PART 1 Antidiabetic And Antioxidant Effects Of Acteoside From Jacaranda Mimosifolia Family Biognoniaceae in Streptozotocin–Nicotinamide Induced Diabetes in Rats

How are the anti-sugar and antioxidant effects of Verbascum glycosides reflected?

Salma A. El-Marasy1, Siham M. El-Shenawy1, Fatma A. Moharram2, Nagla A. El-Sherbeeny3* 1 Department of Pharmacology, National Research Centre, Giza, Egypt; 2 Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Helwan, Egypt; 3 Department of Clinical Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Abstract

BACKGROUND: Acteoside is a phenylethanoid compound isolated from Jacaranda mimosifolia D. Don leaves with a potential antidiabetic effect.

OBJECTIVES: This study was designed to investigate the antidiabetic and antioxidant effects of acteoside in streptozotocin-nicotinamide (STZ-NA)-induced Type 2 diabetes in rats.

METHODS: Diabetes was induced by intraperitoneal (i.p.) injection of a single dose of STZ (52.5 mg/kg), 15 min following i.p. administration of NA (25 mg/kg). Rats were divided into six groups; Group I: Normal rat group received the vehicle, Group II: Diabetic control group, and Groups III-IV: Diabetic rat groups were treated by either oral acteoside (10, 20, and 40 mg/kg) or pioglitazone (30 mg/kg) for 21 consecutive days. Biochemical parameters were assessed in the serum and liver homogenates. Examination of liver sections for histopathology was also carried out.

RESULTS: Acteoside treated rats showed significantly lower levels of blood glucose, glycosylated hemoglobin, total cholesterol, triglycerides, and increased serum insulin compared to control diabetic rats. Furthermore, acteoside treated rats, in comparison to the diabetic control, demonstrated significantly reduced malondialdehyde, increased reduced glutathione liver contents, and attenuated pathological alterations in the liver. These effects were comparable to those caused by the standard antidiabetic drug, pioglitazone. In vitro, acteoside scavenged stable free radical 1,1-diphenyl-2-picrylhydrazyl.

CONCLUSION: Acteoside could be considered as a potential therapeutic agent for type 2 diabetes mellitus. However, studying further mechanisms underlying its antidiabetic effect is recommended.

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Introduction

Diabetes mellitus is a chronic metabolic disorder affecting about 450 million adults around the world. The number is expected to reach 629 million by 2045 [1]. The disease is characterized by hyperglycemia-induced by diminished insulin output from pancreatic beta cells and/or tissues’ resistance to insulin action. Type 2 diabetes is found in 90–95% of all diabetes cases. Diabetes is associated with a high risk of macrovascular and microvascular (nephropathy, neuropathy, and retinopathy) complications [2]. Lifestyle modifications, pharmacological treatments, and careful monitoring are the mainstay for diabetes management. Achieving target glycemic control helps prevent or at least can delay diabetes complications [3]. Despite the benefits of current antidiabetic drugs, every class has undesirable adverse effects. Therefore, searching for new treatments for type 2 diabetes is warranted. New drugs should be efficacious with minimal adverse effects and affordable cost. Diabetic patients use natural remedies that are thought to improve glycemic control, especially in areas where the cost of drugs imposes a real challenge [4]. The genus Jacaranda (Bignoniaceae) is found mainly in tropical and subtropical geographical areas. Jacaranda mimosifolia is native to Brazil but is also cultivated as an ornamental tree in Egypt. Acteoside (verbascoside) was isolated from the leaves of the J. mimosifolia [5]. A variety of promising activities of acteoside was reported in the previous studies including anti-inflammatory [6], [7], hepatoprotective [8], antioxidant [9], antineoplastic [10], and neuroprotective effects [11]. Previous reports have demonstrated the potential anti-hyperglycemic effect of acteoside. For example, acteoside prevented protein glycation in vitro, an activity that is correlated with antidiabetic drugs [12]. Moreover, plant extracts containing acteoside showed anti-hyperglycemic effects in experimental type 2 diabetes [13]. Furthermore, more direct testing of the potential hypoglycemic action of acteoside was reported in the study by Morikawa et al. [14]; after 2 weeks of daily oral acteoside given concurrently with a starch load in mice, glucose tolerance was improved without significant change in weight. However, the antidiabetic effect of acteoside has not been yet explored in experimental diabetes models. Therefore, this work aims to investigate the antidiabetic and antioxidant effects of acteoside in a rat model of Type 2 diabetes induced by streptozotocin-nicotinamide (STZ-NA).

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Materials and Methods

Animals

Male Wistar albino rats (weight 180–210 g) were used in the current study. Rats were purchased from the Animal House Facility of the National Research Centre (Cairo, Egypt). Animals were housed in standardized conditions and allowed to acclimatize for 7 days in the laboratory before starting the experiment. Rats had free access to standard food pellets and water ad libitum. The experimental protocol and all animal procedures were approved by the Ethics Committee of the National Research Centre, Egypt (approval number: 18/042). The committee guidelines are in line with the National Institutes of Health guide for the care and use of laboratory animals.

Materials

STZ was procured from Sigma-Aldrich (Missouri, USA), NA from Bayer Schering Pharma (Switzerland, Europe), and Pioglitazone from Amoun Pharmaceutical Industries Co., (Cairo, Egypt). A pure sample of acteoside was provided by the fourth author. Isolation of acteoside from J. mimosifolia leaves was carried out as previously reported by Moharram and Marzouk [5]. All other chemicals and reagents were of analytical grade.

Diabetes induction

After 12 h fasting, diabetes was induced in rats by a single intraperitoneal dose of STZ (52.5 mg/kg) dissolved in 0.1mol/L citrate buffer (pH 4.5) [15]. STZ was given 15 min after intraperitoneal injection of NA (25 mg/kg) [16]. The next 24 h following STZ injection, a 5% glucose solution was given to rats to overcome the risk of death that may result from hypoglycemic shock. After 48 h of STZ injection, blood glucose levels were estimated in blood samples withdrawn from the tail vein using a portable glucometer. Rats were considered diabetic only if fasting blood glucose was ≥250 mg/dL.

Experimental design

After weighing rats, they were randomly distributed into six groups (six rats per group): Group I: Served as the normal control (given only distilled water); Group II: Diabetic control group; Groups III-V: Diabetic rats treated with oral acteoside for 3 weeks (10, 20, and 40 mg/kg), respectively; and Group IV: Diabetic rats treated with oral pioglitazone (30 mg/kg) for 3 weeks. The vehicle was given to normal and diabetic control rats. Acteoside or pioglitazone treatments were started after confirmation of hyperglycemia, 48 h following STZ injection. Acteoside and pioglitazone doses were selected according to the previously published data by Liu et al. [17] and by Vidal et al. [18], respectively.

Bodyweight changes Initial body weight

was determined by weighing each rat before the beginning of the experiment. Furthermore, the final body weight for each rat was estimated 24 h after the last dose of administration of either vehicle or treatment according to the study design. The percent change in body weight was calculated as follow:

Biochemical analysis

Glucose level

Glucose level (mg/dl) was measured colorimetrically using kits purchased from (Biodiagnostic, Egypt) based on the method by Trinder [19].

Serum insulin level

Serum insulin level (µIU/ml) was measured by enzyme-linked immunosorbent kit Rat Insulin (INS) ELISA (Cusabio Biotech Co., Ltd., Hubei, China) following the manufacturer’s protocol. Glycosylated hemoglobin level (HbA1c) Glycosylated Hb level (ng/ml) was estimated using (Rat [HbA1c] ELISA) purchased from Glory Science, following the manufacturer’s protocol. Serum triglyceride and total cholesterol levels Triglycerides level (mg/dl) and total cholesterol level (mg/dl) were determined using enzymatic methods. Diagnostic kits from Biodiagnostic, Egypt, were used following the method of Fossati and Prencipe [20].

Preparation of tissue homogenate

ice-cold 0.1 M phosphate buffer (pH 7.4). Then, the homogenate was centrifuged at 4000 rpm for 5 min in a cooling centrifuge (2k15; Sigma/Laborzentrifugen). The supernatant was then used for determining the liver contents of malondialdehyde (MDA) and reduced glutathione (GSH). All rats were sacrificed by decapitation under anesthesia, and then their livers were removed. A portion from the liver was homogenized in (20% w/v)

The hepatic lipid peroxide content

Hepatic MDA content (nanomoles/gram of liver tissue) was determined colorimetrically, as described by Satoh [21] using a diagnostic kit purchased from BioDiagnostic Co., Egypt.

Hepatic GSH content

Hepatic reduced GSH content (mmol/g liver tissue) was estimated by a colorimetric method according to Beutler et al. [22] using a kit purchased from BioDiagnostic Co., Egyp

Evaluation of the antioxidant effect (in vitro)

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined following the method described by Peiwu et al. [23]. If the compound is an antioxidant that can donate hydrogen, it will react with DPPH. The reaction will induce a color change of DPPH from deep violet to yellow. This change in color was measured using a spectrophotometer at 517 nm. Ascorbic acid at 0.1 M concentration was the standard [24]. DPPH radical scavenging activity was calculated according to the equation: Radical scavenging activity % = (Ac−At)/ Ac×100 (1) Where Ac and At are the absorbance of the control (DPPH) and acteoside, respectively.

Histopathological examination

Liver tissues were taken from rats and fixed in 10% formaldehyde for 24 h. Then tissues were processed to obtain 4 µm paraffin-embedded sections. The tissue sections were stained by hematoxylin and eosin stain and examined using a light microscope [25].

Statistical analysis

Results are expressed as mean ± SEM for six rats per group. Comparisons between more than two groups were carried out using one-way ANOVA followed by Tukey’s multiple comparisons test. All analyses were done using GraphPad Prism 6.0 statistical package for Windows (GraphPad, San Diego, Calif.). Statistical significance was set at p < 0.05.

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Results

Effect of acteoside on body weight

Figure 1 reveals that diabetes induced by a single i.p. dose of STZ (52.5 mg/kg) 15 min after the i.p. injection of NA (25 mg/kg) led to a significant body weight loss of a percentage of body weight by 11.86 ± 1.32 after 3 weeks of diabetes induction. Meanwhile, normal rats showed a significant gain in % body weight by 22.74 ± 1.85. Acteoside was given orally to diabetic rats in doses of 10, 20, and 40 mg/kg for 21 successive days resulting in a significant gain in the percentage of body weight by 19.05 ± 1.14, 16.92 ± 1.00, and 22.98 ± 1.21, respectively. Similarly, pioglitazone orally administered at 30 mg/kg showed a significant gain in the percentage of body weight by 25.59 ± 2.13.

Effect of acteoside on blood glucose, insulin levels, and HbA1c

As demonstrated in Table 1, diabetic rats showed a significant elevation in blood glucose to a level of 318.70 ± 13.8 mg/dl whereas normal rats' mean level was 81.51 ± 4.15 mg/dl. Three weeks of oral treatment with acteoside (10, 20, and 40 mg/kg) caused a significant reduction of blood glucose to 111.30 ± 0.61, 74.88 ± 3.23, and 75.15 ± 8.45 mg/dl, respectively, versus control value. Furthermore, pioglitazone (30 mg/kg) reduced blood glucose level to 103.00 ± 3.12 mg/dl versus the control value of diabetic rats. Regarding serum insulin levels, control diabetic rats showed a significantly decreased serum insulin level of 1.25 ± 0.07 µIU/ml whereas normal rats 5.32 ± 0.27 µIU/ml. Oral treatment with acteoside (10 mg/kg) resulted in a significant elevation in serum insulin level to be 3.23 ± 0.06 µIU/ml versus control diabetic and normal groups. Acteoside in a dose of 20 mg/kg restored serum insulin level to be 5.38 ± 0.21 µIU/ml versus control value of diabetic rats. Oral treatment with acteoside 40 mg/kg significantly increased serum insulin level to be 6.80 ± 0.20 µIU/ml versus control diabetic and normal values. Pioglitazone treatment resulted in a significant increase in serum insulin level to be 8.00 ± 0.16 µIU/ml versus control diabetic and normal values (Table 1). As depicted in Table 1, control diabetic rats had a significantly elevated HbA1c level of 40.30 ± 3.39 ng/ml as compared to mean normal values of 3.36 ± 0.21 ng/ml. Acteoside in doses of 10 and 20 mg/kg showed a significant elevation in HbA1c levels to be 24.12 ± 1.88 ng/ml and 16.72 ± 1.06 ng/ml, respectively, versus values of control diabetic rats and normal rats. Acteoside (40 mg/kg) significantly reduced HbA1c level to be 10.18 ± 0.92 ng/ml versus control diabetic rats. Similarly, pioglitazone significantly reduced the HbA1c level to be 6.42 ± 0.29 ng/ml versus the values of control diabetic rats.

Effect of acteoside on total cholesterol and triglyceride levels

The effect of acteoside on total cholesterol and triglyceride is shown in Table 2. Control diabetic rats showed a significant increase in total cholesterol to be 139.35 ± 4.28 mg/dl as compared to normal values 94.38 ± 2.38 mg/dl. Oral treatment with acteoside (10, 20, and 40 mg/kg) significantly reduced total cholesterol level to be 96.24 ± 1.08, 90.62 ± 1.60, and 95.14 ± 4.65 mg/dl, respectively, versus control diabetic rats’ values. In the same manner, pioglitazone (30 mg/kg) restored the total cholesterol level to be 96.80 ± 4.17 mg/dl versus the control value of diabetic rats. Concerning triglyceride levels, control diabetic rats showed a significant elevation in triglycerides level to be 153.90 ± 3.33 mg/dl as compared to normal values 107.80 ± 2.00 mg/dl. Oral treatment with acteoside (10, 20, and 40 mg/kg) restored total cholesterol level to be 118.59 ± 6.46, 100.27 ± 4.19, and 91.89 ± 3.20 mg/dl versus control value of diabetic rats. Furthermore, pioglitazone restored triglyceride level to be 108.98 ± 9.14 mg/dl versus control diabetic rats’ value.

Effect of acteoside on MDA and GSH in liver

Results depicted in Figure 2a show that control diabetic rats significantly elevated hepatic MDA content to be 65.27 ± 2.63 nmol/g as compared to normal rats 38.94 ± 1.57 nmol/g. Oral treatment with acteoside (10, 20, and 40 mg/kg) restored hepatic MDA contents to be 32.63 ± 2.14, 36.14 ± 2.75, and 39.40 ± 1.14 nmol/g, respectively, versus control diabetic rats’ value.

Similarly, pioglitazone (30 mg/kg) restored hepatic MDA content to be 39.30 ± 3.49 nmol/g versus control diabetic rats’ value. Regarding hepatic GSH content, control diabetic rats significantly reduced hepatic GSH content to be 6.61 ± 0.17 mmol/g whereas normal values 8.69 ± 0.16 mmol/g. Acteoside (10, 20, and 40 mg/kg) significantly increased hepatic GSH contents to be 8.99 ± 0.37, 8.30 ± 0.21, and 8.56 ± 0.39 mmol/g versus control diabetic rats’ value. Furthermore, oral treatment with pioglitazone (30 mg/kg) restored hepatic GSH to be 9.00 ± 0.30 mmol/g versus control diabetic rats’ value (Figure 2b).

In vitro antioxidant activity of acteoside

In vitro antioxidant activity of acteoside versus ascorbic acid (0.1 M concentration), using DPPH radical scavenging activity method is depicted in Figure 3. After a reaction time of 5 min, different concentrations of acteoside (200, 150, 100, and 50 mg/ml) showed maximum reactive reaction rates of 74.4, 74, 73, and 72.3%, respectively. In the same manner, the reactive reaction rate of L-ascorbic acid was 82.5%.

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Effect of acteoside on histopathologic examination of the liver

In normal rats, no histopathologic alterations were found. Normal histological structure of the central vein and surrounding hepatocytes in the parenchyma was recorded in the normal group (Figure 4a). Pathological changes that were recorded in the liver of control diabetic rats were severe dilatation and congestion of the central and portal vein associated with collagen proliferation and few inflammatory cells infiltration in the periductal tissue surrounding the hyperplastic bile ducts in the portal area (Figure 4b and c). Apoptosis was detected in a few hepatocytes associated with diffuse cells proliferation (Figure 4d). Rats that were orally treated with acteoside (10, 20, and 40 mg/kg), respectively, showed marked reduction of the previously mentioned histopathological lesions (Figures 4e, f, and 5a-d) that were observed in the control diabetic group (Group II). Pioglitazone-treated diabetic group (Group IV) showed few inflammatory cells infiltration in the portal area (Figure 5e). Furthermore, there was a diffuse kupffer cells proliferation in between the hepatocytes (Figure 5f).