1. Introduction
Diabetes mellitus is a chronic metabolic disease that is principally characterized by inflammation, insulin resistance, hyperlipidemia, hyperglycemia, increased formation of reactive nitrogen (RNS) species, and reactive oxygen species (ROS). Chronic hyperglycemia reduces the antioxidant capacity of CAT, SOD, GPx, and erythrocyte glutathione (GSH), provoking insulin resistance through the inhibition of insulin signals [
1]. In type 2 diabetes mellitus, the main cause of mortality and morbidity are premature macrovascular complications which, despite current treatments including healthy nutrition, daily physical activity, monitoring lipid profile and arterial pressure, and oral antihyperglycemic drugs (thiazolidinediones, sulphonylureas, biguanides, and 𝛼-glycosidase inhibitors) are related to adverse side effects or reduction in response after prolonged treatment [
2]. These treatments are inadequate for most patients [
3]; therefore, it is imperative to explore options in medicinal plants and nanomedicine to better manage the disease.
Numerous studies have shown that nanoparticles can adsorb, fix, and carriage other compounds due to their large surface area [
4]. Currently, there is growing evidence of the effectiveness of the use of nanoparticles for type 2 diabetes mellitus (T2DM) management and prevention. Nanomedicine is an emerging form of therapy that focuses on alternative drug delivery and improvement of the treatment efficacy while reducing detrimental side effects to normal tissues [
5].
Selenium is an essential element for humans and can ameliorate the activities of the selenoenzymes and antioxidant enzymes, including GPx [
6], which is associated with the immune system, helping to enhance the immune response in both the innate and acquired immune systems [
7]. Selenium demonstrated insulin-like activities when administered in rats stimulating glucose transport and insulin-sensitive cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) when incubated with rat adipocytes [
8]. Nanoparticles of selenium have received great attention for their unique biological activities and low toxicity and can alleviate hyperlipidemia, hyperglycemia, and in STZ-induced diabetic rats, possibly by eliciting insulin-mimetic activity [
9]. SeNPs establish an attractive platform to transport different drugs to the target of action [
10] due to the good absorptive ability produced by the interaction between -C-N-, -COO, C=O, and -NH2, groups of proteins with the nanoparticles of Se [
7].
Bioactive compounds isolated from herbal plants have been shown to have different pharmacological properties. Selected compounds from plants have been evaluated for their potential in the management of diabetes. Among these naturally occurring products, we find luteolin and diosmin. In this context, luteolin (LU; 3’,4’,5,7-tetrahydroxyflavone) is a bioflavonoid widely distributed in the plant kingdom and consists of a C6-C3-C6 structure that possesses a 2–3 carbon double bond and four hydroxyl groups at carbons 5, 7, 3′, and 4′ [
11]. Luteolin, reduce neuroinflammation and ameliorate brain insulin resistance protecting against the development of Alzheimer’s disease [
12]. Luteolin alleviates oxidative stress, neuroinflammation, and neuronal insulin resistance restoring blood adipocytokines levels in the mouse brain [
13]. Another research demonstrated that LU significantly reduces insulin resistance, improves homeostatic model assessment for insulin resistance (HOMR-IR), glycosylated hemoglobin (HbA1c,) and reduces blood glucose levels [
14]. LU treatment improved malondialdehyde (MDA) content, SOD activity, and the expression of the heme oxygenase-1 (HO-1) protein [
15]. Diosmin, a bioflavonoid glycoside (diosmetin 7-rutinoside) found in citrus fruits, can stimulate the secretion of β-endorphin, associated with the improvement of hyperglycemia by regulating glucose homeostasis and increasing glucose uptake in soleus muscle and reducing hepatic gluconeogenesis [
16]. Previous studies have reported a significant decrease in C-reactive protein (CRP), HbA1c, and levels of plasma lipids such as triglycerides (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL-CHO), very low-density lipoprotein cholesterol (VLDL-CHO), phospholipids, and free fatty acids [
13]. Diosmin increased the activity of antioxidant enzymes, such as GPx, and SOD [
17]. Diosmin possesses neuroprotective effects against long-term potentiation deficits and TBI-induced memory due to a decrease in the pro-inflammatory cytokine TNF-α level in the hippocampus [
18].
The main objective of the present study is to design a new therapeutic strategy for type 2 diabetes where combined SeNPs with flavonoids (luteolin and diosmin) to investigate whether improve the therapeutic effect by a synergic effect on STZ-induced diabetes mellitus in C57BL/6 mice.
3. Discussion
Selenium nanoparticles (SeNPs) were prepared by using ascorbic acid as reducer and gum Arabic (GA) as the stabilizer in a facile synthetic approach where selenium oxyanion (SeO
32) in an aqueous medium which when reacting with the ascorbic acid, is reduced to elemental selenium (Se
0). The zeta potential is an electrokinetic potential associated with the stability of nanosystems used to determine the stability of nanoparticles [
21]. The zeta potential of the synthesized SeNPs and GA/LU/DIO-SeNPs indicated its stability in colloidal solution, suggesting that the repulsion force of bare SeNPs was high and thus much more difficult to aggregate in an aqueous solution. The results demonstrated that nanoparticles did not precipitate over a storage period of 175 days, confirming their stability. The SeNPs without GA precipitates, lost transparency after seven days, supporting that the addition of Arabic gum enhances the stability of the GA/LU/DIO-SeNPs, causing the nanoparticles to remain dispersed during the storage period. The negative charge on the surface of the NPs indicates major stability of gum-stabilized nanoparticles due to the intrinsic capping nanoparticles by proteins and polysaccharides of the gum [
22]. This was verified by utilizing TEM and DLS analysis. The morphology of SeNPs and GA/LU/DIO-SeNPs in TEM analysis showed spherical particles, monodisperse and homogeneous with a relatively smaller diameter. This phenomenon can be due to GA changing the interface of NPs controlling their growth and stabilizing the nanoparticles solution due to their active hydroxyl groups and complex branch structures, indicating that GA, which was adsorbed on the surface of SeNPs, prevents their aggregation.
The nanoparticle solutions showed characteristic absorption peaks in UV/vis spectrum at 270 nm, associated with surface plasmon resonance of SeNPs. These observations agree with previous investigations on spherical SeNPs synthesis [
23]. FTIR analysis was performed to evaluate the interaction between SeNPs and flavonoid molecules. The spectrum of GA/LU/DIO-SeNPs provides evidence that GA and LU/DIO form part of the nanoparticle.
The highest entrapment of 86% suggested a relatively large affinity between luteolin and diosmin and the nanoparticle matrix. Encapsulation efficiency could be ascribed to the accessibility and amount of potential binding sites among the flavonoids and Se. The synthesis process, particle size, and encapsulation reaction are some factors that affect the EE, as well as luteolin and diosmin as core materials also contribute to the EE loaded in SeNPs. In addition, the smaller particle size of GA/LU/DIO-SeNPs with a greater specific surface area and stronger adhesion capacity participated in the greater encapsulation efficiency of 86.1%.
The in vitro release of the drug is considered an important factor in the prediction of drug bioavailability from different formulations. Within the first 60 min, the burst release phase was predominantly caused by adsorbed, or desorption of flavonoids surface bound. In the second phase, the release was slower, associated with nanoparticle matrix degradation or erosion, the channels of the nanoparticles, and diffusion of the flavonoid through the pores [
24].
Diabetes is characterized by the loss or degradation of structural proteins exhibiting a severe loss in body weight caused by proteolysis and an insulin deficiency triggering a reduction in the protein content in the muscular tissue [
25]. GA/LU/DIO-SeNPs prevent loss of weight in diabetic mice group suggesting that they may reduce glycogenolysis, gluconeogenesis, and proteolysis to ameliorate the level of insulin [
25]. In contrast, food intake reduction is associated with neuronal pathways and complex hormonal involving satiety modulations and appetite [
25]. Results indicated that food intake reduction simply reduces energy intake which eventually lowers fat mass and blood glucose [
26]. GA/LU/DIO-SeNPs can regulate water consumption in T2DM mice and alleviates hyperglycemia symptoms.
In the OGTT, was observed an improvement in blood glucose levels in mice treated with nanoparticles as compared to the control group. Treatment with GA/LU/DIO-SeNPs noticeably ameliorated impaired glucose tolerance by enhancing insulin resistance as well as the increase in peripheral utilization of glucose [
27]. Results suggest that GA/LU/DIO-SeNPs can exert a hypoglycemic effect through the insulin-like effect of improving glycogenesis and enhancing cellular uptake of glucose compared to the GBL (sulfonylurea), which activates the release of insulin from the beta cells of the pancreas [
28]. Results agree with previous investigations indicating that many flavonoids produced stimulation of insulin release and, in consequence, exert a hypoglycemic effect [
29]. Results indicated that nanospheres treatment obviously alleviated insulin sensitivity observed in insulin tolerance tests, subsequently promoting glucose metabolism and declining fasting blood glucose levels.
The glucose peak in nondiabetic groups in the experiment maintained normal blood glucose levels, while diabetic animals showed a severe hyperglycemic effect throughout the experimental period. GA/LU/DIO-SeNPs treatment showed significantly fasting blood glucose levels compared with diabetic mice, and blood glucose returned to baseline levels. Findings suggested a significant recovery in the plasma insulin level.
Hemoglobin is the protein that carries oxygen to different cells of the organism. An increased level of glucose in the blood leads to its binding to hemoglobin generating glycated hemoglobin or causing hemolysis of red blood cells by increased lipid peroxide [
30] associated with the production of ROS related to hyperglycemia causes [
31]. Administration of LU/DIO, SeNPs, and GA/LU/DIO-SeNPs reduced HbA1c concentration compared to the untreated diabetic mice. These activities may be associated with the antioxidant effects and their capacity to reduce lipid peroxidation of these treatments, as was demonstrated from this investigation results.
Generally, diabetes is associated with dyslipidemia due to the insufficient or absence of insulin which participates in the regulation of lipid metabolism. Previous investigations have demonstrated that hypertriglyceridemia in diabetes stimulates a change in the oxidant-antioxidant balance and, consequently, increases lipid peroxidation [
32].
In streptozotocin-induced diabetic mice, hypercholesterolemia was observed with an increased level of triglycerides, total cholesterol, and LDL-cholesterol due to an increase in cholesterol biosynthesis and intestinal absorption [
33]. Treatment with nanoparticles decreases triglycerides, total cholesterol, and LDL-cholesterol to a significant level (
p < 0.05); in addition, it produced a significant increase in HDL levels compared with nondiabetic mice. Results demonstrated that NPs have hypocholesterolemic properties due to improving lipid profiles, also causing a decrease in antioxidant enzymes, and protecting the damage to the liver and kidney. An imbalance in the body’s redox homeostasis produces oxidative stress causing damage to cell structures, including nucleic acids, proteins, carbohydrates, and lipids which is an improvement by nanospheres reestablishing the level of lipid profiles towards the normal group.
The administration of STZ to mice causes deterioration in the activities of antioxidant-related enzymes such as CAT, SOD, and GPx. When the activities of these antioxidant enzymes are reduced by the excess of hydrogen peroxide radical and superoxide anion, these stimulate ROS production, and lipid peroxidation. Thus, ROS cause the partial destruction of β-cells playing an important role in the pathology of diabetes mellitus. Supplementation of GA/LU/DIO-SeNPs in STZ-induced diabetic mice to increase the activities of CAT, GPx, and SOD in the kidney and liver. It also showed a significant reduction in kidney and liver MDA levels compared to the diabetic control. The improvement of oxidative stress in diabetic mice may be due to the synergistic ability of antioxidant and anti-diabetic of luteolin, diosmin, and SeNPs together in GA/LU/DIO-SeNPs.
The current research demonstrated liver damage by evaluating AST, ALT, and ALP enzymes. A raised level of these markers in diabetic mice indicated the injury of hepatocytes [
34]. Thus ALT, and AST, liver enzymes, are found in the blood after hepatic cell injury. ALT is more specific to liver damage, while AST and ALP generally are associated with liver injury in cases of muscle damage and cardiac infarction; both markers are linked to hepatic cell function [
35]. Elevation of oxidative stress in the liver is the cause of the increased release of liver markers, AST, ALT, and ALP due to injured hepatocytes. Six weeks of GA/LU/DIO-SeNPs treatment provokes a significant reduction in liver enzyme levels as compared to the diabetic control, suggesting a hepatoprotective effect, repairing liver cells, decreasing liver enzyme leakage into the blood, and reestablishing the level of liver markers towards the normal group.
It has been reported that insulin resistance decreases the capacity of hepatic tissue to synthesize glycogen and utilizes and decomposes glucose; consequently, more glucose is secreted into the blood. Our results demonstrated that GA/LU/DIO-SeNPs could increase hepatic glycogen content in mice, which implied that nanospheres could regulate hepatic glucose. In diabetic conditions, the synthesis of hepatic glycogen content is decreased due to the low level of insulin, which inactivates the glycogen synthase pathway [
36]. The restoration of hepatic glycogen content by the GA/LU/DIO-SeNPs group demonstrated that the combination of flavonoids and Se stimulates the glycogen synthase enzyme by increasing insulin generation from pancreatic cells.
4. Materials and Methods
4.1. Materials
All the chemicals were purchased from Sigma Co.Ltd. (St. Louis, MO, USA). All the solvents in this study were of analytical grade, and aqueous solutions were prepared using Milli-Q water.
4.2. Synthesis of GA/LU/DIO-SeNPs
Briefly, 0.5% Arabic gum (GA) solution was added dropwise into 10 mM Na
2SeO
3 solution, and the mixture was diluted to 100 mL with Milli-Q water. The solution was mixed with 2 mg/mL of a combination of luteolin (LU) and diosmin (DIO; 1:1) under magnetic stirring. After, 40 mM ascorbic acid as the reducing agent [
37] was mixed and kept stirring for 24 h in the dark at 37 °C. The obtained mixture of GA/LU/DIO-SeNPs was dialyzed with a dialysis bag (Mw cut-off: 3 kDa) in Milli-Q water to remove the excess sodium selenite. The GA/LU/DIO-SeNPs were collected by centrifugation at 15,000 rpm at 15 min and were washed twice with H
2O, Milli-Q.
4.3. Characterization of GA/LU/DIO-SeNPs
4.3.1. Zeta Potential and Particle Size Analysis
The zeta potential of nanoparticles and particle size were measured using laser Doppler velocimetry (LDV, Zetasizer Nano ZS, Malvern, UK). Between 1.0 and 1.5 mL of each sample was measured in a polystyrene cuvette at a fixed angle of 173° at 25 °C to obtain zeta potential values to check the bonding mechanism. The non-invasive backscattering (NIBS) assay was used to determine the particle size distribution. The results are reported as the average values of triplicate determinations. Dynamic light scattering (DLS) was used to monitor particle size changes during storage in a Zetasizer Nano ZSP, Malvern Instruments Ltd., Malvern, UK.
4.3.2. Transmission Electron Microscopy (TEM)
The morphology of GA/LU/DIO-SeNPs was observed by TEM (Model HT7700, Hitachi High-Tech, Tokyo, Japan). NPs solution was prepared by sonic oscillation and added directly to carbon-coated copper grids to evaporate the solvent. The micrographs of NPs were then analyzed at an accelerating voltage of 80 kV.
4.3.3. FT-IR Analysis
The FT-IR spectra of LU/DIO, SNPs, and GA/LU/DIO-SeNPs were characterized using a spectrophotometer FTIR-TENSOR27, Bruker, Ettinger, Germany, in the range of 400–4000 cm−1.
4.3.4. UV-Visible Analysis
The UV-visible spectra of the LU/DIO, SNPs, and GA/LU/DIO-SeNPs solutions were obtained using a UV-250 Shimadzu spectrophotometer, Tokyo, Japan. The absorbance was determined in the range of 250–700 nm.
4.3.5. EDX Analysis
The elemental compositions of the GA/LU/DIO-SeNPs were measured using energy-dispersive X-ray spectroscopy (EDS) by SEM-EDX spectrometer (Oxford, UK).
4.3.6. Encapsulation Efficiency
Encapsulation efficiency was measured by quantifying the difference between the total amount of flavonoids used for the preparation of NPs and the amount of free flavonoids in the nanosuspension. Briefly, 2 mL of GA/LU/DIO-SeNPs preparation was centrifuged at 15,000 rpm for 45 min. The flavonoid content in the supernatant was measured by using UV spectrophotometer at 280 nm and determined from the mixture of luteolin and diosmin (LUDIO) standard curve. The encapsulation efficiency of flavonoids was calculated using Equation (1):
4.3.7. In Vitro Release of LU/DIO
Flavonoids-loaded NPs equivalent to 5 mg (LU/DIO) were added with 5 mL of phosphate-buffered saline pH 7.4 (donor medium), then it was explored in a dialysis bag (3500Da, Thermo Scientific, Rockford, IL, USA), immersed in 50 mL of pH 7.4 phosphate buffer which simulated intestinal fluid (SIF) and simulated blood fluid (SBF) and incubated at 37 °C for 30 h. After, at time intervals of 10, 20, 30 and 40 h, aliquots of 1 mL of the sample were used to measure LU/DIO release. The same analysis was performed at pH 5.5 phosphate buffer simulated gastric fluid (SGF; receptor medium). The flavonoid concentration of the aliquots was measured at 280 nm.
The cumulative releasing of flavonoids was calculated using Equation (2):
E: the amount of LU/DIO measured in the release medium
E0: the initial amount of LU/DIO in the NPs
4.4. Stability of the Nanoparticles
The stability of nanoparticles was evaluated as follows: 500 mg of GA/LU/DIO-SeNPs was suspended in 5 mL of phosphate-buffered saline (PBS) and maintained at 37 °C. The shelf life of the nanoparticles was recorded in pre-determined time intervals (every 8 days) using absorbance intensities of the solutions over 5.5 months.
4.5. Experimental Animals
One hundred and forty-four adult male C57BL/6 mice (10 weeks of age) used for this study were obtained from the Central Animal House of the Universidad Anahuac, weighing between 25 to 30 g. The mice were housed in groups of five per cage under a normal 12 h light/dark cycle, controlled humidity (50 ± 5%) and temperature (22 ± 2 °C), and acclimatized to laboratory conditions for one week before starting the experiments, mice had free access to food and water ad libitum throughout the experimental period. The experiments reported in this study followed the guidelines stated in “Principles of Laboratory Animal Care” (NIH publications 85-23, revised 1985) and Mexican Official Normativity (NOM-062-Z00-1999). In addition, all animal experiments were approved by the Animal Experiment Committee of Escuela Nacional de Ciencias Biológicas, IPN (Code: Folio ENCB/CEI/046/2021).
4.6. Induction of Type 2 Diabetic Mouse
Diabetes was induced at 10 weeks of age in males by intraperitoneal injection (i.p.) of a freshly prepared solution of streptozotocin in citrate buffer (40 mg/kg body weight [BW] for 5 consecutive days). Seven days after i.p. injection, blood samples were collected from the tail vein, and blood glucose levels were measured using a glucometer (One Touch Glucometer, Roche, Basel, Switzerland). The mice with blood glucose levels higher than 13.9 mmol/l were considered diabetic. To prevent death from hypoglycemic shock induced by streptozotocin, a 5% glucose solution was administered for 4 days. Supplementation of NPs was carried out for additional 6 weeks, and Metformin (Mtf) was used as a positive control.
4.7. Animal Grouping and Treatment Schedule
Forty-eight C57BL/6 mice were divided into the following 6 groups (n = 8): Group 1: normal control (received distilled); Group 2: STZ diabetic control (received distilled); Group 3: STZ + LU/DIO (10 mg/kg/day); Group 4: STZ + SeNPs (10 mg/kg/day); Group 5: STZ + GA/LU/DIO-SeNPs (10 mg/kg); Group 6: STZ + Metformin (Mtf; 150 mg/kg/day served as positive control). The different concentrations of the samples and Mtf were given daily orally through a cannula.
4.8. Monitoring of Body Weight, Food Intake, and Water Intake
The body weights of mice in different groups were measured and recorded every week. Similarly, changes in the amount of water and food consumed were monitored.
4.9. Biochemical Analyses of Serum of Diabetic Mouse
4.9.1. Assessment of Insulin Resistance, Insulin, Glycated Hemoglobin, and Glucose in Serum
Blood samples were taken from the inferior vena cava and centrifuged at 4000 r/min for 10 min. Blood glucose was determined by the Accu Chek glucometer method (Roche, Basel, Schweizer). The serum insulin levels were detected using a mouse ELISA kit (Thermo-Fisher Scientific, Waltham, MA, USA). The insulin resistance (HOMA-IR) assessment was calculated with reference to existing literature formulas [
38]. HbA1c was determined by ELISA kit assay.
4.9.2. Determination of Lipid Profile
After completion of the experiment, mouse blood was collected, and plasma was separated by a centrifuge at a speed of 13,000 rpm for 10 min (4 °C). For lipid profile analysis of mice in all groups, T-CHO, TG, HDL-CHO, LDL-CHO, and free fatty acid (FFA) were measured using commercial assay kits according to the manufacturer’s indications (Sigma-Aldrich, St Louis, MI, USA).
4.9.3. Liver and Kidney Weight Determination
At the end of the treatment period (6 weeks), the mice were fasted overnight, after which they were sacrificed by cervical dislocation and necropsied. Then liver, and kidney, were removed surgically.
4.9.4. Determination of ALT, AST, ALP, MDA, Antioxidant Enzymes, and Glycogen in the Liver of Mice
Liver tissue was homogenized in PBS to prepare 10% liver homogenate, then centrifugated (4 °C, 3000 rpm, 10 min), and the supernatant was collected. The content of MDA, SOD, GPx, CAT, AST, ALT, and ALP in the supernatant of liver homogenate was determined according to the requirements of the instructions provided in ELISA kit methods (Ela science, Boston, MA, USA).
Liver glycogen content was measured using sodium acetate buffer (100 μL; pH 6), 5 μL of amyloglucosidase, and 20 μL of the homogenate to a final volume of 0.5 mL with double distilled water and incubated at 50 °C for 30 min. A control with everything except amyloglucosidase was also analyzed. A 300 μL aliquot of each sample was then added to 1 mL of glucose oxidase/peroxidase reagent (GOPOD, Megazyme) and incubated at 50 °C for 30 min. The glycogen content was calculated at 510 nm based on a calibration curve constructed by reacting
D-glucose of various concentrations with the same GOPOD reagent [
38].
MDA level was expressed as µmol/mg of protein, GSH-px level was expressed as U mol/g of protein, and SOD activity was expressed as U/mg of protein. CAT activity was expressed as U/mg of protein, and glycogen was expressed as mg/g of tissue.
4.9.5. Determination of Production, MDA, and Antioxidant Enzymes in the Kidney of Mice
Kidney tissue was homogenized in PBS to prepare 10% kidney homogenate, after centrifugated (4 °C, 3000 rpm, 10 min), and the supernatant was collected. The production of MDA, the levels of CAT, SOD, and GPx, in the supernatant of kidney homogenate were measured according to the requirements of the instructions provided in reagent kits.
4.9.6. Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT) on Diabetic Mice
Oral glucose tolerance tests were performed by the following procedure: A total of 48 male 48 C57BL/6 mice were randomly divided into 6 groups of 8 mice in each group:
Group 1 Normal mice (1% Tween 80 in water, 10 mL/kg body weight)
Group 2 Diabetic control (treated with Streptozotocin (STZ)
Group 3 Diabetic + LU/DIO (10 mg/kg body weight)
Group 4 Diabetic + SeNPs (10 mg/kg body weight)
Group 5 Diabetic + GA/LU/DIO-SeNPs (10 mg/kg body weight)
Group 6 Diabetic control + Glibenclamide (5 mg/kg body weight)
LU/DIO, SeNPs, and GA/LU/DIO-SeNPs were orally administered to fasted mice following a period of one hour, all mice were orally administered 2 g glucose/kg of body weight and the blood was sampled at different time-points 30, 60, 90 and 120 min after the glucose administration [
39]. Blood glucose levels were measured by the glucose oxidase method. The same fasting protocol used for OGTT was adopted for the determined insulin tolerance test (ITT). The samples were orally administered to fasted mice for a period of one hour; then, diabetic mice were injected intraperitoneally with insulin (2 U/kg). The glucose level was determined according to the technique previously described at 30, 60, 90 and 120 min after the insulin injection. Blood glucose levels were measured by the glucose oxidase method.
4.10. Statistical Analysis
The results are expressed as the mean ± S.D. (n = 8). One-way analysis of variance (ANOVA) was continued by honest significant difference (Tukey’s HSD) post hoc test for multiple comparisons; data were analyzed using GraphPad Prism 5 software (Version 5.0, San Diego, CA, USA). Post hoc assaying was carried out for intergroup comparisons using the least significant difference test. Statistical significance is displayed as follows: a p < 0.001, b p < 0.01, c p < 0.05 and * p < 0.001, ** p < 0.01.