1. Introduction
Diabetes mellitus is an endocrine disease of multiple aetiologies in insulin secretion. A deficiency in insulin results in hyperglycemia with metabolic disturbances of biomolecules [1]. Weight, obesity, and sedentary lifestyle are the emerging global epidemic of diabetes, and this is considered a degenerative disease of the blood glucose system, characterized by pancreatic beta cells’ deficiency to produce insulin or sufficient insulin, resulting in chronic hyperglycemia, which is associated with long-term microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cardiovascular) complications [2]. Individuals with diabetes have high levels of inflammatory cytokines, activation of leukocytes, and increased tissue fibrosis [3]. Oxidative stress is also a consequence of the imbalance between the oxidative and antioxidant systems that results in the overproduction of reactive oxygen species (ROS), which play a crucial role in the initiation and progression of diabetic nephropathy [4]. Accumulation of ROS leads to oxidative stress, which is associated with increased damage to β-cells and biomolecules [5]. Moreover, because of the relatively low expression of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) in diabetes, pancreatic β-cells may be vulnerable to ROS attack when the system is under oxidative stress situations [6]. Similarly, elevated levels of free radicals, due to inefficiency of the antioxidant defense system, may lead to disruption of cellular function by enhancing the susceptibility of membranes to lipid peroxidation [7]. Due to this increasing prevalence of diabetes and the complications induced by this, in both developed and developing countries, scientists are facing challenges to further conduct research in sourcing potent therapeutic agents from natural sources for more efficient usage in the treatment and management of diabetes as well as the complications arising from diabetes [8]. So, the maximum therapeutic and minimum side effects of herbal remedies have been verified in numerous scientific investigations. Presently, plant materials play major roles in primary health care as therapeutic remedies in many developing countries [9]. This is the reason why plant materials are continuously being scrutinized and investigated for their potential use as hypoglycemic agents.
Plants are well known in traditional medicine for their hypoglycaemic effects and available literature indicates that there are more than 800 plant species showing hypoglycaemic activities in vivo [10]. Moringa oleifera Lam. (Family: Moringaceae) (M. oleifera) is a medium-sized tree, growing in Asia, Africa and tropical areas of the world as a valuable food source. Various parts of M. oleifera tree have been studied for several pharmacological actions. Many reports have described its leaves as antifungal, antimicrobial, antiatherosclerotic, antifertility, relieving pain, central nervous system depressant, antiinflammatory, diuretic and regulating hyperthyroidism. Published research showed that M. oleifera seeds (MOS) have pharmacological activities such as blood glucose-lowering, anti-inflammatory, and antitumor effects and are also used to treat diabetic nephropathy [11]. Herbs are frequently used in conjunction with conventional medications to minimize adverse effects and boost effectiveness at lower dosages.
Diabetic nephropathy can be treated with a variety of oral antidiabetic medications, such as sitagliptin, which can be used alone or in combination for optimal results. It is still difficult to manage diabetes without experiencing any negative side effects because many of these oral antidiabetic medications have a number of major side effects [12]. Diabetic nephropathy was treated with sitagliptin (ST), a dipeptidyl peptidase-4 (DPP-4) inhibitor [13] [14]. However, the increasing resistance to these medications and their many adverse effects have increased the demand for alternative therapies with fewer or even no side effects for diabetics. In recent years, the use of herbal therapies to treat hyperglycemia and other diabetes-related issues has grown in popularity. A previous study assessed the hepatoprotective, hypolipidemic, and antidiabetic effects of Moringa oleifera leaves. In streptozotocin-induced diabetic rats, the current study aimed to evaluate the nephroprotective effects of extracts from Moringa oleifera leaves both individually and in combination with sitagliptin, albeit at a lower dose.
2. Materials and Methods
2.1. Collection of Leaves and Preparation of Extract
For extraction, the maceration technique was used with some modifications [15]. Fresh mature Moringa oleifera leaves were picked from the surrounding countryside, located at 24.2253˚N, 89.6687˚E in Bangladesh. Fresh leaves were collected, rinsed with tap water, and then distilled. For three days, the clean leaves were left to dry in the shade of the sun. An electronic grinder was used to crush the dried Moringa oleifera leaves. The fine powder was soaked at a 5:1 ratio in sealed glass bottles (amber) with 95% ethanol [16]. It was subsequently left to stand at 25˚C - 30˚C for 10 - 12 days while being frequently shaken manually. After quickly passing through cotton, the extract was passed through filter papers (Whatman grade-1). The extract was then allowed to air dry at RT. The crude leaf extract was kept at 4˚C in a glass container that was tightly sealed.
2.2. Chemicals, Reagents and Drugs
For extraction, 95% ethanol from Merck KGaA, Darmstadt, Germany, was used. The active pharmaceutical ingredient Sitagliptin was generously provided by Square Pharmaceuticals PLC, Pabna, Bangladesh. Streptozotocin has been acquired from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. All chemicals and reagents utilized were of analytical grade.
2.3. Experimental Animal
Thirty (30) Swiss Albino male rats, weighing 120.5 g (ranging from 110 g to 150 g) and aged 1.5 months, were procured from a commercial laboratory rat supplier in the Rajshahi district of Bangladesh. The rats were maintained in clear polypropylene cages under a 12-hour light/dark cycle at 24˚C - 26˚C and provided with a regular mouse chow and sterile tap water ad libitum during the study. Rats were utilized for experimentation following a ten-day acclimatization period. The Khwaja Yunus Ali University ethics committee in Bangladesh examined and authorized the animal experiment, with the ethical clearance certificate number KYAU/DEAN/EGC/2024/014. Experiments followed EU guidelines (Directive 2010/63/EU) for pharmacological analysis and experimental design, as well as the WMA statement on the use of animals in biomedical research [17].
2.4. Dose Selection
Dose selection study was performed previously and described clearly in other research work [17].
2.5. Experimental Design
A total of thirty (30) Swiss Albino male rats were utilized in the experiment and separated into five groups by simple randomization. Diabetes was produced in rats, excluding the normal control group, using a jab of streptozotocin (STZ, 45 mg/kg, I.P.), made in 0.1 M citrate buffer at pH 4.5 [17] [18]. The therapy duration was four weeks. At the conclusion of the testing session, rats were anaesthetized and euthanized.
Group Name |
Treatment Features |
NC |
Nondiabetic rats received a balanced diet and tap water ad libitum. |
DC |
Diabetic rats received a balanced diet and tap water ad libitum. |
STG |
Diabetic rats received Sitagliptin (100 mg/70kg BW) orally. |
MoLE |
Diabetic rats received M. oleifera extract (200 mg/kg BW) orally. |
STG + MoLE |
Diabetic rats were received Sitagliptin (50 mg/70kg BW) + M. oleifera extract (100 mg/kg BW) orally. |
2.6. Blood Collection and Kidney Homogenate Preparation
After the experiment, blood samples were taken straight from the heart, and the whole blood was centrifuged for 10 min at 1066 × g to separate the serum [19]. Immediately after sacrifice, both the kidneys were dissected; rinsed with isotonic saline. A homogenate was prepared with 10% (w/v) phosphate-buffered (0.1 M, pH 7.4) using a homogenizer. Homogenates were centrifuged at 15,000 rpm for 10 min at 4˚C and the supernatant was estimated for kidney antioxidant parameters.
2.7. Monitoring Plasma Glucose and Serum Insulin Level
The blood glucose levels of the rats were monitored prior to and following the experiment. Blood samples were drawn from the tail vein, and a glucometer (VivaChek Biotech (Hangzhou) Co., Ltd., China) was used to measure the glucose level. Serum insulin levels were measured using a commercial enzyme-linked immunosorbent assay kit according to the manufacturer’s instruction.
2.8. Estimation of Biomarkers of Oxidative Stress
A portion of the supernatant from the centrifuged kidney homogenate was used to assess endogenous superoxide dismutase (SOD) and enzyme catalase (CAT) activities. Kidney catalase activity was assayed following the method of Kar and Mishra, 1976 [20]. And superoxide dismutase (SOD) activity was assayed according to the method of Marklund and Marklund [6] [21].
2.9. Biochemical Analyses
For determining serum uric acid, urea, creatinine, albumin and calcium, commercial kits (ACCUREX, Biomedical Pvt. Ltd.) were utilized. The biochemical analyses for each component were conducted according to the manufacturer protocols.
2.10. Statistical Analysis
The data were expressed using means ± SEM. For statistical comparisons, p values < 0.05 were deemed significant, and the one-way ANOVA and t-test were employed. For data analysis, GraphPad Prism 9.4.1 (USA) was used.
3. Results
3.1. Glucose and Insulin Level
Figure 1(A), Figure 1(B) demonstrate the outcome of M. oleifera extracts, sitagliptin, and their combination therapy on the plasma blood glucose (PBG) and insulin in STZ-induced DN rats. STZ-evoked DN rats exhibited a marked elevation in PBG and a concomitant decrease in serum insulin concentration compared to control rats, which confirms the progression of a diabetic state in the STZ-evoked DN rats (p < 0.05).
Figure 1. (A) Effect of M. oleifera extracts, Sitagliptin, and their combination therapy on plasma glucose. (B) Insulin level of Streptozotocin-Induced Diabetic Nephropathy in Rats.
The administration of M. oleifera extracts, sitagliptin, and their combination therapy demonstrated an appreciable reduction in the PBG level and restored the level of insulin. This finding demonstrated the potential effect of M. oleifera extracts, sitagliptin, and their combination therapy against STZ-induced DN rats.
3.2. Effects on Renal Function
Effects of M. oleifera extracts, sitagliptin, and their combination therapy on creatinine, Figure 2(B): urea, uric acid, and calcium levels in STZ-induced diabetic rats as shown in Figures 2(A)-(D).
Figure 2. Effects of M. oleifera extracts, sitagliptin, and their combination therapy on (A): creatinine, (B): urea, (C): uric acid, and (D): calcium levels in STZ-induced diabetic rats.
The creatinine, urea, uric acid, and calcium levels were relatively higher in STZ-induced diabetic rats than in the control group. M. oleifera extracts, sitagliptin, and their combination therapy dramatically reduced the levels of creatinine, urea, uric acid, and calcium compared with those levels in the DM group, indicating that renal function improved after intervention.
The serum albumin levels were relatively lower and kidney-body weight ratio in STZ-induced diabetic rats than in the control group. M. oleifera extracts, sitagliptin, and their combination therapy significantly increased the levels of albumin and decreased kidney-body weight ratio compared with those levels in the DM group, as shown in Table 1, also indicating that renal function improved after intervention.
Table 1. Effect of M. oleifera extracts, sitagliptin, and their combination therapy on serum albumin and kidney, body weight ratio in control and experimental animals (Values are given as means ± SEM of five rats in each group. Same superscript letter indicating no significant difference).
Group |
NC |
DC |
STG |
MoLE |
STG + MoLE |
Albumin |
29.66 ± 1.26 |
12.66 ± 1.13a |
21.4 ± 0.88b |
24.86 ± 1.06b,c |
26.1 ± 0.79c |
Kidney-Body Weight Ration |
0.69 ± 0.02 |
1.03 ± 0.05a |
0.78 ± 0.04b |
0.80 ± 0.02b |
0.72 ± 0.02b |
3.3. Biomarkers of Oxidative Stress and Endogenous Antioxidants
The effect of M. oleifera extracts, sitagliptin, and their combination therapy on oxidative enzymes and oxidative stress markers is illustrated in Figure 3. Rats induced with STZ exhibited a marked reduction in SOD and CAT. DN rats treated with M. oleifera extracts, sitagliptin, and their combination therapy elevated the level of SOD and CAT in serum. However, this result indicated that M. oleifera extracts, sitagliptin, and their combination therapy administration ameliorate oxidative stress and endogenous antioxidants in the DN rats.
Figure 3. Effect of M. oleifera extracts, sitagliptin, and their combination therapy on oxidative stress biomarker. (A) Superoxidase (SOD), (B) Catalase (CAT).
4. Discussion
Diabetic nephropathy, a kidney complication of diabetes, is often associated with oxidative stress. In this investigation, the effect of M. oleifera and sitagliptin combination treatment was observed in different parameters related to oxidative stress and renal function measurement of streptozotocin induced diabetic rats. In our investigation, diabetes was produced in rats using streptozotocin (STZ, 45 mg/kg, I.P. injection). Our findings demonstrate that the treatment of STZ-induced diabetic rats with Moringa oleifera leaves extract (MoLE) alone and combination therapy with sitagliptin (STG + MoLE) significantly reduced blood glucose levels and insulin compared to untreated diabetic rats as shown in Figure 1 demonstrating a definite antidiabetic effect.
The increased the levels of creatinine, urea, uric acid, calcium, and kidney-body weight ratio decreased the albumin compared to non-diabetic control rats are major sign of diabetic nephropathy [22]. The results in Figure 2 and Table 1 show significant (p < 0.05) increase in the level of serum creatinine, urea, uric acid, calcium and decrease in plasma albumin in the diabetic rats when compared with respective control rats, while, after the treatment of STZ diabetic rats with M. oleifera extracts (MoLE), sitagliptin (STG), and a combination of sitagliptin (50 mg/70kg BW) and the ethanolic leaves extract of M. oleifera (100 mg/kg BW) (STG + MoLE) the levels of creatinine, urea, uric acid, calcium and kidney-body weight ratio were significantly (p < 0.05) decreased and albumin level was increased. These results are in agreement with other previous studies on the Moringa oleifera Lam. seed extract [11] and leaves extracts [23] on diabetic nephropathy in streptozotocin-induced diabetic rats.
As illustrated in Table 1, a marked (p < 0.05) reduction in plasma albumin level was observed in diabetic rats and this is consistent with the results obtained by Annamalai Prakasam et al. [24]. The decrease in albumin may be due to albuminuria, which are important clinical markers of diabetic nephropathy, and/or may be due to increased protein catabolism [24]. The results of the present study demonstrated that the treatment of diabetic rats with the M. oleifera extracts, sitagliptin, and their combination therapy caused a noticeable elevation in the albumin levels as compared with their normal levels. Such improvement of serum albumin was previously observed after the oral administration of Moringa oleifera Lam. seeds on streptozotocin induced diabetes and diabetic nephropathy in male rats [25].
Moringa oleifera (MO) has rich antioxidant content and diverse therapeutic abilities. Previous investigation identified MO with the ability to prevent the occurrence and complications of diabetic-induced kidney injury through its protective effect on the oxidative status and inflammatory cytokines in the kidneys of diabetic rats. Whereas, an experimental study showed that sitagliptin can restore the GLP-1 by restoring the DPP4 protein in the kidney of diabetic rat [26]. Hyperglycemia-induced oxidative stress has been shown to be actively involved in the onset and progression of diabetes, leading to various complications such as cardiovascular diseases, nephropathy, amputation of limbs and blindness [27]. ROS cause lipid peroxidation, and when this happens, the lipids of the cell membrane undergo catabolism, leading to damage of tissues. CAT and SOD like antioxidant enzymes delay ROS induced oxidative stress, where SOD is responsible for the conversion of superoxide free radical to water and CAT eliminates the synthesizing of hydrogen peroxide and hydroxyl radical [28] [29]. There is also evidence that sitagliptin can significantly diminish the increased ROS generation and restore the antioxidant defense by decreasing the oxidative stress in the kidney of diabetic rats [25] [30]. In this study, rats induced with STZ exhibited a marked reduction in SOD and CAT. DN rats treated with M. oleifera extracts, sitagliptin, and their combination therapy elevated the level of SOD and CAT in serum significantly. Thus, this result indicated that M. oleifera extracts, sitagliptin, and their combination therapy administration ameliorate oxidative stress and endogenous antioxidants in the DN rats.
5. Conclusion
Diabetic nephropathy, a kidney complication of diabetes, is often associated with oxidative stress. The study demonstrated that Moringa oleifera leaf extract and its combination with sitagliptin exhibited antidiabetic activity by decreasing blood glucose and increasing insulin levels, and nephroprotective effects by normalizing albumin and renal functional parameters by minimizing oxidative stress by increasing endogenous antioxidant enzyme. This result suggested protective effects of combination therapy with Moringa oleifera leaf extract and sitagliptin at a lower dose against streptozotocin-induced diabetic nephropathy in rats via inhibition of oxidative stress.
Acknowledgements
The research was conducted using the Animal House and the Phytochemistry & Pharmacognosy Laboratory of the Department of Pharmacy, Khwaja Yunus Ali University. The authors acknowledge the laboratory assistance provided by the Department of Pharmacy, Rajshahi University. We are also grateful to Square Pharmaceuticals Ltd., Bangladesh, for providing sitagliptin for this research.
Abbreviations
STZ |
Streptozotocin |
MoLE |
Moringa oleifera Leaves Extract |
STG |
Sitagliptin |
STG + MoLE |
Sitagliptin + Moringa oleifera Leaves Extract |