues by enzymatic 5’-deiodination of T4 (CitationKelly, 2000). Thyroid hormones in the blood flow are mainly bound to plasma proteins; i.e., albumin, thyroxin-binding globulin (TBG) and thyroxin-binding pre-albumin (TBPA) and they are in equilibrium with free forms which are able to cross cellular membranes and are considered as active forms of the thyroid hormones (CitationKaneco, 1997). Thyroid hormones (THs) stimulate thermogenesis by increasing ATP consumption during TH-dependent processes and decreasing the efficiency of ATP synthesis (CitationOrtega et al., 2007). Thyroid hormones prevent proton coupling by stimulating “uncoupling” protein (UCP) expressions in the inner membrane of the mitochondrion. Therefore, it causes more energy consumption by providing heat release instead of ATP synthesis (CitationAslan et al., 2004).
In the present study there is a significant increase in T3 level and decrease in T4 level which were not statistically significant in the high fat diet group (), and these data were consistent with those of the previous report (CitationKelly, 2000). On the other hand, administration of escin induced significant inhibition of T4 level in untreated (46.8% inhibition) and in high diet treated (36% inhibition) animals. This might possibly be due to the increased T4 deiodination by the high fat diet or increased rate of T4 uptake by target cells (CitationBenvenga & Robbins, 1998). It was demonstrated that fatty acids can regulate pituitary T3 and T4 uptake differently (CitationEverts et al., 1995). Thus, it is possible that a higher T4 uptake caused by the high fat diet results in a normal serum T4 concentration, but in higher serum T3 levels and a higher thyroid hormone action (CitationBrito et al., 2006).
In the study, total cholesterol (p <0.01) and LDL-C (p <0.001) were found significantly high in the high-fat diet group in comparison with those of the controls (); however, the numerical increases in TG and HDL-C levels were not statistically significant. These results seem consistent with the results of a previous study of CitationYang et al. (2006), reporting significant increases in serum HDL, LDL, TG and cholesterol levels of rats fed a high fat diet during a 12-week period. CitationMilagro et al. (2006) also reported that increase in TG was not significant in rats fed a high-fat diet for a 2-month period in comparison to that of the control group.
Saponins (in aglycone or glycoside form) can be dissolved both in fat and water. Besides, saponins have the ability to reduce the surface tension and display properties of detergents, and consequently affect the emulsification of the fat-soluble substances in the digestion system (CitationFrancis et al., 2002). Therefore, it has been reported they affect the formation of mycelia containing bile acids, fatty acids, diglycerides and water soluble and fat soluble vitamins (CitationFrancis et al., 2002). In the present study, a significant rise was observed in the HDL level (p < 0.01) of the escin-treated group in comparison to that of the control group. Significant rise in total cholesterol (p < 0.01), HDL-C and LDL-C (p < 0.001) levels were observed in the high fat diet group of the study, while change in TG level was not statistically significant. Administration of escin in high fat diet-treated animals reduced total cholesterol (p < 0.01) and LDL-C (p < 0.01) levels significantly, however, remained ineffective against rise in TG and HDL-C levels.
In conclusion, the in vivo effect of escin obtained from European horse chestnut on plasma levels of some hormones (leptin, insulin, T3 and T4), lipids (triglyceride, total cholesterol, HDL and LDL) and glucose were investigated in the present study. Results have shown that escins may have beneficial effects in the understanding of obesity by decreasing leptin and total cholesterol levels significantly.
Declaration of interest
The authors confirm that there is no conflict of interest.
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