INTRODUCTION:

Diabetes mellitus (DM) is a multisystem metabolic disorder characterized by a hyperglycemic condition, which is caused by abnormal insulin secretion and insulin performance¹. One of the factors causing diabetes mellitus is obesity, as a consequence of an increase in fat levels in the body, which is drawn by hyperlipidemic conditions and an increase in the cholesterol levels of blood^2,3.

This condition causes the production of reactive nitrogen species (RNS) which oxidize the sulfhydryl groups of proteins, like amino acids in the type of tyrosine; it can increase lipid peroxidation and promote the DNA damage that are able to affect cells which are exposed to ROS and RNS^4,5,6.

One of the negative effects in obesity is the occurrence of insulin resistance which is an inability of insulin to function normally and creates a decrease in the tissue sensitivity toward insulin^6,7,8. Insulin resistance triggers an interference with glucose transporter type 4 (GLUT-4) translocating to the surface of muscle cell membranes and fat cells. The decreasing GLUT4 will disturb glucose uptake into cells and a further increase in the blood glucose levels^9,10,11. The prolonged hyperglycemia conditions can activate the polyol pathway. An excessive activation of polyol pathway in insensitive tissue fostering a lot of glucose being converted into sorbitol which then will be retained in the cell. These changes force the mitochondria in the cell to produce superoxide anions which boost ROS, thus, oxidative stress cells also escalate. The raising production of ROS that exceeds the antioxidant capacity of cells generates an increase in the oxidative stress accompanied by the occurrence of dysfunction and damage to β cells in the pancreas driving a decreased insulin secretion¹².

Antioxidants are substances that can inhibit the effects of free radicals by giving electrons, thus, the damage of lipid, cell membranes, blood vessels, DNA, and other damages catalyzed by the reactive compounds can be prevented^12,13. Furthermore, the indigenous people of Indonesia has relied on medicinal plants for their health need through generations^2,3,14. One of them is Abelmoschus esculentus Moench that has several benefits, precisely for human. One type of antioxidant which has the ability to overcome free radicals is quercetin, a flavonoid compound contained in okra pods (Abelmoschus esculentus Moench)¹⁵. Quercetin compounds have a significant ability to overcome ROS^16,17.

To date, there has been no scientific experiment of the effect of okra pods antioxidant in the streptozotocin-induced type-2 diabetic mice. This study examines the effect of Okra extract on the fasting blood glucose levels, the insulin levels and the activation of GLUT4 receptors on the surface membranes of striated muscle cells, as well as the sensitivity of muscle cells to insulin.

MATERIAL AND METHODS:

Plant Identification:

Taxonomic identification of Okra (Abelmoschus esculentus Moench) fruit was carried out by the Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

Drugs, Chemicals, and Reagent:

The drugs used in this study were acarbose 100 mg (Dexa Medika, Indonesia), streptozotocin (Sigma-Aldrich, USA), and other chemicals such as lard to provide the hyperlipidemic conditions in mice, ethanol (Sigma-Aldrich, USA) for crude extraction and polar fraction of the okra pods extract, n-Hexan (Sigma-Aldrich, USA) for non-polar extraction of the okra pods, ethyl acetate (Sigma-Aldrich, USA) for semi-polar extract of the okra pods, ketamine hydrochloride/xylazine hydrochloride solution (Sigma- Aldrich, USA) for animal anesthesia when taking the intra-cardiac blood and organs from experimental animals, Sodium carboxymethyl cellulose (Sigma-Aldrich, USA), 10% formalin buffer, citrate buffer (Sigma-Aldrich, USA), phosphate buffered saline (Sigma -Aldrich, USA), Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, USA), xylenes (Sigma-Aldrich, USA), paraffin wax (Merck, Germany), poly-L-lysine coated slides (Sigma-Aldrich, USA), hematoxylin-eosin (Sigma-Aldrich, USA), anti-GLUT4 antibody (Sigma-Aldrich, USA). All chemicals and reagents used were of the analytical grade.

Plant Material and Extractions:

There were approximately 20 kilograms of okra pods from the local market in Surabaya, Indonesia. This study used okra pods. Fresh fruit was cut into 2 mm pieces, then dried for 3-5 days, and crushed into powder. Around 500 mg of the powder was put into a bottle, added with 1.5 L ethanol 96%, macerated, shaken 100 times per day for three days constitutively until the solvent was clear. The solvent was evaporated with the Rotavapor^® R-300 (Buchi) at a temperature of around 50 °C until a crude extract was obtained. Next, the crude extract was dried using a freeze dryer. The fractionation process of okra fruit extract was carried out by weighing 500 mg of dried okra fruit and macerated with n-hexane solvent (nonpolar fraction), then shaken 100 times per day for three days until the solvent was clear. After that, the extract was filtered. The filtrate was evaporated while the pulp was macerated again using ethyl acetate (semi-polar fraction), filtered, and then macerated again with ethanol 96% (molar fraction). All filtrates obtained were evaporated with the Rotavapor^® R-300 (Buchi) at a temperature of around 50 °C until three fractions were obtained, they were non-polar, semi-polar and polar fractions.

Experimental Animals and Ethical Clearance:

This study used healthy adult male mice (Mus musculus), strain BALB/C, with the age ranged from 3-4 months old, body weight ranged from 30-40 g, obtained from the Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia. Mice were acclimatized for 2 weeks to provide conditions that were similar to the conditions of the Animal Laboratory, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia. All body weight and blood glucose levels were recorder before and after the administration of lard and streptozotocin. Mice were divided into 7 groups (n = 5 mice), all were in control of environmental conditions (25±5 oC, humidity of 50±10% and 12 light/dark cycle). Mice were fed with standard pellet and drink (ad libitum). All treatment procedures have been tested through Ethical Clearance at the Animal Care and Use Committee, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia (Approval Reference Number: 2.KE.069.04.2018).

Experimental Design:

This study was an experimental study conducted at the Animal Laboratory, Molecular Genetics Laboratory, Animal Histology Laboratory, and Organic Chemistry Laboratory, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia using a completely random design. For induction of diabetic mice, this study used streptozotocin. Fasting blood glucose levels were measured before and after streptozotocin induction on 7^th and 14^th day. The blood glucose level was measured by applying a glucometer to determine the diabetic condition of mice. Only mice with fasting blood glucose levels of more than 130 mg/dL were used as a diabetic group.

Blood Collection:

Blood collection of mice was carried out on 15^th day intracardially. The blood obtained was then inserted into Falcon^TM (Fisher Scientific) and cooled for an hour. After that, the blood serum was taken and centrifuged at 16,000 rpm for 5 minutes.

Blood Analysis:

The blood serum was applied to measure insulin levels employing the ELISA protocol. Whereas, the density of GLUT4 inside the fiber and the surface of skeletal muscle cells was examined using immunohistochemistry. The utilized antibodies were IgG GLUT4 (IF8) monoclonal mouse antibodies, as well as the other material such as polylysine adhesion and ultra-vision HRP polyvalent detection system.

Immunohistochemical Analysis on Glucose Transporter 4 (GLUT4):

This study applied a method based on the previous study of Suarsana et al. for the immunohistochemical evaluation¹⁸. The tissue was fixed for 24 hours in 10% formalin solution and then processed by a standard method using paraffin. First, the slides were deparaffinized in xylene and rehydrated in a descending order of ethanol. Subsequently, tissue samples were incubated in a citrate buffer solution for antigen retrieval. After that, the tissues were incubated with H₂O₂ in methanol for 15 minutes. Furthermore, it was dripped with a 10% bovine serum albumin (BSA) for 45 minutes at 37° C. After washing; the tissue was added with antibody primary monoclonal anti-GLUT4 and then left at the room temperature for an hour. Next, the tissue was mixed with biotinylated IgG for 30 minutes at the room temperature. Then, it was fixed with avidin biotin HRP for 30 minutes. The antigen-antibody reaction results were visualized by using diamino benzidine (DAB) at the room temperature for 5 minutes, then counterstained with HE. The observations were performed under a light microscope with 20 times-magnifications. The results were positively indicated GLUT4 if the tissue turned into brown color.

Statistical Analysis:

Data from this study included the fasting blood glucose level, the blood serum insulin level and GLUT4 expression in the fiber and the surface membrane of mice’s skeletal muscle cell. The data obtained were analyzed using the Static Package for the Social Science (SPSS) program which included normality and homogeneity tests by applying the Kolmogorov Smirnov Test and Levene Test. If the normality and homogeneity tests met the requirements for parametric tests, then it would be continued using One Way ANOVA (α = 0.05). If the data obtained was not homogeneous, then it would be tested by employing the Brown Forsythe Test (α = 0.05). Moreover, an independent t-test was carried out to determine the differences between the 2 groups. If there was a significant difference, it would be followed by Duncan Test at the level of 0.05 or 95%, but if the normality and homogeneity tests did not meet the requirements, then it would be followed by a non-parametric test using the Kruskal Wallis Test (α = 0.05). Then, if it was significantly affected, it would be continued by the Mann-Whitney Test.

RESULTS AND DISCUSSION:

The average data of the mice’s fasting blood glucose level before and after streptozotocin injection are presented in Fig. 1 and the average data of the blood serum insulin level average after VOPE administration is showed in Fig. 2. Whereas, data of the GLUT4 average after VOPE administrations are exhibited in Fig. 3 of which located in the fiber of skeletal muscle cells and in Fig. 4 of which located on the surface of skeletal muscle cells.

The measurement of fasting blood glucose level average before and after streptozotocin induction (see Fig. 1) shows that streptozotocin injection at the dose of 30 mg/kg body weight for 5 consecutive days is able to elevate the fasting blood glucose level significantly from 127,071±14,978 to 189,321±30,867 mg/dL. This indicates that streptozotocin is able to damage the pancreatic islet β-cells and reduce the insulin synthesis as well as elevate the fasting blood glucose level^2,3,6. Streptozotocin can cause DNA fragmentation in pancreatic islet β-cells through the formation of free alkylating agents in order to reduce the cellular nucleotides and their components, such as NAD⁺ which creates a necrosis in pancreatic islet β-cells. Streptozotocin affects glucose oxidation and decreases insulin biosynthesis and secretion¹⁹.

Fig. 1: Fasting Blood Glucose (mg/dL) Before and After Streptozotocin Induction.

In Fig. 2, after the data is analyzed by the Kruskal-Wallis test, there is a significant difference among the treatment groups. Followed by the Mann-Whitney test, the results shows that there are significant differences between KN and KD, but there are no significant differences between KN and KA, EK, NP, and SP. These findings indicate that diabetic mice insulin levels, without administration of okra fruit extract and standard diabetic drug ingredients, reveal a decrease in insulin levels, compared to the normal control group. This is because of the damage to Langerhans β cells which happens in the diabetic control group. Meanwhile, in the diabetic group with VOPE adinistration, it exhibits a significant difference compared to the normal control group because quercetin compounds contained in okra fruit extracts are able to make improvements to the cells of Langerhans β cell, thus, the insulin levels raise close to the normal insulin levels¹⁵.

Fig. 2: The Effect of VOPE toward Mice’s Blood Serum Insulin Levels (ng/mL).

Figure 3 indicates that the diabetic control group (KD) has the highest GLUT4 density in the fiber of skeletal muscle cell compared to the normal control group (KN) and the other groups. The acarbose control group is not different significantly compared to the normal control group and VOPE group. While VOPE group can decrease significantly in GLUT4 density compared to the diabetic control group (KD) which almost reaches the same level as the GLUT4 density in the KN group. Thus, it can be seen that giving VOPE can reduce the GLUT4 density in the fiber of mice skeletal muscle cells. Decreasing the density of GLUT4 in the cytoplasm can overcome the decrease in auto-phosphorylation located in the cytoplasm of skeletal muscle cells, which can reduce the exposure of insulin-sensitive GLUT4 to the cell’s surface. As it is known that GLUT4 is the main glucose transporter located in skeletal muscle cells and fat cells stimulated by insulin, then an insulin resistance can cause a decrease in GLUT4 expression in muscle tissue cells^9,20.

Fig. 3: The Effect of VOPE toward GLUT4 Density in the Fiber of Mice’s Skeletal Muscle Tissue.

From the results of this study, it is known that the administration of VOPE containing quercetin compounds could neutralize the increase of ROS which occurs in the body of experimental animals. Thus, it is expected as well that it can reduce the oxidative stress in DM patients. Quercetin compounds can cause normalization of insulin secreted by pancreatic β cells and stimulate GLUT4 mobility from the fiber to the surface of skeletal muscle cell. This situation can increase insulin sensitivity so that glucose uptake into cells becomes normal¹⁸.

Fig. 5: The Effect of VOPE toward GLUT4 Density on the Surface Membrane of Mice’s Skeletal Muscle Tissue.

Fig. 5 shows that VOPE significantly increases in the density of GLUT4 located in the surface membrane of skeletal muscle cell compared to the KD group. The increase in GLUT4 density almost reaches the same level as the GLUT4 density in the KN group. Thus, it can be concluded that giving VOPE can increase the GLUT4 density on the surface membrane of the mice’s skeletal muscle cell. The raising GLUT4 density on the surface membrane of skeletal muscle cell can increase the muscle cell sensitivity to insulin and improve the insulin sensitivity. The GLUT4 density from the highest order is expressed in the EK, NP and EP groups. Insulin sensitivity is the ability of insulin in reducing the blood glucose concentrations, by stimulating glucose used in fat and muscle tissue as well as suppressing glucose production by the liver¹⁸. Furthermore, Patel and Kori stated that the usage of the plant extracts for the treatment of diabetes and isolated fraction of Ammania baccifera exhibited anti-hyperglycemic activity when compared with standard hypoglycemic agent²¹.

CONCLUSION:

In sum, the study presented that the administration of VOPE could ameliorate the fasting blood glucose, the insulin levels, and the GLUT4 density. It may therefore be suggested that VOPE showed a promising antidiabetic agent in streptozotocin-induced type-2 diabetic mice.

ACKNOWLEDGEMENT:

Authors would like to thank the Head of Institute of Innovation and Research in Universitas Airlangga for the opportunity which was given to conduct this study funded by the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia. Moreover, special thanks to PMDSU Scholarship - Batch III by The Ministry of Research, Technology, and Higher Education of the Republic of Indonesia which was awarded to Arif Nur Muhammad Ansori, Suhailah Hayaza, and Raden Joko Kuncoroningrat Susilo.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.02.2019 Modified on 19.04.2019

Research J. Pharm. and Tech 2019; 12(8):3703-3708.

DOI: 10.5958/0974-360X.2019.00633.4