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Momordica charantia nanoparticles potentiate insulin release and modulate antioxidant gene expression in pancreas of diabetic rats
Egyptian Journal of Medical Human Genetics volume 23, Article number: 63 (2022)
Green synthesis of nanoparticles with medicinal plants has inherent potential in the management of diabetes mellitus. This study synthesized Momordica charantia nanoparticles using silver nitrate to investigate the antidiabetic properties of this extract and the synthesized nanoparticles in vivo.
The M. charantia nanoparticles used were synthesized biologically under ambient conditions from methanolic leaf extract of M. charantia using 1 mM concentration of aqueous silver nitrate and characterized using spectroscopic methods, FTIR and scanning electron microscopy. In vivo, the antidiabetic activity of M. charantia nanoparticle was assessed in streptozotocin-induced (65 mg/kg) rats. Rats were treated with M. charantia nanoparticle (50 mg/kg), aqueous leaf extract (100 mg/kg) metformin (100 mg/kg) and silver nitrate nanoparticle (10 mg/kg) for 21 days. Following treatment, rats were killed for biochemical analysis. Also, reverse transcript-polymerase chain reaction analyses of Takeda-G-protein-receptor-5, glucagon-like peptide-1, Insulin, superoxide dismutase, catalase and Nuclear factor-erythroid factor 2-related factor 2 (NRF2) - were carried out in the pancreas. A significant reduction in blood sugar levels was noted in rats treated with M. charantia nanoparticles. A reduction (p < 0.05) of pancreas alanine transaminase, aspartate aminotransferase and alkaline phosphatase was observed when compared with diabetic untreated rats. M. charantia nanoparticles significantly increase the antioxidant enzymes in diabetic rats when compared with diabetic untreated rats. The decrease in the level of triglyceride, cholesterol and low-density lipoprotein was observed when compared with diabetic control rats and also a significant increase in the expression of Takeda-G-protein-receptor-5, glucagon-like peptide-1, insulin, superoxide dismutase, catalase and NFE2-related factor 2 genes was observed when compared with diabetic untreated rats.
Momordica charantia nanoparticles exhibited potential antidiabetic activity in the rat model of diabetes and thus may serve as a therapeutic agent that could be developed for medical applications in the future.
Diabetes mellitus type 2 (T2DM) is now the world's fifth leading cause of mortality due to its alarming rise across the globe . Diabetic cases have risen from 153 to 368 million worldwide in the previous three decades. Presently, 382 million cases of diabetes are reported worldwide, and this figure is expected to reach 592 million . Type 2 diabetes can result in a number of secondary complications, including diabetic neuropathy, peripheral vascular disease and diabetic neuropathy . Years of scientific research and clinical experience have demonstrated that abstention from a sedentary lifestyle, along with therapeutic intervention, can significantly reduce the course of diabetes and its consequences. One of the most cost-effective methods of managing type 2 diabetes in developing nations is through the use of medicinally active plants [4, 5].
Metal nanoparticle synthesis is one of the most promising goals of researchers today in terms of producing biocompatible nanomedicine. Silver nanoparticles (AgNPs) in particular have been extensively studied due to their remarkable physical, chemical and antibacterial characteristics and are thus used in biosensing, catalysis, the food industry, optics, electronics and medicine [6, 7]. Numerous physical and chemical approaches for the manufacture of AgNPs with precise control over particle size have been established.
Momordica charantia (M. charantia) is a well-acknowledged medicinal plant belonging to the cucurbitaceae family found in tropical and subtropical regions of the world. The name itself delineates its distinguishing bitter taste. It is beneficial in the folkloric amelioration of stomach pain, diabetes, fevers, leprosy, snake bite, cancer, menstrual disorders, infections and hypertension. It also demonstrates antiallergic, antibiotic, anticancer, antitumor, antihelmintic, antileukemic as well as antioxidant properties [8, 9]. The use of medicinally active herbs in the treatment of type 2 diabetes mellitus (T2DM) is widespread . Clinical investigations using human patients, on the other hand, are few and of low significance in design. There has been an increase in the use of nanoherbal treatments since they are more bioavailable and have a lower dose. Previous findings have portrayed the significant role of metals in glucose metabolism and the affiliation of their inadequacy with diabetes [11, 12]. M. charantia nanoparticles (50 mg/kg) have been shown in earlier research to lower blood sugar levels in STZ-induced diabetic rats when compared to the plant extract [11, 13]. Hence, the quest of the present study is to investigate the antidiabetic prowess of M. charantia nanoparticle alongside its natural extract on STZ-induced diabetic rats.
Collection of plants and preparation of silver nanoparticles
Bitter melon leaves (M. charantia) were collected within Akure, Nigeria, air-dried and powdered. The bitter melon nanoparticles were synthesized according to previously described procedures . Briefly, the filtrate from the plant extract was added to a 1 mM concentration of aqueous silver nitrate solution (ratio 1: 9 (v/v)) and allowed to interact for 24 h leading to color change from dark green to light brown, which is an indication of nanoparticle formation. The bioreduction of Ag+ ions to Ag0 by M. charantia phytochemicals was monitored using a UV–Vis spectrophotometer (430 nm). The resulting solution (nanoparticles solution) was freeze-dried and stored at room temperature. Further characterization of the nanoparticles was done using a scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) .
Experimental design and animal treatment
Forty-seven (47) adult male Wistar rats were obtained from “blinded for peer review to ensure anonymity” and housed at room temperature (22–25 °C) with a 12-h light–dark cycle, accessible clean food and water ad libitum. The experiments were performed in accordance with the National Guidelines for Experimental Animal Welfare and with approval of the Animal Welfare and Research Ethics Committee at the Federal University of Technology, Akure, Nigeria. Acclimatization of the animals was done for two weeks after which diabetes mellitus was induced by intraperitoneal (i.p) injection of freshly buffered (0.1 M citrate, pH 4.5) solution of STZ (65 mg/kg) to overnight fasted rats. Blood glucose was monitored and animals with blood glucose ≥ 200 mg/dl after 72 h were selected and used for the experiments and divided into six different groups.
Group 1: Normal Control Group.
Group 2: Diabetes control (STZ (65 mg/kg)).
Group 3: STZ (65 mg/kg) + M. charantia nanoparticles (50 mg/kg).
Group 4: STZ (65 mg/kg) + silver nitrate (10 mg/kg).
Group 5: STZ (65 mg/kg) + metformin (100 mg/kg).
Group 6: STZ (65 mg/kg) + M. charantia aqueous extract (100 mg/kg).
The treatment of the rats was done for 11 days, and the fasting blood sugar level of the rats was monitored all through the treatment period.
Determination of serum enzyme activities
The activities of serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined using Randox diagnostic kit according to the manufacturer’s protocol.
Determination of serum lipids
According to the manufacturer’s protocols, the total serum cholesterol, triglycerides (TG) and high-density lipoprotein cholesterol (HDL cholesterol) were determined using the Randox diagnostic kit. Low-density lipoprotein cholesterol (LDL cholesterol) was estimated according to the Friedewald equation . All the serum lipid concentrations were expressed in mg/dl.
Determination of lipid peroxidation level
The level of lipid peroxidation in the heart and aorta homogenates was determined using thiobarbituric acid reactive substance (TBARS) as previously described . Briefly, the reaction mixture consists of 0.8 ml of 0.1 M TRIS-KCl buffer (pH 7.4), 0.25 ml of 30% TCA and 0.25 ml of 0.75% TBA (prepared in 0.1 M HCL) and 0.2 ml of the sample homogenate. The results were expressed as nmol/mg protein.
RNA isolation and quantification
RNA was isolated from the pancreas using TRIZOL reagent and RNA extraction kit (Zymo Research, USA). The purity of the isolated RNA was determined by the ratio of absorbance at 260 nm and 280 nm. The concentration of the isolated RNA was determined at an absorbance of 260 nm.
Complementary deoxyribonucleic acid (cDNA) synthesis by reverse transcriptase reaction
One microgram of the total RNA was used to synthesize cDNA by reverse transcriptase reaction using Protoscript II First Strand cDNA Synthesis Kit (Biolabs, New England) according to the Manufacturer’s protocol in a three-step reaction condition: 65 °C for 5 min, 42 °C for 1 h and 80 °C for 5 min.
Gene amplification by polymerase chain reaction
Amplification of genes of interest was done by polymerase chain reaction (PCR) as previously described . PCR was performed using Master Mix reagent kit (Thermo Scientific, USA) and appropriate primers (Table 1) for desired amplifications in a Multigene Labnet International machine. The amplified genes were separated on 0.1% agarose gel electrophoresis in a 1 × Tris-borate-EDTA (TBE) buffer, and the bands were quantified with “ImageJ” software. The β-actin gene was used to normalize the relative expression level of the respective gene.
Statistical analyses were performed using GraphPad 5 Prism (GraphPad Software, USA) by one-way analysis of variance (ANOVA) and data were expressed as mean ± SEM. Multiple comparisons between groups were made with Bonferroni post hoc test. Differences were considered statistically significant at a p-value of < 0.05.
Characterization of the nanoparticles
The presence of silver nanoparticles was evident by the formation of UV–visible absorbance maxima around 430 nm (Fig. 1). SEM analysis revealed tubular clusters of silver nanoparticles with uneven surface morphologies and dimensions (Fig. 2a–c). FTIR spectroscopy results revealed intense bands around 3690.1, 3280.1, 2922.2, 2851.4, 1543.1, 1625.1, 1401.5, 131.95 and 1028.7 which corresponds with O–H, N–H,C–H, C = C, C–O stretches (Fig. 3). This indicates the possible role of phenol, flavonoids and amine functional groups in stabilizing the nanoparticles.
Effect of M. charantia nanoparticles on fasting sugar level of diabetic rats
As demonstrated in Fig. 4, the STZ induction of diabetes in the experimental rats increases the fasting blood of the rats relative to control; this signifies the onset of hyperglycemia in the STZ-induced diabetic rats. The results showed that oral administration of M. charantia nanoparticles (50 mg/kg), metformin (50 mg/kg) and M. charantia extract (100 mg/kg) to diabetic-induced rats significantly (p < 0.05) reduced the glucose level of rats.
Effect of M. charantia nanoparticles on serum ALT, AST and ALP
The level of serum ALT, AST and ALP was significantly (p < 0.05) increased in the diabetes untreated group compared to the control group. The serum level of ALT, AST and ALP, however, was significantly decreased following the oral administration of M. charantia nanoparticles (50 mg/kg) for 11 days. The bar charts representing the activity of ALT, AST and ALP in treated and untreated groups are shown in Fig. 5a–c.
Effect of M. charantia nanoparticles on lipid peroxidation and antioxidant markers
As opposed to the diabetic control, the group treated with M. charantia nanoparticles alleviated lipid peroxidation as shown by a significant decrease in the level of serum MDA (Fig. 6a). Also, results from this study showed that the level of serum SOD and CAT were significantly decreased in the diabetes control group compared to the control group. The antioxidative properties of M. charantia nanoparticles were confirmed after treatment of rats with the plant nanoparticles elevated the level of serum SOD and CAT (Fig. 6b, c).
Effect of M. charantia nanoparticles on serum lipids
The bar chart representing the effect of M. charantia nanoparticles, metformin, of M. charantia on serum triglyceride (Fig. 7a), serum cholesterol (Fig. 7b), serum HDL (Fig. 7c) and rats. There were a significant decrease in the level of serum triglyceride, cholesterol and LDL, and a significant increase in serum HDL following oral administration of M. charantia nanoparticles, relative to diabetes control.
Effect of M. charantia nanoparticle on CAT, SOD and NRF2 mRNA expression
From the gene expression study, the mRNA expression of CAT, SOD and NRF2 was significantly downregulated following the induction of diabetes in the experimental rats (Fig. 8a–c). M. charantia nanoparticles were able to modulate the gene expression of CAT, SOD and NRF2 by upregulating the mRNA expression of these genes.
Effect of M. charantia nanoparticles on insulin-sensitive genes
The gene expression study denotes that the mRNA expression of TGR5, GLP-1 and INSULIN was significantly downregulated in disease state (diabetes control) compared to non-diabetic rats. After the oral treatment of the STZ-induced rats with M. charantia nanoparticles for 11 days, there was upregulation of TGR5, GLP-1 and INSULIN in the pancreatic tissue of diabetic rats (Fig. 9a–c).
The present study shows the insulin-potentiating effect as well as the genetic modulating abilities of M. charantia nanoparticle (NPs) and M. charantia plant extract (PE) against the pathophysiological occurrences following DM induction in experimental rats. The results show that STZ administration brought about a strident rise (p < 0.05) in blood glucose when compared with rats in the non-diabetic group. Though a previous study has reported the glucose-lowering effect of M. charantia in DM treatment, this study showed that oral administration of M. charantia NPs reduces the glucose level in diabetic rats. This claim is supported by previous findings [11, 13].
Glucose homeostasis is primarily regulated in the liver, which is a critical organ for energy metabolism [16, 17]. In patients with hepatic insulin resistance, liver dysfunction indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) have been found to be a good indication of liver health and type 2 diabetes risk [18, 19]. They have been established as a particular marker for liver damage . The results of the study illustrated that there is an increased in the activity of serum AST, ALT and ALP in diabetic rats, which signifies the onset of liver injury. Treatment of diabetic rats with M. charantia NPs and PE conversely mitigated this misdemeanor, causing a marked decrease (p < 0.05) in the serum levels of AST, ALT and ALP. This could possibly be due to its sufficient phenolics and saponins content  which have, in previous times, been associated with antidiabetic activities [22, 23].
Oxidative stress occurs as a result of overproduction and accumulation of oxygen reactive species (ROS) which overwhelm the endogenous antioxidant ability to neutralize these highly reactive chemical compounds ; it has been thought that oxidative stress plays a big part in how diabetes progresses and how it gets worse . Malondialdehyde (MDA) is one of the final products of lipid peroxidation , and it is a significant marker of oxidative stress.
This study showed that STZ induction of diabetes in experimental rats prompted a drastic increase in the level of MDA. This result corroborates with previous studies [27, 28]. Also, Weydert et al.  established that SOD and CAT enzyme play a major role in cellular regulation of ROS; therefore, decreased activity of these enzymes may increase the vulnerability of the cells to oxidative stress, which consequently leads to cell death. In agreement with this study, the study showed a significant decrease in the serum levels of SOD and CAT in the diabetic model. Treatment with M. charantia NPs and PE invariably attenuated the pathophysiology and oxidative damage occasioned by STZ administration by decreasing the serum level of MDA and subsequently increasing serum levels of SOD and CAT. These biochemical assay results corroborate gene expression profiling of antioxidant genes (SOD, CAT and Nrf2) in the pancreas of STZ-treated rats, where there were significant upregulation of SOD and CAT mRNA expression and downregulation of Nrf2 mRNA expression following the treatment of the experimental rats with M. charantia NPs and PE. M. charantia's antioxidant properties may be due to the high content of polyphenols and other groups of phytochemicals found in the plant [30, 31].
Furthermore, this study takes into consideration the effect of M. charantia NPs on glucose metabolizing genes in the pancreas of STZ-induced diabetic rats. They include the Takeda G protein-coupled receptor 5 (TGR5) gene, glucagon-like peptide-1 (GLP-1) and insulin. The first demonstration of the stimulatory impact of M. charantia NPs on GLP-1 secretion via TGR5 activation has been made in the current work, which is the first of its kind. The current investigation demonstrated that M. charantia NPs increased the glucose-lowering impact in STZ-treated rats by upregulating the pancreatic mRNA expression of GLP-1 via TGR5 activation and a consequent upregulation of insulin mRNA expression. The development of diabetes in animal models has been shown to be delayed by GLP-1. GLP-1 analogues have been used to treat patients with diabetes because they reduce energy intake in people [32, 33]. Furthermore, GLP-1 agonists have been shown to have a minimal risk of hypoglycemia and to help obese people lose weight . It has been proved in humans that GLP-1 inhibits endogenous glucose synthesis directly . As a result, the upregulation of pancreatic GLP-1 mRNA via TGR5 activation by M. charantia NPs in diabetic rats is beneficial in lowering blood glucose levels.
Plasma lipid levels are often elevated in diabetes mellitus, and this elevation is associated with an increased risk of coronary heart disease . In STZ-induced diabetic rats, hypercholesterolemia and hypertriglyceridemia have been thoroughly reported . According to the findings of this study, the total blood cholesterol, triglycerides and LDL cholesterol levels of diabetic rats all increased significantly, but the HDL cholesterol levels of diabetic rats decreased significantly. Plasma lipid concentrations are unusually high due to an increase in the mobilization of free fatty acids from peripheral depots, which is the primary cause of the elevated levels . Treatment with SNPs and PE, however, mitigated the physiological misdemeanor and hypercholesterolemic activities (Fig. 4a–d) in the serum of diabetic rats prompted by STZ administration. The levels of these hyperlipidemic parameters were normalized and a better ameliorative effect was seen in rats treated with M. charantia NPs than PE and those given metformin (MET). Reduced cholesterol absorption, its binding with bile acids within the digestive tract and increased bile acid excretion could all M. charantia NPs' lipid-lowering properties [39, 40]. It could also be due to a decrease in cholesterol biosynthesis and/or an increase in LDL receptors [41, 42].
Summarily, the present study showed that oral administration of M. charantia NPs potentiates insulin release and glucose-lowering effect and increases the antioxidant status in diabetics via activation of TGR5 via upregulation of GLP-1 mRNA expression and upregulation of SOD and CAT mRNA expression, and elevates serum activities of CAT and SOD. Finally, fat-lowering capacity is also demonstrated by M. charantia NPs.
This study demonstrated the hypoglycemic and antioxidative effect of M. charantia nanoparticles through the modulation of genes in the pancreas of diabetic rats.
Availability of data and materials
All the data is included in the manuscript.
Serum alanine aminotransferase
Takeda G protein-coupled receptor 5
Nuclear transcription factor
- M. charantia :
Type 2 diabetes mellitus
Scanning electron microscope
Fourier transform infrared spectroscope
Thiobarbituric acid reactive substance
Polymerase chain reaction
Farnesoid X receptor
Brown adipocyte tissues
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The experiments were performed in accordance with the National Guidelines for Experimental Animal Welfare and with approval of the Animal Welfare and Research Ethics Committee at the Federal University of Technology, Akure, Nigeria. The Committee reference number is not available.
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Elekofehinti, O.O. Momordica charantia nanoparticles potentiate insulin release and modulate antioxidant gene expression in pancreas of diabetic rats. Egypt J Med Hum Genet 23, 63 (2022). https://doi.org/10.1186/s43042-022-00282-0
- Momordica charantia
- Medicinal plants
- Silver nanoparticles
- Green synthesis