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The activity of curcumin combined with ZnCl2 on streptozotocin-induced diabetic rats: An anti-diabetic, anti-hyperlipidemic study

Suhailah Saud Al- Jameel1*

1Department of Chemistry, College of Science, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia.

Correspondence: Suhailah Saud Al- Jameel, Department of Chemistry, College of Science, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia. [email protected]


ABSTRACT

Diabetes plays a direct role in the development of oxidative stress. Numerous molecules have been evaluated to see whether they can improve diabetes. Curcumin (CUR) and Zinc Chloride (ZnCl2) have various health-beneficial properties. The present study aims to assess the efficacy of CUR combined with ZnCl2 on oxidative changes in Streptozotocin(STZ)-induced diabetes in rats. Forty-eight Albino rats were treated with STZ (60 mg/kg b.w. intraperitoneally) and divided into six groups (G1-G6). G1 was the negative control, while G2 was the positive control. G3,G4 received STZ+CUR (100, 200 mg/kg b.w, respectively). G5 and G6 received (15 mg/kg b.w STZ+ZnCl2 +100 mg/kg b.w CUR) and (15 mg/kg b.w STZ+ZnCl2 +200 mg/kg b.w CUR), respectively. Serum levels of glucose, insulin, lipid profile, liver transaminases (ALT, AST), and lipid peroxidation (GSH, SOD, TBA, and CAT) were determined. Furthermore, morphological changes of the liver were studied. Results: The results showed that glucose, TC, TG, LDL-C, ALT, AST, SOD, TBA, and CAT levels increased, while insulin, HDL-C, and GSH decreased in the STZ group. Treatment with (200 CUR +15 mg/kg b.w ZnCl2) ameliorated these changes with superior results than other groups. The study confirmed that CUR+ZnCl2 improved the oxidative stress condition in rats' serum and tissues; therefore, they could work effectively as preventive agents against STZ- induced diabetes.

Keywords: Curcumin, Streptozotocin, Oxidative stress, ZnCl2, Diabetic rats


Introduction  

Diabetes mellitus (DM) is a metabolic disorder with a variety of etiologies [1, 2]; it is characterized by chronic hyperglycemia and disturbance in protein, fat, and carbohydrate, metabolism due to a lack of insulin excretion/or insulin action. DM has become a public health threat around the globe [3-5]; it is also a major health issue in the Kingdom of Saudi Arabia. The progression of diabetic hepatosclerosis is triggered by oxidative stress. It is caused by an excess of reactive oxygen/nitrogen radicals, as well as a reduction in endogenous antioxidant systems, which leads to the development of degenerative diseases [6, 7]. Although there are several anti-diabetes drugs that regulate hyperglycemia, treatment options that target DM sequelae, including oxidative stress and dyslipidemia, are limited. Therefore, the focus of the current research is to explore new factors and strategies that would curb the spread of the diabetes epidemic and its fatal consequences. A combination of anti-diabetic drugs supplied with other chemicals has emerged as an interesting strategy for managing hyperglycemia and other DM disorders. Curcumin (CUR) is a South Asian spice and is a highly pleiotropic molecule that is a widely-known antioxidant [7] and has been demonstrated to have anti-bacterial, [8] anti-inflammatory, anticancer [9], antiatherosclerotic, antimicrobial [10], wound healing [11], and other pharmacological actions. Moreover, it has the ability to scavenge reactive oxygen species (ROS), including alkoxy radicals, hydroxyl radicals, and superoxide radicals, directly in the living cells [12], completely degrades superoxide through mimicking superoxide dismutase [13, 14], and acts as a protective barrier in conditions when high levels of peroxynitrite and hydrogen peroxide molecular oxidants are produced inside the cells [15].

Zinc, an essential trace element, has been found to improve glucose absorption and transport in numerous tissues while suppressing pancreatic insulin release and raising pancreatic insulin content [16]. Evans' research [17] has also revealed that zinc supplementation has a beneficial influence on the progression of age-related macular degeneration; individuals who took antioxidants and zinc supplements were less likely to lose visual acuity. Furthermore, studies have found promising results when curcumin is used in combination with anti-diabetic drugs [18] or other phytochemicals [19] to regulate glycemia and reduce other diabetic problems.

The aim of this research is to study how CUR combined with ZnCl2 affects the levels of oxidative stress biomarkers and serum lipid profile; and how the combination exerts defensive actions against morphological changes in liver tissue in STZ-induced diabetic rats. (Figure 1) This would benefit in determining the role of the combination, as well as alleviating the deteriorating effects of diabetes worldwide.

 

Figure 1. Enhancing curcumin's bioavailability by combining it with ZnCl2 - application on rats

 

Materials and Methods

Chemicals and experimental procedures

High purity analytical grade chemical reagents were procured from Sigma, Merck, Aldrich. All kits were manufactured by Biosystems (Alcobendas, Madrid, Spain), Sigma (St. Louis, MO, USA), and Biodiagnostic (Cairo, Egypt). Seventy-two Albino Wister male rats, in the weight range of 200g to 205g, were purchased from Animal House, King Faisal University. Rats were kept in an individual cage in a room at a controlled temperature of 25°C, 50% humidity, 12 h of light and dark cycle with screened bottoms. These rats were fed for 10 days with the basal diet containing corn starch (70%), casein (10%), corn seed oil (10%), cellulose (5%), a mixture of salt (4%), and vitamins (1%). Then, rats were weighed and separated into six groups, with 12 rats in every group. The assigned 6 diet groups were as follows: G1 signified the negative control (NC), G2 designated the positive control (PC) in which rats were injected by STZ (60 mg/kg BW), G3 enclosed the rats that received STZ (60 mg/kg BW) + CUR (100 mg/kg BW), in G4 rats were treated with STZ (60 mg/kg BW) + CUR (200 mg/kg BW), rats in G5 were treated with [STZ (60 mg/kg BW) + CUR (100 mg/kg BW)+ ZnCl2 (15 mg/kg BW)] and those in G6 were treated with [STZ (60 mg/kg BW) + CUR (200 mg/kg BW) + ZnCl2 (15 mg/kg BW)]. Decapitation was used to kill all the rats at the end of the experiment (21 days). Following Schermer [20], blood samples were obtained from the orbital plexus using heparinized capillary glass tubes. First, a dry clean centrifuge tube was utilized to place each blood sample and then centrifuged at 4°C with the revolution of 1500g for 30 min to acquire the blood serum. An approval (lRB-2018-10-216) from the University was obtained to perform the study.

Biochemical analysis

ALT, AST, glucose, insulin, and lipid profile assay

ALT, AST activities [21], Glucose [22], Insulin [23], Total cholesterol [24], Triglycerides [25] and HDL-C and LDL-C [26]  concentrations were estimated by using standard kits.

Thiobarbituric acid (TBA) assay

Lipid peroxidation was estimated according to Hommouda et al.’s method [27] and upon the TBA reactivity principle. Briefly, 2.5mls of 10% trichloracetic acid (TCA) and 0.5ml of plasma were mixed in test tubes. After 15 minutes of incubation at 90°C and then cooling via a cold water bath, the mixture was centrifuged for 10 minutes at 3000 rpm. Then 2mls of the supernatant was added to 1ml of 0.675% TBA. The tubes were capped and incubated for 15 minutes at 90°C and then allowed to cool down at room temperature. The optical density was measured at 532nm by a spectrophotometer (Schimadzu, AA6800, Tokyo, Japan).

Superoxide dismutase (SOD) assay

The SOD activity of erythrocytes was determined in the hemolysates via commercially available kits from Randox Laboratory, Crumlin, Ireland. Hemolysis of erythrocytes was done by using ice-cold deionized water followed by strong vortexing. SOD activity was determined through the superoxide radicals that are generated from xanthine and xanthine oxidase, which in turn react with 2-(4- iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to produce a red formazan dye. The degree of inhibition of such a reaction is correlated to SOD activity [28].

Catalase (CAT) assay

CAT activity was determined via Aebi method [29], which employs a UV spectrophotometer at 240nm. To determine the ceruloplasmin activity in serum, 5ml of phenylenediamine substrate was added to 2 tubes (test and curve tubes). Then, 1ml sodium azide (NaN3) solution was added only into the curve tube while 100μls of serum was added to both tubes. Both tubes were mixed and kept for 15 minutes at 37°C. Finally, 1ml of NaN3 was added to the test tube only, and a spectrophotometer was used to measure the OD at 546nm.

Glutathione (GSH) assay

Concentrations of serum GSH were measured according to Beutler et al. method [30].

Statistical analysis

Data were analyzed by comparing the results of various treatment groups to the results of control groups. All data were presented as mean±SD. ANOVA with posthoc least significant difference test (LSD) at (p 0.05) were integrated and used to examine significant differences between values.

Histopathology

 Liver specimens were fixed in 10% formalin, paraffinized, sectioned (4µm thickness), and stained using hematoxylin and eosin dyes. Light microscopy was used to demonstrate hepatic pathological alterations such as degeneration, atrophy, cell distortion, and necrosis, as well as the effectiveness of micronutrients in alleviating these pathological aspects [31].

Results and Discussion

Table 1 shows the effect of CUR+ZnCl2 on serum glucose during 21 days in STZ-induced diabetes in rats. The level of serum glucose increased significantly (p≤0.05) in PC group (STZ-treated); and reduced significantly (p≤0.05) in CUR (100 & 200 mg/kg b.w) and CUR coupled with ZnCl2 (15 mg/kg b.w) treated groups, concerning the PC.

 

 

Table 1. Effect of curcumin combined with ZnCl2 on serum glucose level in diabetic rats

Time

Treatments

Glucose (mg/dl)

Day 0

Day 7

Day 14

Day 21

G1 (NC)

99.907a ±0.100

100.513f±0.320

99.955e±0.027

100.297f±0.258

G2 (PC)

99.752a±0.187

391.685a±0.624

435.090a±0.535

502.443a±0.258

G3 (STZ+ CUR 100mg/kg b.w)

99.756a±0.182

222.407b±0.300

178.485b±0.310

163.107b±0.368

G4 (STZ+ CUR 200mg/kg b.w)

99.982a±0.035

218.027c±0.341

177.987b±0.328

153.540c±0.262

G5 (STZ+ ZnCl2 15 mg/kg b.w + CUR 100 mg/kg b.w)

99.912a±0.073

202.167d±0.364

163.450c±0.217

111.118d±0.303

G6 (STZ+ ZnCl2 15 mg/kg b.w + CUR 200 mg/kg b.w)

99.742a±0.208

193.217e±0.274

151.457d±0.408

101.993e±0.140

LSD

0.437

1.122

0.990

0.790

Results for 12 rats in every group are shown as mean±SD; statistical significance at p≤0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

 

 

The levels of insulin were given in Table 2. The results demonstrated that the level of serum insulin was reduced significantly (p≤0.05) in PC when compared with NC. Administration of CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl2 (15 mg/kg b.w) led to a marked increase (p≤0.05) in insulin level when compared to PC. The treatment of 200 mg/kg b.w CUR + ZnCl2 15mg/kg b.w gave results near to the NC after 21 days.

The results of TC, TG, HDL-C, and LDL-C were given in Table 3. The data indicated that serum levels of TC, TG, and LDL-C were significantly (p≤0.05) elevated in PC; however, HDL-C was significantly (p≤0.05) reduced compared to NC and other treated groups. A significant (p≤0.05) inhibition in TC, TG, and LDL-C levels was observed in CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl2 treated groups while HDL-C levels were elevated, compared to PC. Moreover, the group that was treated with CUR 200 + ZnCl2 15mg/kg b.w, gave the best results.

 

 

 

Table 2. Effect of curcumin+ZnCl2 on serum insulin level in diabetic rats

Time

Treatments

Insulin (pg/ml)

Day 0

Day 7

Day 14

Day 21

G1 (NC)

45.852a±0.088

46.067a±0.394

46.128a±0.147

45.850a±0.086

G2 (PC)

45.882a±0.094

11.205e±0.176

8.347f±0.112

6.255f±0.121

G3 (STZ+ CUR 100mg/kg b.w)

45.967a±0.037

18.865d±0.169

27.002e±0.160

31.977e±0.031

G4 (STZ+ CUR 200mg/kg b.w)

45.985a±0.035

22.298c±0.278

28.935d±0.223

36.218d±0.139

G5 (STZ+ ZnCl2 15 mg/kg b.w + CUR 100 mg/kg b.w)

45.960a±0.041

22.710c±0.223

30.285c±0.208

38.730c±0.186

G6 (STZ+ ZnCl2 15 mg/kg b.w + CUR 200 mg/kg b.w)

45.930a±0.058

28.280b±0.260

32.843b±0.275

42.140b±0.153

LSD

0.184

0.754

0.564

0.374

Results for 12 rats in every group are shown as mean ± SD with statistical significance at p ≤ 0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

 

 

Table 3 shows the effect of CUR (100 & 200 mg/kg b.w) and CUR + ZnCl2 15mg/kg b.w on serum AST and ALT enzymes. ALT and AST activities significantly (p≤0.05) increased in PC group compared with NC and other treated groups. The CUR and CUR + ZnCl2 treated groups showed a lower compared to PC, especially at CUR 200 + ZnCl2 15mg/kg b.w treated group.

STZ exposure significantly (p≤0.05) elevated the levels of SOD, TBA, and CAT, while GSH was significantly (p≤0.05) decreased as compared with the NC group. Administration of CUR (100 & 200 mg/kg b.w) and CUR combined with ZnCl2 15mg/kg b.w significantly (p≤0.05) lowered SOD, TBA and CAT levels (Table 4). CUR 200 + ZnCl2 15mg/kg b.w treated group improved the levels of GSH and SOD close to NC.

 

 

Table 3. Effect of curcumin combined with ZnCl2 on serum AST and ALT concentration in diabetic rats

Treatments

ALT (U/L)

AST (U/L)

G1 (NC)

63.395f±0.143

134.097f±0.080

G2 (PC)

153.623a±0.191

326.940a±0.041

G3 (STZ + CUR 100mg/kg b.w)

107.103b±0.135

218.202b±0.104

G4 (STZ + CUR 200mg/kg b.w)

92.367c±0.208

198.118c±0.063

G5 (STZ + ZnCl2 15mg/kg b.w + CUR 100mg/kg b.w)

86.407d±0.297

172.235d±0.186

G6 (STZ + ZnCl2 15mg/kg b.w + CUR 200mg/kg b.w)

67.025e±0.076

141.173e±0.123

LSD

0.544

0.318

Results for 12 rats in every group are shown as mean±SD with statistical significance at p≤0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

 

Table 4. Effect of curcumin+ZnCl2 on serum GSH, SOD, TBA, and CAT concentration in diabetic rats

Treatments

GSH

Mmol/g

SOD

U/ml

TBA

(nmol/L)

CAT

nmol/mg protein

G1 (NC)

23.847e±0.091

6.497e±0.138

31.473f±0.123

21.392f±0.220

G2 (PC)

15.482a±0.196

17.415a±0.137

67.492a±0.146

51.308a±0.162

G3 (STZ + CUR 100mg/kg b.w)

43.772b±0.129

11.433b±0.151

52.413b±0.203

39.970b±0.045

G4 (STZ + CUR 200mg/kg b.w)

38.723c±0.103

9.355c±0.176

43.445c±0.169

32.604c±0.107

G5 (STZ + ZnCl2 15mg/kg b.w + CUR 100mg/kg b.w)

30.775d±0.134

9.350c±0.118

41.243d±0.176

31.150d±0.052

G6 (STZ + ZnCl2 15mg/kg b.w + CUR 200mg/kg b.w)

23.868e±0.086

7.122d±0.061

33.295e±0.165

22.260e±0.159

LSD

0.370

0.390

0.478

0.419

Results for 12 rats in every group are shown as mean±SD with statistical significance at p ≤ 0.05 than NC (ANOVA was obtained via Fischer's LSD test). The values with distinct superscript letters (a, b, c, and d) differ significantly from each other at p ≤0.05.

 

 

Histopathological investigations revealed that the diabetic liver had a high degree of hydropic degeneration accompanied by coagulative necrosis (Figure 2). Treatment with CUR (100 & 200 mg/kg b.w) and their combination with ZnCl2 15mg/kg b.w showing improvement appeared in the hepatocytes, Kupffer cells nuclei, and blood sinusoids (Figure 2).

 

 

a)

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