INTRODUCTION:
Diabetes mellitus (DM) is a multisystem disorder, which affects about 6% of the world’s population. Hyperglycemic condition characterizes DM because of insulin secretion, insulin function, or both1. One of the factors causing DM is obesity, due to the increase levels of fat in the body caused by the hyperlipidemic conditions and the increase of blood cholesterol2. The rise of cholesterol levels can be seen from the increasing levels of free fatty acids, which contributes in the growth of mitochondria production and risk of the reactive oxygen species (ROS) exposure.
The increase of superoxide production will lead to a rise in nitric oxide (NO) which is caused by NO synthase induction. As the consequence, the condition triggers the production of reactive nitrogen species (RNS). RNS has the ability to oxidize the sulfhydryl proteins, amino acids such as tyrosine, and the increased lipid peroxidation as well as the DNA damage which can occur to the cells that are exposed to ROS and RNS3,4,5.
Once the human body reaches the stage of obesity, there is one negative effect that follows which is body resistance to insulin. It means that insulin is no longer has the ability to work properly. It decreases tissue sensitivity to insulin2,3. Insulin resistance could interfere with the translocation of glucose transporter 4 (GLUT4) to the surface of muscle cell membranes and fat cells. The decrease in GLUT4 will disrupt the glucose uptake and, further, could increase the blood glucose levels6. The prolonged hyperglycemic condition can activate polyol pathways. The excessive activation of polyol pathway in the insulin investitive tissues causes a lot of glucose being converted into sorbitol which can be retained in the cell. These changes force mitochondria in the cell to produce superoxide anions which increase ROS; thus, the oxidative stress of the cell also rises. This increase in oxidative stress will trigger lipid peroxidation in the cell membrane. MDA is a lipid peroxidation product that has been recognized as one of the biological markers for reliable oxidative stress. MDA concentrations in serum, blood plasma and tissue as a way to determine the antioxidant activity which also acts as a major indicator of potential oxidative stress in the diabetic patient. The increased ROS production which exceeds the cell antioxidant capacity leads to a rise in the oxidative stress accompanied by the dysfunction and β cell damage in the pancreatic tissue7. As a result, it decreases the insulin secretion. Moreover, the prolonged hyperglycemic condition can cause the level of RNS to go up. ROS and RNS can oxidize right away and damage DNA, proteins, and lipids8,9.
Any harmful effects of free radicals can be hindered by antioxidants. Electrons were produced by antioxidants thus it put a stop to any further damages in lipids, cell membranes, blood vessels, DNA, as well as other damages triggered by reactive compounds such as ROS and RNS. Basically, a body has a defense and an immune system. This defense deals with free radicals by producing the endogenous antioxidants in the form of superoxide dismutase (SOD), glutathione peroxidase (GSH-x) and catalase (CAT). SOD is an enzyme catalyzing the dismutation of superoxide such as superoxide anions to O2 and H2O2. The performance of endogenous antioxidants that have been produced by the body is activated by donating electrons10. The amount of ROS, produced by the body cells, allows an imbalance between endogenous antioxidants and ROS levels in these cells. The imbalances could reduce the level of endogenous antioxidants such as SOD, GSH-x, and CAT. It is important to increase the amount of antioxidant to avoid the harmful effects of free radicals. In order to do so, many types of fruits and vegetables are encouraged to be consumed since they contain many extra exogenous antioxidants, such as vitamin E, vitamin C, and other high antioxidants. In addition to vitamins E and C, there is also alpha-lipoic acid (ALA), polyphenol and flavonoid which can be used as antioxidants. Flavonoids and polyphenols could improve the cell sensitivity to insulin11.
The indigenous people of Indonesia has relied on medicinal plants12 for their health need through generations13. One of them is Abelmoschus esculentus Moench that has several benefits, precisely for human14. One of the antioxidants used to overcome free radicals is quercetin. Quercetin is a flavonoid compound contained in the seeds of Abelmoschus esculentus Moench. Quercetin compounds have a significant ability to reduce ROS, hydrogen peroxide and protein oxidation by donating hydrogen atoms and significantly stabilizing the free radicals. It is important to remember that Quercetin componds do not easily participate in other radical reactions15,16. In addition to neutralizing the free radicals, reducing oxidative stress are also expected to be owned by these antioxidants, especially in numerous affected cells due to the prolonged hyperglycemic conditions, such as the islets of Langerhans, hepatocytes, and renal proximal tubular cells8,9,17. Up until now, there is no scientific explanation about the antioxidant potential of okra pods extract. This study was intended to explore the antioxidant potential of Abelmoschus esculentus Moench pods extracts and to overcome the high free radicals in mice so it can be used to improve their tissue sensitivity to insulin.
MATERIAL AND METHODS:
Plant Identification:
Taxonomic identification of okra (Abelmoschus esculentus Moench) by the Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.
Materials and Tools:
The used materials were crude extract of okra pods and its fractionation (non-polar, semi-polar, and polar), streptozotocin (Sigma-Aldrich), citrate buffer pH 4.5, and phosphate-buffered saline (PBS), solvent carboxymethylcellulose (CMC), standard antidiabetic drugs (Acarbose 100mg/kg body weight), lard, anesthetic solutions (xylazine and ketamine), and 10% D-glucose in distilled water. This study used several main tools such as standard mouse cages, drinking bottles, feed, husk, microscope, Petri dish, analytic scales with accuracy of 4 numbers behind the comma, injection needles, 1mL insulin injection needles for induction of diabetes, glucometer and glucostrips (Accu-Chek® Active Test), blood cholesterol strips, rotary vacuum evaporator (Buchi), and spectrophotometer (Bio-Rad Laboratories).
Plant Extractions:
There were approximately 20 Kg 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:
Adult male mice (Mus musculus) with good physical state were used in this study, strain BALB/c, with various age range started from 3-4 months old, and body weight ranged from 30-40 g. The mice were obtained from the Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia. Before being moved to the Animal Laboratory of Faculty of Science and Technology, Universitas Airlangga, the mice were acclimatized for 2 weeks to provide similar conditions of the Animal Laboratory. 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°C, 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:
A completely randomized design was used in this experimental study. The experiments took place in Molecular Genetics Laboratory, Animal Laboratory, Animal Histology Laboratory, and Biochemistry Laboratory of the Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia. Streptozotocin was used to induce diabetic condition in mice. Fasting blood glucose levels were measured before and after streptozotocin induction on 7th and 14th day. The blood glucose level was measured to determine the diabetic condition of mice. Mice that were categorized as the diabetic group were those whose fasting blood glucose reached more than 130 mg/dL. These experimental animals were divided into several groupings. The first group was the non-diabetic mice that were used as normal control group (KN) while the second group was the diabetic mice that were induced by streptozotocin. The diabetic mice was further divided into another two control groups which were diabetic control group (KD) and VOPE treatment group. As much as 100 mg/kg body weight dose of Acarbose (KA) was given to the diabetic control group. Meanwhile, VOPE treatment group consists of crude extract group and fraction extract groups. Crude extract group (EK) was given 100 mg/kg body weight of the crude extract. Fraction extract groups were divided into non-polar group (NP), semi-polar group (SP), and polar group (EP). The dose of each fraction groups was based on the conversion of crude extract dose. NP dose was 20.04 mg/kg body weight, SP was 28.39 mg/kg body weight, and EP was 54.11mg/kg body weight. All of the treatments were administered for 14 days.
Measurement of SOD Level:
Blood collection of mice was carried out on 15th day intracardially. The blood obtained was then inserted into FalconTM (Fisher Scientific) and cooled for an hour. After that, the blood serum was taken and centrifuged at 16,000 rpm for 5 minutes. In order to measure the levels of SOD employing the ELISA protocol, the blood serum was applied. SOD test was also done on the 15th day. Blood serum was taken from mice heart and SOD level was checked using the SOD Kit.
Oral Glucose Tolerant Test:
The oral glucose tolerance test (OGTT) was performed on the 15th day, to know the tolerance of body tissue toward glucose. All mice were administered 10% D-glucose. The given unit of D-glucose solution was adjusted to the weight of the mice, which was 1 unit for 1 g BB. Blood glucose level test was performed, started from 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after 10% D-glucose oral administration. The result was then converted into the area under curve (AUC). Trapezoid or Trapezoid Rules was used to measure the AUC, using the formula:
AUC = 
a and b are two parallel sides, which consist of blood glucose levels between two measurement periods (mg/dL), t values indicate the interval of measurement (minutes).
Statistical Analysis:
Data from this study included the level of fasting blood glucose, the blood serum SOD level and the oral glucose tolerant test result. Data with normal distribution and homogenous variation was analyzed using one-way variance analysis continued with Duncan test. Data with normal distribution and non-homogenous variation was analyzed using the Brown-Forsythe test proceeded with a t-test. Meanwhile, the Pearson test was conducted to analyze the relationship between the islets of Langerhans diameter and the fasting blood glucose level in mice. All statistical test was conducted at α = 0.05.
RESULTS AND DISCUSSION:
Fig. 1 presented the mean of mice’s fasting blood glucose level before and after STZ induction while Fig. 2 presented the mean result of SOD level after VOPE treatment. In the meantime, Fig. 3 and Fig. 4 presented the mean data of OGTT and AUC.
Fig. 1. Fasting blood glucose (mg/dL) before and after STZ induction. A significant difference is indicated by the different letter.
Fig. 2. SOD (U/mL) changes of each mice group after treatments. The different letter indicated a significant difference.
Fig. 3. Blood glucose level (mg/dL) changes of all groups after treatment, performed by OGTT. The different letter indicated a significant difference.
Fig. 4. AUC (mg/dL/mins) differences of all mice groups. The different letter indicated a significant difference.
Fig. 1 explains the average of fasting blood glucose level measurement before and after STZ induction. It is important to note that for 5 consecutive days with 30 mg/kg body weight, STZ injection was able to substantially uplift the fasting blood glucose level from 127.071±14.978 to 189.321±30.867 mg/dL. The data provided the insight that STZ was capable of damaging pancreatic islet β-cells and reducing insulin synthesis. STZ was also capable of lifting up the level of fasting blood glucose2,8.
The existence of free radicals can be neutralized by antioxidants. The formation of free radicals is caused by the presence of oxidants that enter the body of living things. One of the oxidant compounds that often causes an oxidative stress and reduces SOD level is STZ17. 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+ resulting in a necrosis in pancreatic islet β-cells18. STZ affects glucose oxidation and decreases insulin biosynthesis and secretion. In this study, STZ was used as an oxidant. The treatment groups, induced by STZ, were KD, KA, EK, NP, SP and EP groups. After injection of 30mg/kg body weight multiple low-dose STZ, the 4 groups (EK, NP, SP, EP) were given various fractions of okra pods extract as an antioxidant to neutralize the oxidative stress caused by STZ.
Based on the results of SOD levels in Fig. 2, the lowest SOD levels were found in the KD group. It was because this group was a diabetic group that has no administration of exogenous antioxidant. While the group with the highest SOD level was SP which was treated by a semi-polar okra pods extract, all of the EK, NP, SP and EP groups were able to increase the SOD level as well. This showed that the four fractions of okra fruit extract contain the exogenous antioxidants that can increase the levels of endogenous antioxidants such as SOD levels. It was because the okra pods contain flavonoid which worked as an H atom donor that would reduce ROS levels as well as played role as a chelating agent. Flavonoids such as quercetin bound to the SOD enzyme so that the SOD enzyme could bind back to the cofactor (Zn or Cu)9,15. On the other hand, Asghar et al. and Ansori et al. reported that Onosma hispidum wall root extracts and Garcinia mangostana pericarp extract have antidiabetic properties, respectively9,19.
The EK, NP, SP, and EP fractions contained flavonoids and other antioxidant compounds that worked to reduce ROS levels so that SOD activity could be increase. In the semi-polar fraction, okra extract played the role as chelating agent and H atom donor, thus, it could reduce ROS levels and reactivate SOD enzyme. Whereas in the EK and EP groups, SOD activities were able to return to KN. This occurred because those two extracts had reduced ROS levels, especially superoxide ions. For this reason, the substrate for SOD enzyme was reduced and SOD activity was equivalent as normal. Based on the pathway analysis, all of VOPE were able to significantly affect SOD enzyme activity. ROS levels, such as superoxide ions, could affect SOD activity because the superoxide ions acted as the substrates for SOD enzymes. The mechanism of okra pods extracts as an antioxidant is by donating H atoms which will reduce the number of ROS20.
The OGTT is a test performed after the administration of glucose. This test is used to examine the cell resistance to insulin, damage to pancreatic islet β-cells, hypoglycemia, acromegaly, and impaired carbohydrate metabolism. Measurement of glucose levels was performed for two hours, with five measurements. Each measurement took 30 minutes. In this study, red okra pods extract was used with 4 different fractions, they were crude, non-polar, semi-polar and polar fractions. The aim was to find out which fraction is the most effective way in increasing the tissue tolerance to glucose. In the EK group, the glucose levels were increasing at the 30th minute from 183.75mg/dL to 233.75 mg/dL. This proved that 30 minutes after the injection of D-glucose, mice experienced hyperglycemic conditions. The same event was experienced by the other groups. At the 30th minute, NP experienced an increase of 117 to 229.75 mg/dL, SP increased from 154.5 to 218 mg/dL, and EP also experienced an increase from 161.25 to 199.25 mg/dL. Later at the 60th minute, all groups experienced a decrease in the blood glucose levels. After being analyzed by AUC and Duncan test (see Figure 3), it could be concluded that the most effective dose was performed by EP with AUC value of 21127.5. KN group had a significant difference with all treatment groups. The EK, SP, and KA groups did not differ significantly compared to one group or another, but these three groups differed significantly with NP and EP groups.
It is common when the level of free fatty acids increases accordingly with the increase of blood glucose level. As the consequence, it triggers the production of mitochondrial superoxide and therefore strengthen the risk of cell exposure by ROS21. This increase in superoxide production can also lead to an increase in NO caused by the enzyme NO synthase induction22. This condition can interfere with insulin function and secretion, which later can increase the complications of DM23. In the okra pods extract fractions, the reducing blood glucose levels was caused by the presence of antioxidant activity in okra pods in all of the EK, NP, SP, and EP groups. This event is also supported by the statement of Evans et al. that antioxidant administration can improve cell sensitivity to insulin11.
CONCLUSION:
In summary, the administration of VOPE was proven to significantly increase SOD levels and tissue glucose tolerance in diabetic mice.
ACKNOWLEDGEMENT:
The support of this study was granted by The Ministry of Research, Technology, and Higher Education of the Republic of Indonesia. We are also delighted to deliver our gratitude to the PMDSU Scholarship (Batch III) that was being awarded to Arif Nur Muhammad Ansori, Raden Joko Kuncoroningrat Susilo, and Suhailah Hayaza.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 11.05.2019 Modified on 14.06.2019
Accepted on 01.07.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(12): 5683-5688.
DOI: 10.5958/0974-360X.2019.00983.1