Antidiabetic and Antihyperlipidemic Effects of Ethanol Extract of Rosa canina L. fruit on Diabetic Rats

Preparation of Extract

Rosa canina fruits were purchased from a local herb store in Kashan, Iran and powdered by a grinder. A voucher specimen (No. R116) of the flower was identified and authenticated by the taxonomist at the Barij Research Institute, Kashan, Iran.

Two kilograms of this powder was mixed with 5 L ethanol + water (70:30) mixture and was placed on a shaker for 48 hours. Then, the supernatant fluid of mixture was separated and filtered and placed in a sterile container. ¹⁵ The extract was concentrated under reduced pressure in a rotary evaporator at 30°C to 40°C. Then, it was stored until the analysis.

Determination of Total Phenolic Content

The total phenolic content in extract was determined using the Folin-Ciocalteu reagent and gallic acid was used as standard. Calibration line was y = 0.0017x − 0.0016 and R ² = 0.9995. The sample (25 µL) and 1.25 mL of sodium carbonate (75 g/L) were added to 2.5 mL of Folin-Ciocalteu reagent (10% v/v). After 1.5 hours of incubation at room temperature, the absorbance was measured at 765 nm by spectrophotometer. The results were given as gallic acid equivalent per 100 g of extract.

Animals

Male Wistar albino rats aged 2 to 3 months wiith a body weight ranging from 180 to 200 g, were purchased from Central Animal House, Physiology Research Center, Kashan University of Medical Sciences, Iran and were maintained in an air conditioning room (25°C ± 1°C) with a 12-hour light/12-hour dark cycle. Food and water was provided ad libitum. All the animal experiments were approved by the Animal Research Ethics Board at Kashan University of Medical Sciences (No. 9144).

Induction of Diabetes

Diabetes was induced in male Wistar albino rats by intraperitoneal administration of streptozotocin (single dose of 55 mg/kg body weight). Streptozotocin was dissolved in freshly prepared 0.01 M citrate buffer, pH 4.5. ¹⁸ In order to prevent the initial drug-induced hypoglycemic mortality, 20% glucose solution was given to streptozotocin-injected animals for 24 hours. Seventy-two hours after the streptozotocin injection, the overnight fasting blood glucose levels were taken from tail arteries of rats. Animals with fasting blood glucose levels higher than 250 mg/dL were selected as diabetic rats. All the animals were allowed free access to water and pellet diet and were maintained at room temperature (25°C ± 1°C).

Experimental Design

Rats were divided into 5 groups (n = 10 in each group). Diabetes was induced in all groups except the normal control group.

Group I: Normal nondiabetic control

Normal and diabetic controls rats were fed with distilled water, whereas Rosa canina extract or glibenclamide was administered orally to treated rats once a day for 6 weeks.

Biochemical Analysis

Fasting blood glucose levels and body weights were measured on the days 0 and 42. At the end of the experimental period, rats were fasted for 12 hours with free access to drinking water and then were sacrificed by anesthetic ether and further cervical dislocation. Blood samples were collected into sterile plastic tubes and allowed to clot at room temperature for 20 minutes, and were then centrifuged for 15 minutes and the platelet-free unhemolyzed serums were collected and were stored at −30°C for later measurements. Pancreas and liver were immediately removed and placed in labeled beakers in 10% formaldehyde solution. Stored serum samples were analyzed for glucose levels by glucose oxidase-peroxidase method. Cholesterol was measured by enzymatic colorimetric method. The amount of triglycerides was determined by colorimetric method (enzymatic hydrolysis of triglycerides). All the biochemical analyses were performed using commercial kits (Parsazmun Company, Iran).

Histopathological Examinations

The pancreases were excised, fixed in 10% formaldehyde solution and processed in an Auto-Technicon machine and paraffin blocks were prepared. Microscopic sections (5 µm) were stained with hematoxylin and eosin and examined under a light microscope with a computer-connected camera.

Statistical Analysis

The results were expressed as mean ± standard error of mean. The statistical significance was estimated by 1-way analysis of variance followed by Tukey’s post hoc test. P values less than .05 were considered as significant.

The Total Phenolic Content of Rosa canina Extract

Total phenolic content of extract was determined as gallic acid equivalent using Folin-Ciocalteau reagent. Total phenolic content was 5.9% ± 0.4% gallic acid equivalent/100 g fraction.

The Effect of Rosa canina Extract on the Level of Fasting Blood Glucose

The effect of Rosa canina extracts on the fasting blood glucose levels in streptozotocin-induced diabetic rats is summarized in Figure 1. Fasting blood glucose levels were significantly elevated after the induction of diabetes in comparison with normal rats (P < .001). Administration of Rosa canina extract significantly decreased fasting blood glucose levels at the dose of 250 mg/kg body weight (94 ± 18.19 vs 317.71 ± 59.28 mg/dL) and at the dose of 500 mg/kg body weight (117.87 ± 15.41 vs 317.71 ± 59.28 mg/dL) in comparison with control (P < .001 for both).

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Figure 1.

Fasting blood glucose levels in different animal groups during the experimental period. NC, normal control; DC, diabetic control; RC 250 and 500, Rosa canina at dose 250 and 500 mg/kg body weight; G, glibenclamide. Values are means ± standard error of the mean. *P < .05. **P < .01 compared with DC group. ^† P < .001 compared with NC group.

Effect of Rosa canina Extract on Serum Lipid Profile

The effect of Rosa canina extract on the serum lipid profile in streptozotocin-induced diabetic rats has been reported in Table 1.

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Table 1.

Effect of Ethanol Extract of Rosa canina on the Lipid Profile Levels in Streptozotocin-Induced Diabetic Rats.^a

Groups	TG (mg/dL)	Cholesterol (mg/dL)	HDL (mg/dL)	LDL (mg/dL)
NC	83.37 ± 10.19	69.77 ± 2.79	23.11 ± 1.18	28.00 ± 3.70
DC	90.11 ± 12.49	66.22 ± 2.42	24.55 ± 1.44	23.64 ± 2.19
D + RC (250 mg/kg)	59.08 ± 8.83b	65.00 ± 5.43	25.83 ± 1.76	27.35 ± 3.18
D + RC (500 mg/kg)	69.37 ± 9.67	70.25 ± 4.34	27.12 ± 1.63	29.25 ± 1.79
D + G (0.6 mg/kg)	86 ± 7.58	66.75 ± 3.01	23.81 ± 1.36	26.37 ± 2.42

Abbreviations: DC, diabetic control; D + G, diabetic + glibenclamide; D + RC, diabetic+ Rosa canina; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NC, normal control; TG, triglycerides.

^aValues are means ± standard errors of the mean.

^b P = .041.

The levels of serum triglycerides significantly decreased by the administration of Rosa canina extract at a dose of 250 mg/kg body weight (59.08 ± 8.83 vs 90.11 ± 12.49 mg/dL) compared with diabetic control group (P = .041). Serum cholesterol, low-density lipoprotein, and high-density lipoprotein levels did not change significantly in treated rats compared with control group.

Histopathological Findings

Microscopic examinations of the pancreas sections showed the normal histology of the pancreas in normal nondiabetic rats of control group (Figure 2). The islets of Langerhans in diabetic rats showed sever necrotic changes. In the necrotic islets, changing in nucleus of cell, disappearing of nucleus, karyolysis were seen and in some places residue of destructed cells were visible (Figure 2).The improving effect of Rosa canina extract (250 mg/kg body weight) on diabetic rats was clearly obvious and the pancreas histology of the extract treated diabetic rats did not show a significant difference compared with the normal nondiabetic rats (Figure 2). Under the light microscope, most extract treated rats (250 mg/kg body weight) showed many small islets of Langerhans with normal histology. Although, the higher dose of extract (500 mg/kg) also could reverse the diabetic effects of streptozotocin (Figure 2), but the slides related to low dose (250 mg/kg body weight) of extract had more normal cells than that of high dose (500 mg/kg) of extract (Figure 3).

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Figure 2.

The pancreas microscopic sections of normal (A), diabetic (B), diabetic rats treated with Rosa canina extract 250 mg/kg body weight (C), and 500 mg/kg body weight (D) (hematoxylin and eosin, 100×).

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Figure 3.

Higher magnification of pancreas microscopic sections of normal (A), diabetic (B), diabetic rats treated with Rosa canina extract 250 mg/kg body weight (C) and 500 mg/kg body weight (D) (hematoxylin and eosin, 400×).