Anti diabetic property of aqueous extract of Stevia rebaudiana Bertoni leaves in Streptozotocin-induced diabetes in albino rats

Diabetes mellitus is a group of metabolic diseases characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both [1]. According to World Health Organization Diabetes mellitus will become the seventh leading cause of death worldwide in 2030 [2]. Through proper diet, exercise and pharmacologic interventions, the incidence of diabetes can be overcome [3]. The pharmacological drugs used for the treatment of diabetes, are either too expensive or have certain adverse side effects. Therefore, for the treatment of diabetes mellitus many traditional plants have been preferred as natural source of drugs [4] because they are considered to be safe, less toxic than synthetic ones [5] and have strong antioxidant activities due to which these plants become more effective against diabetes [6]. Stevia rebaudiana Bertoni as a traditional plant is famous due to its sweet taste and beneficial effects in blood glucose regulation. Stevia rebaudiana Bertoni (family Asteraceae) popularly known as stevia, sweet weed, honey leaf and sweet herb of Paraguay [7]. Stevia leaves contained complex mixture of diterpene glycosides including stevioside, steviolbioside, rebaudiosides (A, B, C, D, E) and dulcoside A but the major sweet constituents are stevioside and rebaudioside A [8, 9]. Natural non-caloric sweetener stevioside (a major component of stevia) is 100–300 times sweeter than sucrose and have been extensively used as a non-caloric sugar substitute in many kinds of foods, medicine, beverage, cosmetics, wine making, household chemical industry and other food industries [10]. It possesses anti-hyperglycaemic, anti-hypertensive, anti-oxidant, anti-tumor, anti-diarrheal, diuretic, gastro and renal-protective and immunomodulatory properties [11]. The anti-hyperglycemic effect of S. rebaudiana was investigated in both rats and humans by [12, 13]. They mentioned that stevioside demonstrates a positive effect on hyperglycemia through decreasing the absorption of glucose in duodenum, glycogenolysis and gluconeogenesis.

As the synthetic drugs used for the treatment of diabetes result in many complications. Hence the use of natural source (Stevia rebaudiana Bertoni) for the treatment of diabetes is safe and non-carcinogenic [8, 9]. Hence, the present experiment was undertaken to study the antidiabetic effect of S. rebaudiana in albino rats.

Means values for feed and water intakes in different groups of rats (per rat/day) have been shown graphically in Figs. 1 and 2. The results demonstrated that administration of stevia sweetener reduced the feed and water intakes in diabetic rats than N₀ and D₀. The highest feed and water intakes 14.57 g/rat/day, 29.82 mL/day and 13.14 g/rat/day, 28.95 mL/day respectively were observed in N₀ (non-diabetic control) and D₀ (diabetic control). While stevia sweetener at dose of 500 ppm/kg b.w showed the lowest amounts of feed and water intake (13.38 ± 0.98 g/rat/day) and (24.38 ± 0.58 mL/day) followed by 200 ppm/kg b.w (15.20 ± 1.00 g/day), (26.26 ± 0.53 mL/day), 300 ppm/kg b.w (14.81 ± 0.97 g/rat/day), (25.68 ± 0.60 mL/day) and 400 ppm/kg b. wt (13.82 ± 0.99 g/rat/day), (25.10 ± 0.64 mL/day) of stevia extracts respectively (Figs. 1 & 2).

Effect of administration of stevioside on the body weight of rats has been shown in Table 2. It is apparent from the results that the highest body weights (154.60 ± 4.02–185.90 ± 5.87 g/rat) and (153.22 ± 4.22–179.32 ± 4.55 g/rat) were observed in N₀ (normal control) and D₀ (diabetic control). While the lowest body weight was recorded in D₄ (diabetic rats received 500 ppm/kg b.w aqueous stevia extract) (148.60 ± 7.02–120.81 ± 7.80 g/rat) as followed by D₁ (152.12 ± 5.01–132.78 ± 4.32 g/rat), D₂ (150.30 ± 6.33–128.70 ± 4.54 g/rat) and D₃ (149.82 ± 6.88–124.32 ± 6.10 g/rat) during study period from 1st week to 8 weeks.

Regarding the body weight gain %, highest percentage (23.27%) and (19.48%) was observed in N₀ and D₀. When diabetic rats were given stevia sweetener then their body weight gain % decreased by − 13.84, − 15.89, − 18.75 and − 20.55% respectively after 8 weeks (Table 2).

Table 3 represents the random blood glucose levels of normal and diabetic rats, affected by different levels of stevia aqueous extracts. From the results, it was observed that random blood glucose (RBG) levels of N₀ and D₀ increased from (82.73 ± 2.91 to 89.25 ± 2.76 mg/dL) and (342.1 ± 1.22 to 391.22 ± 1.65 mg/dL) respectively at the beginning of the study to end of trail respectively. However, the RBG levels of D₁, D₂, D₃ and D₄ decreased from 332.23 ± 1.34, 330.23 ± 1.44, 327.88 ± 1.23 and 326.44 ± 1.65 mg/dL at 1st week to 94.43 ± 1.23, 93.29 ± 1.54, 91.22 ± 1.87 and 90.77 ± 1.27 mg/dL respectively at 8th week (Table 3). Stevia extract decreased the random blood glucose % levels of groups D₁, D₂, D₃ and D₄ by − 71.74, − 72.25, − 73.08 and − 73.24% respectively after 8 weeks (Table 3).

As presented in Table 4, there was significant (P < 0.05) increase in the fasting blood glucose level of the diabetic control group rats, relative to the normal control group. However, this was significantly (P < 0.05) restored toward normal in the diabetic rats given stevia aqueous extract, as indicated by the decrease in their fasting blood glucose levels from the 1st week to the 8th week. According to results highest level of fasting blood glucose (306.4 ± 2.65 mg/dL) was recorded in D₀ (diabetic control group rats). While fasting blood glucose levels of diabetic rats received stevia aqueous extract significantly decreased from 90.70 ± 2.98 (D₁) to 88.22 ± 2.97 (D₄) mg/dL. The fasting blood glucose % levels of groups D₁, D₂, D₃ and D₄ decreased by − 64.87, − 65.28, − 65.96 and − 66.09% respectively after 8 weeks (Table 4).

The glycosylated hemoglobin (HbA1c) level of the rats is shown in Fig. 3. According to results HbA1c level (9.27 ± 1.09%) of D₀ significantly (P < 0.05) increased than N₀ (5.92 ± 1.02%). But as compared to the D₀, diabetic groups D₁, D₂, D₃ and D₄ received stevia aqueous extract had significantly (P < 0.05) lower HbA1c levels (6.22 ± 1.11%, 6.06 ± 1.08%, 5.77 ± 1.06% and 5.32 ± 1.00%) respectively; indicating that the stevia extract decrease the glycosylation of hemoglobin.

The insulin levels of diabetic and normal rats are shown in Fig. 4. According to results the insulin levels of diabetic D₀ (15.89 ± 1.22 μIU/mL) control group decreased as compared to N₀ (18.02 ± 1.44 μIU/mL). The results of this study concluded that diabetic rats given stevioside mixed in distilled water increased the levels of serum insulin. The results further demonstrated that given stevia aqueous extracts at different dose levels improved significantly (P < 0.05) from 16.04 ± 1.24 to 17.82 ± 1.33 μIU/mL (D₁ to D₄) (Fig. 4).

The liver glycogen level of the rats is shown in Fig. 5. In this study, glycogen level of D₀ (17.07 ± 1.35 mg/g) decreased significantly (P < 0.05) compared to the N₀ (45.22 ± 2.22 mg/g) (Fig. 5). However, the diabetic rats received stevia aqueous extracts (200, 300, 400 and 500 ppm/kg) significantly (P < 0.05) increased the liver glycogen levels (35.27 ± 2.12, 37.43 ± 2.14, 42.66 ± 2.20 and 45.02 ± 2.24 mg/g) (Fig. 5).

In this study, we evaluated the anti-diabetic activity of aqueous extract of stevia in diabetic albino rats as previous researches confirmed its pharmacological importance due to presence of glycosides like stevioside in it. Administration of aqueous stevia extract orally at different concentrations (200, 300, 400 and 500 mg/kg) for 8 weeks, significantly decline the feed and water intakes of diabetic albino rats. Stevia a low-caloric sweetener may reduce the feed and water intake and not promote weight gain because they do not stimulate the appetite [24]. Similarly, stevia sweetener at doses of 25, 250, 500 and 1000 mg/kg b. w may also reduce the feed intake in adult female wistar strain rats [25].

The results indicated that aqueous extract from leaves of Stevia rebaudiana, produced a significant (P < 0.05) dose-dependent reduction in body weight (Table 2) and body weight gain percentage of the rats treated with Stevia extract (Table 3) as compared to N₀. The highest gain in body weight was noticed in N₀ while lowest was recorded in D₄. Stevioside in the diet lowered the blood glucose level. The body weight of rats might reduce due to lower metabolisation of diet glucose or decrease amount of rat’s food consumption [26]. This reduction in weights of rats receiving stevia extract may be due to high amount of stevioside that reduced the food intake of rats [27]. This finding is collaborated with the previous researches which proved a positive association between the decrease of body weight gain percent and the decline in feed intake and dose of stevioside given to the rats [28‐30].

This study depicted that different concentrations of stevia extract had a good efficacy in controlling diabetes with an excellent control of random and fasting blood glucose level in diabetic rats at study period of 8 weeks. Previous study showed that stevioside was able to regulate blood glucose levels by enhancing not only insulin secretion and sensitivity but also insulin utilization in insulin deficient rats which was due to decreased PEPCK gene expression in rat liver [31]. According to another study, stevia extract may contain some biomolecules that may sensitize the insulin receptor to insulin or stimulates the β-cells of islets of langerhans to release insulin which may finally lead to improvement of carbohydrate metabolizing enzymes towards the reestablishment of normal blood glucose level [32]. Stevia rebaudiana leaves extract decreased the random and fasting blood glucose levels of rats by revitalizing the β-cells of pancreas thus reactivated the glycogen synthase system by improving insulin secretion and liver glycogen level [25, 27, 33, 34]. These results are in agreement with Awney et al. [27]; Abo Elnaga et al. [25]; Assaei et al. [30] and Akbarzadeh et al. [34] who also observed that stevia aqueous extract lowered the random and fasting blood glucose levels in diabetic rats due to more insulin secretion and increased glycogen level.

The diabetic rats treated with stevia aqueous extract exhibited HbA1c values near normal levels (≥6.5% (48 mmol/mol) as a result of improved glycemic control due to initiation of glycogen production framework of the extract. The decrease of HbA1c showed that the ability of extract to control the diabetes [33].

These results are in accordance with Prasad et al. [35] and Rao et al. [36] who demonstrated the anti-diabetic effects of ethanolic extract of the roots of Chonemorpha fragrans and combination of herbal product (Curcuma longa and Eugenia jambolana) in streptozotocin- induced diabetic rats and concluded that both have a good efficacy in controlling diabetes.

The serum insulin level in the diabetic control group decreased due to STZ that resulted in diabetes by the rapid depletion of β-cells, which reduced the insulin release. An insufficient release of insulin causes hyperglycemia, which results in oxidative damage by the generation of reactive oxygen species and the development of diabetic complications [37]. When stevia aqueous extracts at different dose levels were given to the diabetic albino rats then their insulin levels improved significantly (Fig. 4) due to the presence of natural components (stevioside) in stevia leaves that are related to inhibition of hepatic expression of phosphoenolpyruvate carboxykinase and gluconeogenesis coupled with stimulation of hepatic glycogen synthesis that resulted in increase of insulin secretion and insulin sensitivity [38]. Evidence from other studies revealed that stevia aqueous extract elevate the insulin level due to stevioside that acts on pancreatic tissue, exerts beneficial anti-hyperglycemic effects through the PPARγ-dependent mechanism [34, 30].

The results are in collaborations with the studies conducted by Shivanna et al. [39]; Saleh et al. [40] and Abou khalil et al. [41] who concluded that aqueous extracts of Stevia rebaudiana leaves and Desert date (Balanites aegyptiaca) and Parsley (Petroselinum sativum) stevioside normalize the pancreatic cell function by restoring the insulin immune reactivity in STZ-induced diabetic rats.

The reduction in insulin release and liver glycogen level of diabetic control group rats are due to STZ (a known diabetogen) used for induction of diabetes in rats that brings about the destruction of β- cells of the islets of Langerhans [42]. However, the diabetic rats received stevia aqueous extracts (200, 300, 400 and 500 ppm/kg) were able to significantly (P < 0.05) improve the liver glycogen levels (Fig. 5). Stevioside (sweetener) present in stevia extract acts directly on pancreatic beta cells and resulted in increase of insulin secretion [12]. Increased level of Insulin enhances intracellular glycogen deposition by stimulating activities of glycogen synthase and inhibiting glycogen phosphorylase [38].

Similar results were observed by previous researchers who found that Plectranthus esculenthus extracts and Mangifera indica kernel flour-supplemented diet restored the liver glycogen levels in STZ induced diabetic rats [42, 43].

The present study suggests that aqueous extract from stevia leaves may decrease the random blood glucose level and fasting blood glucose and glycosylated (HbA1c) hemoglobin while insulin and liver glycogen levels significantly increased of the diabetic rats, compared with the diabetic and non-diabetic control rats after 8 weeks study period. It is concluded that aqueous extact of stevia with concentration 500 ppm/kg body weight of rats showed best results of all the parameters determined. It is understood from the results that stevia extract has anti-diabetic effects in albino rats, and therefore could be used as natural anti-diabetic drug for the treatment of diabetes and its associated complications.