Research Article
Effect of Flax Seeds and Date Palm Leaves Extracts on Serum Concentrations of Glucose and Lipids in Alloxan Diabetic Rats
Department of Biochemistry, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Diabetes Mellitus (DM) is a chronic metabolic disease that characterized by impairment of glucose metabolism and utilization. The prevalence of the disease all over the world is in a continuous increase expecting to reach 221 millions by 2010 (Carter, 2004; Wild et al., 2004). Most of diabetic patients have health complications or risk factors such as high blood pressure and cholesterol that increase one's risk for heart disease and stroke. When combined with DM, these risk factors add up to big trouble, however, vascular complications are the most common causes of morbidity and mortality in diabetic patients (Guo et al., 2005).
An intensive research work is conducted in herbs that could have a protective effect against DM due to the side effects associated with the oral hypoglycemic agents for the treatment of the disease (Kim et al., 2006). Although, Subash Babu et al. (2007) demonstrates that cinnamaldehyde, isolated from cinnamon, possesses hypoglycemic and hypolipidemic effects in streptozotocin-induced diabetic rats, however, an opposing findings reported that cinnamon has no effect on adolescents with type 1 diabetes (Altschuler et al., 2007). A survey study conducted in south-eastern Morocco, concluded that 78% of the patients regularly use medicinal plants, Phoenix dactylifera was among the most frequently used plants to treat diabetes and hypertension (Tahraoui et al., 2007). Haliga et al. (2007) reported that dietary flax seed supplementation improved serum lipid concentrations and reduced diabetic macro vascular complications in streptozotocin diabetic hamster. Several investigators reported the benefits of flax seeds administration in the reduction of the risk factors of atherosclerosis (Prasad, 2005, 2008; Mandasescu et al., 2005). Antioxidant activities were reported in many plants, among them is date palm fruit (Phoenix dactylifera) which possesses a potential antioxidant compounds that capable of scavenging free radicals (Vayalil, 2002; Mansouri et al., 2005; Biglari et al., 2008).
The present investigation attempts to study the effect flax seeds (Linum usitatissimum) and date palm (Phoenix dactylifera) leaves extracts on serum concentration of glucose and lipids in alloxan diabetic rats.
This study was conducted between November 20, 2008 and March 12, 2009.
Chemicals and reagents: Alloxan for diabetes induction was obtained from CDH laboratory reagent (New Delhi, India). Methanol and other chemicals were of analytical grade and obtained from BDH (Poole, United Kingdom). Kits for the assay of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), triglycerides (TG) and glucose were purchased from Human GmbH (Wiesbaden, Germany).
Plant material and extraction: Date palm leaves (DPL) were collected from Riyadh city. At first, DPL were washed from dust with tap water and then rinsed with distilled water and distributed in a clean benches and allowed to stand until dryness at room temperature. Dry DPL were cut into small pieces and immersed in liquid N2.
Flax Seeds (FS) were purchased from Riyadh local market. One hundred grams powder of each plant were prepared by using a blender (FS powder was prepared without immersion of seeds in liquid N2 prior blending). The powdered samples were then extracted with 300 mL methanol-water (4:1, v/v) at room temperature for overnight using an orbital shaker. Filtrates were collected through glass wools and residues were extracted again with the same above mentioned protocol. Pooled filtrates were collected and centrifuged at 8000 rpm for 10 min and the supernatants were concentrated under reduced pressure at 40°C using a rotary evaporator. Yields of DPL and FS were 8.4 and 6.2 g, respectively.
Animal groups and dosage: Male Wistar rats (180-205 g) were obtained from the animal house, Faculty of Pharmacy, King Saud University, Riyadh, Saudi Arabia. All animals were housed in cages with 12/12 h light/dark cycles at 22°C. Animals were given rat chow and water ad libitum. Diabetes was induced by an injection of alloxan (150 mg kg-1, i.p., dissolved in physiologic saline). The animals were kept under observation for one week prior to the start of treatment. All animal experiments were carried out in accordance with King Saud University Ethical Committee Acts. Extracts of FS and DPL were administered orally (0.2 mL) at a concentration of 100 mg KBW-1 by using oral gavage. Treatment with extracts was continued on regular daily basis over a period of 30 days.
Rats were divided into 6 groups (10:14 rats/each). Group 1 consisted of normal control rats (NC). Group 2 consisted of normal control rats treated with FS (NC+ FS). Group 3 consisted of normal control rats treated with DPL (NC+DPL). Group 4 consisted of diabetic control rats (DC). Group 5 consisted of diabetic rats treated with FS (D+FS). Group 6 consisted of diabetic rats treated with DPL (D+DPL).
Body weights and food intake of controls and diabetic treated rats were recorded daily. Aliquots of blood samples from all groups were collected weekly in plain tubes. Serum samples were separated from cells by centrifugation at 5000 g in a refrigerated centrifuge.
Biochemical assays: Glucose, Total Cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were estimated in serum samples by standard enzymatic methods using Human kits (Bartham and Trinder, 1972; Richmond, 1973; Schettler and Nussel, 1975; Izawa et al., 1997; Okada et al., 1998), respectively. The concentrations of these parameters were measured in a Humalyzer model 3000.
Data analysis: The data presented in this investigation are expressed as means±standard error of the mean (SEM) and comparison between experimental and corresponding control rats was made by using students t-tests. The probability value of <0.05 was considered significant.
Body weight and food intake of control and diabetic rats treated with FS and DPL extracts: Table 1 shows that gain in body weight and food intake in alloxan diabetic rats was markedly reduced (p<0.001) compared to their matched control rats. However, normal control and alloxan diabetic rats that were treated with FS or DPL did not show any significant changes in the same parameters.
Effect of FS and DPL extracts on serum glucose and serum lipids concentration of normal control (NC) rats: Table 2 shows that serum glucose concentration of NC rats after two weeks treatment with FS or DPL was not significantly different from that of NC rats without treatment. However, after four weeks treatment with FS extract, NC rats showed significant decrease in serum glucose concentration (p<0.03). Treatment of NC rats with FS extract reduced TC significantly by 37 and 55% after two and four weeks of treatments, respectively. This finding coincided with the report of Bhathena et al. (2002). On the other hand, DPL extract showed significant decrease in serum TC concentration of NC rats after four weeks of treatment (p<0.05). The concentration of TG in both FS and DPL groups were not significantly changed from their matched NC group. Although, present results showed that FS had no significant effect on serum TG concentration is contrasting with the report of (Bhathena et al., 2002), however, this could be due to the used concentration of FS. Serum concentration of LDL-C was significantly decreased after four weeks treatment of NC rats with either FS or DPL (p<0.02 and 0.01), this decrease was 21% and 29% respectively. A significant increase (p<0.02) was observed in serum HDL-C concentration when NC rats were treated for four weeks with DPL extract as the concentration increased from 0.33 to 0.56 mmol L-1.
Table 1: | General characteristics of control and diabetic rats treated with flax seeds and date palm leaves extracts |
Data are expressed as means±SEM, with number of rats given in parentheses. As compared with matched groups, *p<0.001 (student t test) |
Table 2: | Serum concentrations of glucose, Total Cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein -cholesterol (HDL-C) in control rats treated with flax seeds and date palm leaves extracts |
Data are expressed as means±SEM, with number of rats given in parentheses. As compared with matched groups, *p<0.01, **p<0.02, p<0.03, p < 0.05 (student t test) |
Table 3: | Serum concentrations of glucose, Total Cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein -cholesterol (HDL-C) in diabetic rats treated with flax seeds and date palm leaves extracts |
Data are expressed as means±SEM, with number of rats given in parentheses. As compared with matched groups, *p<0.01, **p<0.02, p<0.03, Ip<0.003, p<0.001 (student t test) |
Effect of FS and DPL extracts on serum glucose and serum lipids concentration of alloxan diabetic rats: Table 3 shows that serum glucose concentration of diabetic rats treated with FS extract for 2, 3 and 4 weeks was significantly decreased reaching a value of 8.14 mmol L-1 at fourth week (p<0.001). In contrast, no any significant changes were noticed in the same parameters of diabetic rats treated with DPL extract throughout the study period. Serum TC concentration of diabetic rats treated with either FS or DPL extract showed significant decrease throughout the weeks of study. Our results obtained with DPL extract agreed with Al-Maiman (2005) who reported that date seeds fiber at a concentration of 1.5% significantly reduced plasma TC in rats. Although, present finding in that FS extract decreases serum TC concentration of diabetic rats was coincided with that reported by Haliga et al. (2007) however, the degree of this reduction was different, this could be due to many factors include species differences, diabetes induction procedure, dose concentration as well as the way of FS administration.
Both of FS and DPL extracts were significantly reduced serum TG levels of diabetic rats (Table 3). With regard to FS, our finding contrasted with those reported by Haliga et al. (2007) who stated that FS supplementation had no significant effect on serum TG concentrations of diabetic hamsters. Treatment of diabetic rats for two weeks with either FS or DPL extract resulted in a significant reduction of serum LDL-C concentration (p<0.001).
The decrease in serum LDL-C level after treating diabetic rats with FS extract could be accounted to the presence of Secoisolariciresinol diglucoside (SDG), a compound of an antioxidant activity, isolated from FS as reported by Prasad (1999). Although, Chaira et al. (2007) reported that flesh and pit extracts of date palm fruit have free radical scavenging activities, however, the significant effect (p<0.001) (third and fourth weeks) of DPL extract on serum LDL-C level could be attributed to the antioxidant potentials of DPL extract as it was investigated in our laboratory (unpublished work). Another rationale explanation for the effect of DPL extract on LDL-C concentration in diabetic rats could be due to prevention of oxidative stress, from this view, present findings support the report of Saafi et al. (2010).
Both of FS and DPL extracts have no significant effect on serum HDL-C concentration of diabetic rats during the study (Table 3). We have no explanation for these results at present, however, we can propose that if the study duration is extended, a positive increase in serum HDL-C concentrations could be obtained to associate the significant decrease in LDL-C concentration.
The findings of the present investigation showed that FS had a significant decrease of both serum concentration of TC and LDL-C, from this view, our findings supported the suggestions reported by several investigators that FS has a hypocholesterolaemic effect (Lucas et al., 2004; Yang et al., 2005). DPL extract also showed a significant reduction of serum concentration of TC and LDL cholesterol in diabetic rats (Table 3).
The current investigation concludes that both of FS and DPL extracts have a hypocholesterolaemic effect and could have a protective effect against diabetes complications as well as hyperlipidemia through improvement of lipid profile.
The author would like to thank the research center, college of science for supporting this project (No. Bio/2008/67).