European Journal of Cardiovascular Medicine

T2DM is predominantly due to the failure of the bodily tissues to respond to insulin or synthesize enough insulin [1,2]. Diabetes mellitus (DM) is a metabolic disorder that leads to chronic hyperglycemia, a pathogenesis condition that may include defects in insulin secretion and/or action [3,4]. Medicinal plants or plant-based medicine have been used cost-effectively throughout the world to prevent and/or treat diabetes. The most common form of DM is type 2 diabetes mellitus (T2DM), which accounts for approximately 90% of DM cases. Momordica charantia L. (MC), commonly referred to as bitter gourd or bitter melon, is a tropical and subtropical plant that is a member of the Cucurbitaceae family. Rich in phytochemicals, the fruits and leaves of the Momordica species offer nutritional and nutraceutical components that may have a multitude of health-promoting effects. The plant has long been recognized and utilized for a variety of medical purposes, including as the treatment of T2DM, hypertension, obesity, cancer, bacterial and viral infections, and even AIDS [5], in numerous traditional and folk medicines [6].

Bitter melon, or karela, has been utilized for thousands of years in Ayurvedic therapy. All plant parts, including the seeds, roots, leaves, and especially the immature fruits, are said to possess its medicinal qualities [7]. The juice's diuretic, laxative, and anti-helminthic properties made it useful for treating a wide range of ailments. It is used to treat jaundice, persistent fever, joint pain, and illnesses involving the liver or digestive tract. It is used topically to treat burns, boils, and rashes as well as chronic skin conditions. For the treatment of type 2 diabetes, eating the entire plant is advised [7].

Based on nutritional study, this plant is the most nutrient-dense of all the cucurbits; it's a rich source of fiber, proteins, carbs, vitamins, and minerals. Fruits are made up of 93.2% water, 18.02 percent protein, and 0.76% fat, depending on their dried weight [8]. Fresh and green fruits contain vitamin C, A, and P, thiamine, riboflavin, niacin, and minerals [9]. The dietary supplementation of Momordica charantia has been extensively researched to cure a variety of illnesses, including type 2 diabetes, dyslipidemia, obesity, and cancer. This indicates that MC extracts have hypoglycemic and lipid-lowering characteristics, despite the fact that the outcomes of clinical trials undertaken thus far have been equivocal [10].

Chronic systemic inflammation in diabetics raises blood glucose levels and is associated with an increased risk of obesity and cardiovascular disease. Numerous disorders have a definite link with chronic inflammation: obesity, metabolic syndrome, cardiovascular disease, T2DM, neurological diseases, and cancer have all been linked to chronic inflammation [11]. There is much evidence suggesting that oxidative stress contributes to chronic inflammatory disorders. As a result, inflammation and oxidative stress are closely connected pathophysiological processes that have the potential to trigger one another [12].

Based on above evidences, this study was conducted to further explore the efficacy of momordica charantia ethanolic extract on blood sugar reduction in streptozotocin induced diabetic rat.

Ethical Approval: The study protocol was approved by the Central Animal Ethics Committee of Banaras Hindu University via Letter No: Dean/2017/CAEC/248.

Study Site: The study was conducted in the Department of Pharmacology, IMS BHU Varanasi. Rat (Charles- Foster, weight=60g to 80g) was procured from animal house of IMS BHU. They were then feeded with appropriate food under standard condition till they gain weight of 150g to 200g.

Animals:

Inbred Charles-Foster (CF) albino rats weighing 150-200 g, belonging to either gender, were procured from the Institute of Medical Sciences, central animal house at Banaras Hindu University in Varanasi. For one week prior to and during the trials, they were housed in the departmental animal house at 26 ± 20 C and 44-56% relative humidity, with light and dark cycles lasting 10 and 14 hours, respectively. The animals were fed a conventional mouse pellet diet (Pashu aahar), although water was available at all times. The food was removed from the animals 18 to 24 hours before to the experiment. The principles outlined in "Principles of laboratory animal care" (NIH publication no. 82-23, amended 1985) guideline were followed 13. Prior to beginning the experimental investigation, approval was obtained by the Institutional Animal Ethical Committee (Letter No: Dean/2017/CAEC/248).

Collection and preparation of extract:

Momordica charantia's dried seeds were obtained from B.H.U. Varanasi's botanical garden and identified with the Dravyaguna department of the Institute of Medical Sciences at Banaras Hindu University, Varanasi.

Preparation of ethanolic extract:

The 500 grams of dried seeds from Momordica charantia were broken up and put in a glass jar with enough ethanol for 72 hours before being filtered out. Momordica charantia'sethanolic extract was vacuum-dried and kept at -20°C until needed. The yield of the extract was 27.24 grams.

Standard medication chosen for the study: The comparison group was given 500 mg of metformin (Gluconorm).

Treatment plan: Four groups of animals were randomly assigned, and 6 (six) animals in each group were chosen for the study.

• Group I: control rats (0.5% CMC)
• Group II: STZ+CMC
• Group III: Metformin+ STZ (7.4mg/kg)
• Group IV: STZ+MC (250mg/kg)

The usual anti-diabetic medication metformin, along with the extracts, were administered orally once daily, suspended in 0.5% carboxymethyl cellulose (CMC).

Twenty-one days later, the experiments were carried out. Only 0.5% CMC was given to the control rats. Momordica charantia ethanolic extract, given orally, was investigated for its hypoglycemic effect. A dose of Momordica charantia that works best was selected for more research. The medication and extracts were administered orally to the animals via an oro-gastric tube at a rate of 10 milliliters per kilogram of body weight. The course of treatment lasted for 21 days.

Methodology:

Induction of diabetes: Severe hyperglycemia was induced in adult rats (150–200 g) using a single intraperitoneal dose of streptozotocin (STZ, 70 mg/kg) dissolved in citrate buffer (pH 4.5) [14]. Blood glucose levels were measured after 21 days, and rats with fasting blood glucose levels >225 mg/dL were selected.

Blood glucose estimation: Blood glucose levels were determined using the glucose oxidase-peroxidase (GOD-POD) method [15]. Serum samples were mixed with working reagent, incubated at 37°C for 15 minutes, and absorbance at 505 nm was measured.

Total cholesterol estimation: Blood samples were centrifuged to extract serum, and total cholesterol levels were measured using the CHOD-POD method [16].

Triglyceride estimation: Triglyceride levels in serum were determined using the GPO-POD method [17].

Free radical generation: Tissue homogenates (muscle, pancreas, liver) were prepared, centrifuged, and various parameters estimated [18].

Lipid peroxidation (LPO): Malondialdehyde (MDA) levels were measured as a unit of lipid peroxidation using a mixture of tissue homogenates and reagents, incubated at 95°C for 60 minutes. Absorbance was measured at 532 nm [19].

Nitric oxide (NO): Reactive nitrogen intermediates were assessed using the Griess reagent. Absorbance was measured at 540 nm [20].

Enzymatic antioxidants:

• Superoxide dismutase (SOD): Reduction of nitro blue tetrazolium was measured at 560 nm [22].
• Glutathione: Tissue samples were processed with EDTA and DTNB, and absorbance was measured at 412 nm [23].

Statistical analysis: Every outcome value is displayed as the mean ± standard error of the mean (SEM). Student's t-test was performed to assess the statistical analysis involving two groups, while Dunnett's multiple comparison post-test and one-way analysis of variance (ANOVA) were utilized to compare the control and different treatment groups statistically. At <0.05 values, statistical significance was deemed acceptable.

Diabetes mellitus was induced in adult rats (150-200 g) by injecting streptozotocin (STZ; 70 mg/kg, 14.0 mg/ml, 1 ml/200g body weight in citrate buffer, pH 4.5) intraperitoneally (ip) and the blood glucose levels were estimated at 0 days, 3rd days, 7th day and 21st day of STZ injection. The blood glucose level at 0 days was 95.16 ± 13.24 mg% which increased to a level of 285.16 ± 82.27% at day 3 of STZ administration. Thus, the adult Charles-Foster strain rats showed diabetes (mean fasting blood glucose level >200 mg%) with STZ (70 mg/kg, ip). For future study therefore, rats showing blood glucose level greater than 200 mg% at 3rd day of STZ administration were selected for respective studies.

Study of diabetic parameters

Effect of diabetes was studied on various biochemical parameters like blood glucose (BG), total cholesterol (TC), triglycerides (TG), after 21 days of STZ administration in rats. The results were compared with respective control+STZ groups. Day 3 and day 7 blood glucose result was also compared with respective control+STZ groups.

Effect on blood glucose

Ethanolic extracts of dried seed of Momordica charantia (300mg/kg) were given orally (po), once daily for 21 days starting from day 3rd and last dose was given on the day of experiment to 18 h fasted rats, one hour before the experiment to normal rats (NR) and 3 weeks diabetic rats. Blood was collected from the retrobulbar plexus of rats, and blood glucose was estimated in all the groups following the standard procedure mentioned earlier. Momordica charantia showed a decrease in blood glucose level of 4.7% at day 7 and 52% at day 21. Similarly, Metformin showed a decrease in blood glucose level of 50.27 at day 21.

Blood glucose level

Table 1: Plant Momordica charantia showed a statistically significant decrease in blood glucose level at day 21 (p - 0.039). At day 7, the plant was showing slight decrease in blood glucose level, which was not statistically significant. It is concluded that the plant is effective on long-term administration, while we observe no beneficial effect on short-term use.

Table 1: Effect on blood glucose level

Blood Triglycerides and Cholesterols level

TABLE 2: shows the effect of plant extract on blood TGA and Cholesterol level at day 21. Plant Momordica charantia did not show any statistically significant reduction in Cholesterol level (P 0.357), but there was a significant reduction in blood TGA level (p< 0.005).

TABLE 2: Effect on Blood Triglycerides and Cholesterol Levels

Table 3: shows that after 21 days of treatment, the value of GSH was decreased in the streptozotocin-treated group in liver, kidney, muscle, and Pancreas homogenate compared to the Control group. Momordica charantia group showed significantly increased value of GSH in kidney (p 0.02) and muscle (p 0.04) homogenate. These indicate the antioxidant activity of Momordica charantia.

TABLE 3: Effect on Blood Glutathione Level

Table 4: After 21 days of treatment, the value of SOD was decreased in streptozotocin treated group in liver, kidney, muscle and Pancreas homogenate compared to Control group. Momordica charantia group showed significantly increased value of SOD in kidney (p 0.004), pancreas (p 0.01) homogenate. These indicate the anti-oxidant activity of Momordica charantia.

TABLE 4: Effect on Blood SOD Level

Table 5: After 21 days of treatment, the value of NO was increased in the streptozotocin-treated group in the liver, kidney, muscle, and Pancreas homogenate compared to the Control group. Momordica charantia group showed a significant decrease value of NO in Liver (p 0.01) and pancreas (p 0.03) homogenate. These indicate the anti-oxidant activity of Momordica charantia.

TABLE 5: Effect on Blood NO Level

Table 6: After 21 days of treatment, the value of LPO was increased in the streptozotocin-treated group in the liver, kidney, muscle and Pancreas homogenate compared to the Control group. Momordica charantia group showed a significantly decreased value of LPO in Liver (p 0.020), kidney (p 0.02), pancreas (p 0.01), and Muscle (p 0.03) homogenate. These indicate the antioxidant activity of Momordica charantia.

TABLE 6: Effect on Blood LPO Level

The prevalence of diabetes in adults increased to 8.8% of the global population in 2017, and by 2045, it is predicted to reach 9.9% of the population. According to estimates, there were 424.9 million diabetics globally in 2017; by 2045, that number is expected to rise by 48% to 628.6 million [24]. Many natural remedies have been promoted for the management of diabetes mellitus, and a number of them have been clinically and experimentally proven effective. Aegle marmelose (root, bark, and leaf), Allium cepa, Alium sativum, Azadirachta indica (leaf), Brassica juncea (whole plant), Coccinia indica (whole plant), Eugenia jambolana (seeds), Ficus bengalenesis (bark), Gymnemasylvestre (leaf), Mongifera indica (leaf), Momordica charantia (seed, leaf), Musa sapientum (fresh flower), Ocimum sanctum (leaf), Phyllanthus niruri (whole plant), Punica granatum (flower), Trigonella foenumgraecum (whole plant and seed), Tinospora cordifolia (root), and Zingiber officinale roscoe (rhizome) [25]. The most often utilized models for screening antidiabetic medicines in diverse studies were the oral glucose tolerance test, streptozotocin, and alloxan-induced diabetic mice or rat models. There are theories regarding how they affect the activity of beta cells in the pancreas, how they raise insulin sensitivity, or how plant extracts mimic insulin. Additional mechanisms could also be at play, including increased peripheral glucose utilization, increased hepatic glycogen synthesis or decreased glycogenolysis, inhibition of intestinal glucose absorption, decreased glycaemic index of carbohydrates, and decreased glutathione effect [26]. Glycogen synthase is activated by synthase phosphatase, which leads to glycogenesis. In STZ-diabetic mice, this activation appears to be impaired [27,28]. In patients with STZ-insulin-dependent diabetes, this inhibition of synthase phosphatase is nearly complete after 1–2 weeks. Diabetes appears to alter the lipid composition of cell membranes, as shown by elevated levels of lipid peroxidation, non-enzymatic glycation, and the ratio of cholesterol to phospholipids [29]. Increased plasma levels of phospholipids, free fatty acids (FFA), total cholesterol (TCH), and triglycerides (TC) were seen in STZ diabetic rats.

The present study was therefore intended to examine the effect of the alcoholic extract of Momordica charantia on streptozocin-induced diabetic albino rats, using metformin as a reference drug. In the study, we selected Momordica charantia, commonly known as bitter melon, on the basis of its hypoglycemic and ulcer protective effects. This outcome is consistent with previous authors' findings that different plant leaf extracts significantly lowered rats' blood glucose levels. After 21 days of treatment, the glucose level was 52% lower in the alcoholic extract of M. charantia. With the use of cell-based tests, animal models, and human clinical trials, numerous investigations have shown that M. charantia possesses strong antidiabetic properties [30]. Activating AMPK, reducing insulin resistance, preserving islet β-cells, boosting insulin secretion, increasing hepatic glucose clearance and decreasing gluconeogenesis, and blocking intestinal α-glucosidase and glucose transport are some of the potential mechanisms of M. charantia that have been documented. Numerous studies have shown that M. charantia extract (MCE) has pharmacological effects through mechanisms that include modulating incretin activity, promoting insulin secretion and release, rejuvenating beta Langerhans islets, stimulating enzymes involved in glucose utilization, and reducing small intestine absorption of glucose and fatty acids. It is well known that the entry of food into the gut triggers the release of hormones that control the incretin function, or the production and secretion of hormones by pancreatic islets. These attributes are shared by GLP-1 and GIP [31]. This implied that endocrine L-cells secrete more GLP when exposed to extract from MC leaves. Peroxisome proliferator-activated receptor (PPAR) expressions can be upregulated and activated by MCE. The number of β cells in diabetic rats was considerably raised after 9 weeks of MCE juice administration. Conversely, the bitter-tasting components of BG may cause the L cells' bitter taste receptors to become active, which would increase the release of GLP-1. After half an hour, a single oral dose of MCE dramatically reduced blood glucose and raised insulin and GLP-1 levels. Exendin-9, a GLP-1 receptor antagonist, was administered beforehand to eliminate this acute hypoglycemia [32].

According to a 2018 study by Bhat et al., the increased GLP-1 levels in the normal and diabetes treatment groups might be the result of L-cell proliferation and regeneration, which attach to L-cell receptors and undergo conformational changes that activate a number of signal transducers. By increasing the intracellular Ca2+ concentration, the polar molecules of M. charantia also depolarize the L-cell, causing the release of GLP-1. Insulin secretion and beta-cell proliferation are subsequently increased by GLP-1. The enzyme is more active during diabetes, which also causes an increase in lipolysis and the release of additional fatty acids into the bloodstream. With diabetes, there is an increase in fatty acid content, which also leads to an increase in fatty acid β-oxidation, which produces more cholesterol and acetyl Co-A. Under normal circumstances, insulin enhances the clearance of LDL cholesterol from receptor mediators, while decreased insulin activity during diabetes results in hypercholesterolemia. Lipid breakdown may be the reason for the elevated free fatty acid content, which could lead to an increase in NADPH-dependent microsomal lipid peroxidation. Rats with diabetes caused by alloxan showed an increase in phospholipids. A lipid bilayer that functions as an interface between the non-polar lipoprotein of the lipoprotein core and the polar plasma environment is formed by phospholipids, which are found in cell membranes and make up the great majority of surface lipoprotein [33,34]. Elevated levels of phospholipids were seen in the tissues of streptozoctocin-induced diabetic rats.

The current study's findings on M. charantia and metformin showed a triglyceride reduction of about 16%. In a different study, liver lipid measures in hamsters given diets rich in cholesterol and low in cholesterol are improved by extracts from bitter melon (Momordica charantia) [35]. In contrast to normal rats, the SD rats in this study displayed elevated amounts of oxidative free radicals, including LPO and NO, in a variety of homogenates, including the liver, kidney, muscle, and pancreas. Both normal and SD animals' levels of free radicals (LPO and NO) dropped after receiving MCE treatment. The SD group in the current study had lower levels of SOD and glutathione. The SD rats' levels of SOD and GSH increased after receiving MCE treatment.

Numerous studies have shown that, in experimental settings, M. charantia is a good natural source of antioxidants; both in vitro and in vivo, it exhibits efficacy against oxidative damage. Several in vitro models were used to evaluate which solvent extracts had the strongest anti-oxygenic activity: bitter gourd pulp and its extracts, seed powder, and its ethanol/water extracts [36]. When M. charantia is added to diabetic rats, the levels of TBARS are considerably reduced and the activities of antioxidants (SOD, CAT, and GST) are dramatically increased. In alloxan-induced diabetic rats, oral treatment of lyophilized M. charantia powder is crucial for lowering serum TBARS and preserving GSH levels. A wild variant of bitter gourd's alcohol extract and aqueous extract have been shown to have the ability to remove the 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) radical. One of the most potent antioxidants and free radical scavengers found in M. charantia is flavonoids. The quantity of flavonoids increased progressively with the antioxidant capacity [37]. This study provided scientific validation for the widely reported ethnomedicinal usage of M. charantia in the management of diabetes. Because M. charantia is rich in phytochemicals, its use as a medicinal plant has been enhanced. Rats with streptozocin-induced diabetes show a hypoglycemic response to alcoholic extracts of M. charantia. The reference medication, metformin, produced a similar result during treatment, pointing to a similar mechanism of action. In order to manage diabetes mellitus, there is currently an increasing interest in assessing herbal medicines, which are said to be less toxic and have insignificant side effects. This is especially true in nations where access to traditional diabetes treatment is limited. Therefore, as M. charantia seems to be usually safe, its usage is advised.

Based on the current findings, our research suggests that Momordica charantia alcoholic extract has good long-term anti-hyperglycemic efficacy with a considerable drop in blood TGA level. After 21 days of administration of an alcoholic extract of Momordica charantia, our study shown a substantial difference in anti-oxidant parameters, i.e., increased GSH & SOD while decreased NO, LPO in several tissue homogenate (muscle, kidney, liver, and pancreas), demonstrating its anti-oxidant characteristics. Thus, we can conclude that Momordica charantia alcoholic extract might be a good candidate/ cost cost-effective/ rational /anti-oxidant treatment in cases of type 2 DM.

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