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Introduction
Diabetes mellitus is characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. Cardiovascular disease (CVD) is a major complication and the leading cause of premature death among patients with diabetes (Centers for Disease Control and Prevention, 1999). The primary risk factor for CVD among patients with type 1 diabetes is hyperglycemia, although other risk factors, such as hypertension and dyslipidemia, may occur secondary to uncontrolled hyperglycemia (Bate & Jerums, 2003). In contrast, the pathophysiology of cardiovascular complications of type 2 diabetes involves elements of hyperglycemia, dyslipidemia, hypertension, and obesity. It was also reported that tight control of hyperglycemia and aggressive therapeutic treatment of diabetic dyslipidemia are associated with the reduced risk of CVD in diabetic patients (American Diabetes Association, 2003). Although there have been enormous improvements in medication, strategy to cure diabetes mellitus completely has not been established so far. Therefore, numerous studies have been carried out to evaluate natural products, including plant materials, as alternative treatments for diabetes to be used in addition to conventional treatments (Bailey & Day, 1989).
In line of remedy from natural substances, it was reported that Salicornia herbacea L. could exert hypoglycemic and hypolipidemic effects. S. herbacea is an annual herb growing in the salt mashes and on the muddy seashores. Ethanol extract of S. herbacea was found to prevent the onset of the hyperlipidemia induced by high fat diet in mice (Park et al., 2006). S. herbacea powder supplementation decreased blood glucose and triglyceride levels in streptozotocin (STZ)-induced diabetic rats (Bang et al., 2002; Kim, 2007a). Consumption of water extract of enzyme-treated S. herbacea showed hypocholesterolemic effect in rat fed high cholesterol diet (Kim et al., 2006). S. herbacea was also reported to have strong antioxidative effect in vitro (Lee et al., 2004; Oh et al., 2007) and in vivo (Bang et al., 2002; Jang et al., 2007; Kim, 2007b). Since oxidative stress generated by free radical species plays a major role in the progression of diabetes (Kaneto et al., 2005), S. herbacea could be beneficial for the prevention and treatment of diabetes via its antioxidative effects. However, the beneficial effects of S. herbacea on carbohydrate and lipid metabolism in type 2 diabetes were not studied. Therefore, the present study was aimed to investigate the effect of S. herbacea on hyperglycemia and hyperlipidemia in animal model of type 2 diabetes mellitus.
Materials and Methods
Reagents
Assay kits for glucose, cholesterol, and triglyceride were purchased from Asan Co (Seoul, South Korea) and a glycated hemoglobin (HbA1C) assay kit from BioSystems (Barcelona, Spain). Cornstarch was acquired from Daesang Co. (Seoul, Korea). Casein, L-cystine, mineral mixture, and vitamin mixture were purchased from ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA) and tert-butyl hydroquinone from Fluka Co. (Milwaukee, WI, USA). Sucrose and soybean oil were obtained from Cheiljedang Co. (Seoul, Korea). Acarbose and orlistat were purchased from Bayer Korea (Seoul, Korea) and Roche Korea Co. (Seoul, Korea), respectively. Yeast α-glucosidase, porcine pancreatic lipase, p-nitropheny-α-D-glucopyranoside, 4-methylumbelliferyl oleate (4-MU oleate), and all other reagent grade chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA).
Preparation of the desalted ethanol extract
S. herbacea was obtained from a local market in Busan, Korea. S. herbacea was freeze-dried, powdered and extracted with ten volumes of 70% ethanol for 12 h three times at room temperature. The solvent was removed by rotary evaporation at 50℃. The dry extract was redissolved in 200 volumes of 100% ethanol and filtered using filter paper (No. 2, Whatman Inc., Florham Park, NJ, USA). The solvent was removed by rotary evaporation at 50℃. The yield of the desalted ethanol extract of S. herbacea was 4.2%. The dry extract was used for in vivo study and redissolved in dimethylsulfoxide (DMSO) to be used as a test material for the in vitro study.
Animals and experimental design
Three-week-old male C57BL/KsJ db/db mice (n=16) were purchased from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea). After 1 week of adaptation during which the animals had free access to commercial chow, they were randomly divided into a control and a S. herbacea group. The mice in the control group were offered a standard AIN-93G diet, which contained 39.8% cornstarch, 20% casein, 13.2% dextrinized cornstarch, 10% sucrose, 7% soybean oil, 5% Alphacel, 3.5% mineral mixture, 1% vitamin mixture, 0.3% l-cystine, 0.25% choline bitartrate, and 0.0014% tert-butyl hydroquinone, whereas the S. herbacea group was offered the same diet supplemented with 1% (wt/wt, final concentration) of ethanol extract of S. herbacea ad libitum for 6 weeks. The mice were housed individually in plastic cages and located in a room where temperature (23-27℃), humidity (50-60%), and lighting cycle (0600-1800 hr light and 1800-0600 hr dark) were controlled. Body weight and food intake were measured three times a week. At the end of the experimental period, the mice were sacrificed by heart puncture after an overnight fast. Blood HbA1C levels were measured by the chromatographic method using a commercial assay kit. Blood samples were centrifuged at 3000 ×g for 15 min; plasma was removed and frozen at -70℃ for further analysis. Plasma glucose, triglyceride, and total cholesterol were measured by enzymatic methods using commercial assay kits. The experiments were performed according to the guidelines of animal experimentation approved by the Animal Resource Center at Inje University, Korea.
Enzyme inhibition assay
Yeast α-glucosidase inhibitory activity was measured by the chromogenic method developed by Watanabe et al. (1997) using a microplate reader (model 550, Bio-Rad, Hercules, CA, USA). Yeast α-glucosidase (0.7 U) dissolved in 100 mM phosphate buffer (pH 7.0) containing 2 g/L bovine serum albumin and 0.2 g/L NaN3 and 5 mM p-nitrophenyl-α-D-glucopyranoside in the same buffer (pH 7.0) were used as an enzyme and a substrate solution, respectively. The final concentration of the S. herbacea extract and acarbose, a positive control, was 0.5 mg/mL. The measurement was performed in triplicate.
The inhibitory activity against pancreatic lipase was measured by using 4-MU oleate as a substrate (Bitou et al., 1999). Porcine lipase (25 µL) was added to the reaction mixture which consisted of 50 µL of 0.1 mM 4-MU oleate, 20 µL of a McIlvane buffer (0.1 M citrate-Na2 HPO4, pH 7.4) and 5 µL of a sample solution. After the final volume was adjusted to 0.1 mL, the reaction mixture was incubated at 37℃ for 10 min. The amount of 4-MU released by the lipase was measured using fluorescence multi-detection reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 320 nm and an emission wavelength of 450 nm. The final concentration of the S. herbacea extract and orlistat, a positive control, was 0.25 mg/mL. The measurement was performed in triplicate.
Results
Body weight and food intake of the mice are shown in Table 1. Chronic consumption of S. herbacea extract at the level of 1% of the diet did not significantly influence body weight, food intake and feed efficiency ratio in db/db mice. The effect of S. herbacea extract on glycemic control is shown in Fig. 1 and 2. The fasting plasma glucose level of the S. herbacea group (509.7 ± 40.9 mg/dL) tended to be lowered compared with the control group (535.6 ± 35.4 mg/dL, Fig. 1). Blood glycated hemoglobin level of S. herbacea group (7.8 ± 1.0%) was not significantly different from the control group (8.1 ± 1.2%, Fig. 2). The effect of S. herbacea extract on lipid profile is shown in Fig. 3 and 4. Consumption of S. herbacea extract significantly decreased plasma triglyceride (152.7 ± 21.0 mg/dL, Fig. 3) and total cholesterol levels (205.7 ± 22.1 mg/dL, Fig. 4) compared with the control group (180.7 ± 26.7 mg/dL and 233.5 ± 25.2 mg/dL, respectively, p<0.05).
The inhibitory activities of the ethanol extract of S. herbacea against yeast α-glucosidase and porcine pancreatic lipase are shown in Table 2. The ethanol extract of S. herbacea inhibited yeast α-glucosidase activity by 10.2% at the concentration of 0.5 mg/mL in vitro. Acarbose, an α-glucosidase inhibitor, which is used for an oral hypoglycemic agent inhibited the enzyme activity by 31.8%. Inhibitory activities of S. herbacea extract against porcine pancreatic lipase were 56.2% at the concentration of 0.25 mg/mL. Orlistat, a pancreatic lipase inhibitor, showed inhibition of 95.4% at the same concentration.
Discussion
S. herbacea has been shown to exert hypoglycemic and hypolipidemic effects in rodents fed a high fat diet or in animal models of type 1 diabetes (Bang et al., 2002; Kim et al., 2006; Kim, 2007a). However, its hypoglycemic and hypolipidemic effects in animal model of type 2 diabetes have not been reported. Achieving near-normal glycemic control and lowering plasma lipid levels lead to a decrease in the risk of cardiovascular complications of diabetes which is the leading cause of premature death of patients with type 2 diabetes (UKPDS, 1998). In this study we determined antidiabetic effect of S. herbacea in db/db mice which exhibit type 2 diabetes and obesity. Consumption of ethanol extract of S. herbacea led to a significant decrease in plasma triglyceride and cholesterol. S. herbacea extract showed strong inhibitory activity against porcine pancreatic lipase in vitro. Inhibitory activity of S. herbacea extract against pancreatic lipase was 59.0% of that of orlistat at the concentration of 0.25 mg/mL in vitro. Inhibitor of pancreatic lipase could interfere with digestion of dietary triglycerides and retard absorption of fatty acids (Ballinger & Peikin, 2002). Chronic consumption of orlistat, a potent inhibitor of pancreatic lipase, reduced body weight, plasma triglyceride and cholesterol in obese patients (Lucas et al., 2003). In this study, S. herbacea with pancreatic lipase inhibitory activity showed triglyceride and cholesterol-lowering effect but did not influence body weight significantly in db/db mice. Dioscorea nipponica Makino (Kwon et al., 2003) and onion skin extract (Kim, 2007c) with pancreatic lipase inhibitory activity also decreased blood triglyceride and cholesterol in rats fed high fat diet. Orlistat has been reported to show side effects such as diarrhea (Ballinger & Peikin, 2002). Thus natural product with lipase inhibitory activity without side effects such as S. herbacea could be useful hypolipidemic agents which could reduce risks for diabetic complications. Plasma glucose and blood glycated hemoglobin levels of S. herbacea group tended to be decreased compared with the control group, but the differences between the two groups were not significant. It was reported that S. herbacea powder supplementation at the level of 5% of the diet showed hypoglycemic effect in STZ-induced diabetic rats (Bang et al., 2002). Kim reported supplementation of S. herbacea powder at the level of 20% of the diet significantly decreased blood glucose in STZ-induced diabetic rats and suggested that dietary fiber contained in S. herbacea could be responsible for the hypoglycemic effect (2007a). However, consumption of ethanol extract of S. herbacea did not affect glycemic control in db/db mice in this study. S. herbacea extract showed mild inhibitory activity against yeast α-glucosidase in vitro. Inhibitory activity of S. herbacea extract against α-glucosidase was 31.9% of that of acarbose at the concentration of 0.5 mg/mL in vitro. It appears that the hypoglycemic effect of S. herbacea could be stronger than that of S. herbacea extract if dietary fiber is the active component exerting hypoglycemic effect.
In conclusion, ethanol extract of S. herbacea reduced plasma triglyceride and cholesterol in animal models of type 2 diabetes and the hypolipidemic effect could be partly due to inhibitory activity against pancreatic lipase.
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