Antihyperlipidemic and Histopathological Pancreas Analysis of Muntingia calabura L. Fruit Extract on Alloxan-Induced Diabetic Mice

 

Tridiganita Intan Solikhah1,2*, Gahastanira Permata Solikhah3

1Division of Veterinary Clinic, Department of Veterinary Science,

Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia.

2School of Health and Life Science, Universitas Airlangga, Indonesia.

3Cahaya Petclinic, Veterinarian, Mojokerto, Indonesia.

*Corresponding Author E-mail: tridiganita-intan-s@fkh.unair.ac.id

 

ABSTRACT:

Hyperlipidemia is clinically manifested by high levels of Total Cholesterol (TC), Triglycerides (TG), Low Density Lipoprotein (LDL) and low levels of High Density Lipoprotein (HDL) in the blood. Antihyperlipidemic drugs available currently have adverse effects. One of the medicine plants with antihyperlipidemic properties with no side effects is Muntingia calabura fruit. The sample used for this research were 30 male mice were divided into 5 groups, i.e., a negative control, a diabetic control, a positive control group, and two treatment groups which were given 100 and 300mg/kg of M. calabura fruit extract every day for 14 days. Examination of TC, TG, HDL, LDL and histopathological pancreas were determined after administration of the extract orally for 14 days. The results demonstrated an administration of glibenclamide and M. calabura fruit could effectively reduce TC, TG and LDL and increase HDL compared to the diabetes control group (P<0.05). In the diabetes control group, the mice given alloxan 150mg/kg showed a decrease in Langerhans islet density, vascularity, and islet injury compared to the normal control group. Mice given M. calabura fruit extract showed a significant increase in pancreatic Langerhans cells granulation and cell density. The conclusion of this research is M. calabura fruit extract showed improvements in lipid profile and pancreatic Langerhans cell regeneration. Therefore, the fruit extract of M. calabura is a potential antihyperlipidemic drug.

 

KEYWORDS: Alloxan, Antihyperlipidemic, Diabetes, Histopathological pancreas, Muntingia calabura.

 

 


INTRODUCTION: 

Hyperlipidemic conditions are frequently observed in diabetes mellitus (DM)1,2. Hyperlipidemia in diabetes mellitus causes atherosclerosis, which is the formation of plaque on the aortic and coronary artery walls, which narrows the lumen and lowers the elasticity of blood vessels3. Blood flow is obstructed when the lumen of blood vessels becomes narrowed, causing coronary heart disease (CHD)4,5. Various research have found that the main causes of atherosclerosis and CHD are hyperglycemia, hypercholesterolemia, and LDL oxidation. In order to reduce the mortality risk from CHD, it is therefore possible to lower blood sugar, cholesterol, and LDL oxidation levels6.

 

 

In Indonesia, the prevalence of hypercholesterolemia in the 25-34 years age group is 9.3%, while in the 55-64 years age group is 15.5%7,8. The risk factors include genetic, diet pattern and lack of exercise. Cardiovascular disease (CVD) is the largest cause of death worldwide, accounting for over 17.5 million deaths each year, with 80% of deaths occurring in low and middle-income nations9.

 

The pancreas is a crucial glandular organ in the body which contains both exocrine and endocrine tissues. The pancreas's responsibility is to produce pancreatic juice, which contains the enzymes trypsinogen, amylase, and lipase. These enzymes combine with food items in the duodenum and perform digestive processes in the intestine; the exocrine portion is composed of acinar cells which discharge enzymes into the duodenum via a duct. The islets of Langerhans, whose role is to create the hormone insulin, which is then absorbed by the blood, help compensate the endocrine system. Insulin is required for metabolism of carbohydrates, fats, and proteins. Chemical substances which get into the body in high doses can damage islet of Langerhans cells. This damage will result in reduced insulin production, which will cause in hyperglycemia and hyperlipidemia10,11.

 

There are many antihyperlipidemic drugs available recently. However, these drugs have adverse effects12. Therefore, herbal medicines are much developed13–15, with almost no side effects, affordable and available in almost all areas16,17. One of the medicinal plants with antihyperlipidemic properties is Kersen (Muntingia calabura), which is rarely in demand but provides a high potential to repair lipid profile in hyperlipidemia. M. calabura fruit contains several compounds such as riboflavin, fiber, thiamine, beta-carotene, niacin and ascorbic acid that are beneficial to the human body. M. calabura fruit also contains several compound groups that are useful as antihyperlipidemic agents, namely alkaloids, flavonoids, saponins, steroids, sterols, tannins, phenols and terpenoids15,1820.

 

The administration of Kersen juice does not significantly reduce cholesterol level, significantly reduces triglycerides and does not significantly increase HDL level 18. In Addition, Puspasari et al. (2016)22 stated that the administration of Kersen leaves extract has been proven to improve lipid profile (total cholesterol, TG, LDL, and HDL) in male white mice (Mus musculus) induced by used cooking oil. Diabetes-induced hyperlipidemia is caused by an excess mobilization of fat from the adipose tissue as a result of glucose underutilization23. Alloxan is diabetogenic because it destroys beta-cells in the islets of Langerhans, resulting in a substantial drop in insulin release and hyperglycemia. Insulin insufficiency causes a variety of metabolic changes in animals, including increased blood glucose, cholesterol, alkaline phosphate, and transaminases, among other things24. Based on this background, no research has been conducted using ethanol extract of cherry fruit, so the author is interested in studying the effectiveness of the antihyperlipidemic and pancreatic histopathology of Kersen fruit extract (M. calabura) on alloxan-induced diabetic mice.

 

MATERIALS AND METHODS:

Chemicals:

Alloxan monohydrate was purchased from Nitrakimia, Indonesia. The alloxan monohydrate solution was prepared by freshly dissolving in 0.9% NaCl. TC, TG, HDL, and LDL standard kits. Extraction using 96% ethanol and dissolved using Carboxyl Methyl Cellulose (CMC) before being given to mice. 10% formalin salt solution, paraffin for pancreatic histology examination.

 

Preparation of M. calabura fruit extract:

Fruit from M. calabura rinsed with clean water, dried in an oven set to 60°C, then ground and sifted to create a dry powder. Fruit powder from M. calabura was steeped in 96% ethanol at a ratio of 1:7 for 24 hours while remaining at room temperature. The M. calabura fruit pulp was remacerated with the addition of 96% ethanol at a ratio of 1:4 after the maserate had been separated after 24 hours of filtering. All macerates were filtered, then evaporated at 60°C until a thick extract was produced. To protect the concentrated extract from deterioration, it was put in a beaker glass, wrapped with aluminum foil, and kept in the freezer. To create the necessary extract, Carboxyl Methyl Cellulose (CMC) was used as the solvent at a concentration of 0.5%.

 

Animals:

30 male mice weighing between 25 and 35g were utilized as the sample in this study. They were separated into 5 groups, each with 6 mice. The institutional, national, and ethical rules regarding live animals were all rigorously followed. The Health Research Ethical Clearance Commission Faculty of Dental Medicine, Universitas Airlangga granted ethical clearance for the use of animals in this study under number 507/HRECC.FODM/XI/2020.

 

Antihyperlipidemic activity:

Alloxan monohydrate with a dose of 150mg/kg body weight was dissolved in 0.9% NaCl and injected peritoneally to all mice except the negative control group. Alloxan monohydrate solution was only injected once and waited for 5 days to achieve hyperglycemic condition. Mice that had blood glucose higher than 200 mg/dL were used for the experiment.

 

The animals were divided into 5 groups, i.e., a negative control which included non-treated white mice, a diabetic control group which included white mice injected with 150mg/kg body weight of alloxan peritoneally, a positive control group which was given 600µg/kg body weight of glibenclamide, and two treatment groups which were given 100 and 300mg/kg of M. calabura fruit extract every day for 14 days via intragastric gavage in the morning. Blood samples were taken from the heart to check for cholesterol level, triglycerides, HDL and LDL.

 

Histological test:

At the end of the experiment was finished, the pancreatic tissues were taken from the abdomens of all groups of mice after they had been given anesthesia. Separated and let to dry for 48 hours in a 10% formalin salt solution, the pancreatic tissue was then fixed. Following sample dehydration and infiltration, the samples were embedded in paraffin and stained with hematoxylin-eosin (H&E) on a glass slide. The glass object was examined under a light microscope and photographed with a digital camera23.


Table 1: Effect of M. calabura fruit on serum lipid marker in diabetic mice.

Groups

TC (mg/dL)

TG (mg/dL)

LDL (mg/dL)

HDL (mg/dL)

Negative control

86.8 ± 5.76

83.2 ± 4.09

25.6 ± 7.63

61.6 ± 6.02

Diabetes control

175.2 ± 4.82*

229.6 ± 6.73*

92 ± 6.20*

39.6 ± 3.71*

Glibenclamide 600 µg/kg

118.8 ± 4.55

94.6 ± 2.79*#

29 ± 1.58*#

67.4 ± 5.13*#

M. calabura fruit extract 100 mg/kg

132.8 ± 3.63*#

110.6 ± 2.41*#

30.6 ± 2.41*#

83.8 ± 2.39#

M. calabura fruit extract 300 mg/kg

166.6 ± 2.7*#

138.6 ± 6.27*#

40.6 ± 2.41*#

73.8 ± 1.92*#

*p < 0.05 (compared with normal control group); #p<0.05 (compared with diabetes control group).


 


Data analysis:

The SPPS 22 program was used to evaluate all of the research data. The significance was assessed using a One-Way ANOVA test. The Tukey test was used to determine whether there were group differences. At P<0.05, a difference was judged to be significant.

 

RESULTS:

Effect of M. calabura fruit extract on lipid profile (mg/dL) in alloxan-induced diabetic mice. (Table-1).

 

The findings showed that alloxan induction significantly raised total cholesterol, triglycerides, and LDL when compared to the normal control group (P<0.05). Additionally, compared to the normal control group, alloxan induction significantly reduced HDL (P<0.05). Furthermore, glibenclamide and M. calabura fruit administration may significantly lower total cholesterol, triglycerides, and LDL while increasing HDL in comparison to the diabetes control group (P<0.05).

 

Histological findings:

Figure 1 displays a representative photomicrograph of the histological analysis of pancreatic tissue from normal control groups (without treatment), diabetes control groups given alloxan 150mg/kg, two groups of diabetic mice given M. calabura fruit extract at different concentrations (100mg/kg and 300mg/kg), and positive control mice given glibenclamide at a dose of 600g/kg. In comparison to the mice who received alloxan 150 mg/kg in the diabetic control group, these animals had lower Langerhans islet density, vascularity, and islet damage levels. Pancreatic Langerhans cells granulation and cell density significantly increased in mice administered M. calabura fruit extract. In comparison to the control group of alloxan-induced mice models, pancreatic Langerhans cells were seen to improve and regenerate in the treatment group of diabetic mice given extract at varied concentrations.

 



Figure 1: Photomicrographs of histopathological changes in pancreatic islet cells treated with M. calabura fruit extract 100mg/kg (A), M. calabura fruit extract 300mg/kg (B), glibenclamide 600µg/kg (C), diabetes control group (D) and normal control group without treatment (E). (H & E stain, 400X).


 

DISCUSSION:

Cardiovascular disease is the leading cause of death in some countries, with around 17.5million deaths every year8. One of the cardiovascular diseases includes hyperlipidemia, which is characterized by an increase in serum lipid. High LDL cholesterol level is a risk factor for coronary heart disease24.

 

Animals induced with alloxan showed damage to the pancreatic β cells20,25, which lead to a decrease in insulin level. Reduced insulin levels will cause an increase in hormone-sensitive lipase. This causes an increase of free fatty acids and the level of fatty acid in plasma increases two folds. Reduced insulin levels will lead to cells lacking energy. Therefore, pancreatic α cells respond by secreting glucagon26. Glucagon will affect the mobilization of free fatty acids. It has an antagonistic effect on insulin and increases hormone-sensitive lipase in adipose tissues. An increase in hormone-sensitive lipase causes an increase in triglyceride hydrolysis, thus increasing the level of free fatty acids.

 

The administration of M. calabura fruit at 100 and 300 mg/kg BW to mice resulted in a decrease in TC, TG, LDL-C, as well as an increase in HDL-C levels. Furthermore, therapy with M. calabura fruit at a higher dose (300mg/kg) and glibenclamide (600μg/kg, standard medicine) significantly reduced cholesterol levels in hyperlipidemia mice's serum, with almost comparable outcomes. Kersen fruit extract acts in reducing LDL levels by a double mechanism from flavonoid, saponin, polifenol, and alkaloid27,28. Flavonoid works by reducing the level of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase which will show an effect of reduced cholesterol levels in the body29. Flavonoids also increase the activity of Lecithin Cholesterol Acyltransferase (LACT) which can reduce the level of free cholesterols in blood30,31. Flavonoid substance has the mechanism to increase the level of HDL cholesterol by increasing the release of cholesterol from macrophages and increasing the expression of ATP-Binding Cassette (ABC) transporter A1 and increasing apolipoprotein A1, which is the base material for HDL30,32.

 

Saponin works by bonding with bile acids to form large mixed micelles. Thus, the cholesterols in the micelle cannot be absorbed by microvilli on the surface of intestinal epithelial cells. This results in the reduction of LDL and total cholesterol levels. Saponins can reduce the amount of triglycerides in the blood by preventing their absorption in the intestine. Saponins limit cholesterol absorption in the intestine, preventing cholesterol against being absorbed and finally expelled with feces. Saponins attach to bile acids and enhance the excretion in the stool, causing an increase in the conversion of cholesterol to bile acids in an effort to preserve bile acid storage. As a consequence, LDL receptors from the liver will be enhanced, resulting in an increase in LDL absorption and a decrease in plasma cholesterol levels. Polyphenols are antioxidants that could also stabilize free radicals by compensating for electron deficits and suppressing the formation of free radicals. Alkaloids can work by lowering gluconeogenesis, which lowers blood glucose levels and diminishes the need for insulin33,34.

 

The observation results in Figure 1 indicated that alloxan-induced mice showed damage to the pancreatic cells. In alloxan-induced diabetic mice, histopathological analysis of the pancreas revealed a decrease in the Langerhans islets density, vascularity and injury. There used to be a significant of necrosis and degradation in the diabetic group. This demonstrates that alloxan administration can harm pancreatic endocrine cells, particularly beta cells, resulting in a reduction in insulin release into blood vessels. The islet of Langerhans has a high prevalence of necrosis and degeneration, which is indicated by the presence of empty spaces in the center of the islet. Beta cell necrosis causes empty spaces inside the islets of Langerhans. Necrosis is characterised by injury to the structure and functionality of the cells as a whole, characterized by cell lysis and tissue inflammation. Necrosis is the result of deadly damage. A decrease in the number of pancreatic beta cells implies a problem with insulin metabolism in the pancreas, which leads to a reduction in the volume of beta cells in the islets of Langerhans35.

 

Alloxan works by producing reactive oxygen species, which are hydrogen peroxide (H2O2) and free hydroxyl radicals (OH) which are highly reactive in attacking biological molecules, which damages pancreatic cells. The histopathology results of the normal control group showed a difference, There was no or very little necrosis in the normal control group, and the cell nucleus was very dense, with no edematous cells (swelling), signifying that now the islet of Langerhans was in normal condition (no damage).

 

The pancreatic islets of langerhans of mice given M. calabura fruit extract showed improvement and regeneration of pancreatic langerhans cells. This histopathological analysis of the pancreas proved the ability of M. calabura fruit extracts to regenerate alloxan-damaged pancreatic Langerhans cells. Unfortunately, the Langerhans islet's condition has not yet returned to normal. It is hypothesized which the repair of pancreatic cells with ethanol extract of cherry fruit lasts longer than two weeks. This observation is in agreement with other research that suggested diabetic pancreas histological improvements increase after treatment with medicinal plants with high phenolic content36. Antioxidant action which can reduce cell damage, particularly in pancreatic beta cells, and stimulate insulin production,  because it includes arabinogalactan type II, which regulates the cAMP-Akt insulin production pathway 37,38. Other compounds, such as flavonoids and saponins, enhance pancreatic β cell regeneration39. The presence of bioactive substances in the cherry fruit ethanol extract, such as alkaloids, flavonoids, saponins, and polyphenols, was assumed to be responsible for the enhancement in the cherry fruit ethanol extract treatment group. These bioactive compounds have the potential to serve as antioxidants. Antioxidants have a significance in the regeneration of damaged cells. Cell damage caused by the presence of free radicals could be minimized by the presence of antioxidants, which act as reducing agents and diminish oxidizing chemicals before damaging cells, hence minimizing cell damage40. Histological results in the positive control, which was given glibenclamide also showed an improvement, this is in line with a theory that glibenclamide works by helping the regeneration of β cells and helping to increase insulin secretion.

 

CONCLUSION:

The findings of this study indicate that the fruit extract of M. calabura showed improvements in lipid profile and pancreatic Langerhans cell regeneration. Therefore, the fruit extract of M. calabura is a potential antihyperlipidemic drug. Further studies are needed to evaluate the antihyperlipidemic activity of M. calabura fruit with various extraction methods and solvents.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 07.10.2022            Modified on 21.01.2023

Accepted on 28.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4841-4846.

DOI: 10.52711/0974-360X.2023.00785