Nephroprotective Activity of Okra Pods Extract
(Abelmoschus esculentus L.) in Sodium Nitrite-Induced Mice
Sri Puji Astuti Wahyuningsih, Baiq Naili Dewi Atika, Elma Sakinatus Sajidah, Dwi Winarni
Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, 60115, Indonesia
*Corresponding Author E-mail: sri-p-a-w@fst.unair.ac.id
ABSTRACT:
This aim study was conducted to determine the effect of antioxidants from okra pods methanol extract (OPM) in sodium nitrite-induced mice based on total phenolic and flavonoid test, superoxide dismutase (SOD) activity, catalase (CAT) activity, levels of blood urea nitrogen (BUN) and creatinine (Cre), and kidney histology. Okra pods were extracted using methanol solvent. Thirty adult male BALB/c mice (8-10 weeks, ±30g) were divided into six groups: normal control, negative control (sodium nitrite exposure), and treatment group (sodium nitrite exposure and administration of OPM at doses of 50, 100, 200, and 400mg/kg BW). These mice were exposed to sodium nitrite 50mg/kg BW and were administrated with extract OPM for 19 days through gavage. Subsequently, serum was collected to evaluate the activity of SOD and CAT, the levels of BUN and Cre. Kidneys were removed for histopathology studies. The results revealed that sodium nitrite toxicity induced a significant increase in the levels of BUN, Cre, moreover a significant decrease in the activity of SOD and CAT. Sodium nitrite also altered kidney histopathology (necrosis of tubules) compared to the negative control. However, the administration of OPM significantly reduced kidney necrosis, decreased the levels of BUN and Cre, and also significantly increased the activity of SOD and CAT as normal. From the results of this study was concluded that OPM could be exhibiting nephroprotective effect against sodium nitrite induced nephrotoxicity in mice.
KEYWORDS: Okra pods, sodium nitrite, enzyme antioxidant, enzyme kidney marker, kidney histology.
INTRODUCTION:
In the body, NaNO2 will be absorbed through the gastrointestinal system and into blood vessels that allow it to pass through tissues and organs. Nitrite will then be converted into nitrosonium ions which react with amines to form N-nitrosamines7. N-nitrosamines formed induces an increase in reactive oxygen species (ROS) and causes lipid peroxidation8. Increased lipid peroxidation disrupts cell homeostasis9.
Okra (Abelmoschus esculentus L.) is a vegetable from the family Malvaceae which is grown in tropical and sub-tropical regions. Okra comes from Ethiopia and Sudan and has been widely distributed from Africa to Asia, Southern Europe, and America. In Indonesia, okra has been cultivated as a vegetable since 1877 in West Kalimantan10, reported that okra pods have a relatively high scavenger activity against superoxide compared to other vegetables11. Okra has been widely known as a compound that contains high antioxidants. Antioxidants are chemical components that can protect body cells from damage due to ROS. An imbalance between antioxidants and ROS in the body results in oxidative stress which can cause premature aging, atherosclerosis, and cancer12. Chemical components that act as antioxidants are flavonoid and poly-phenolic groups13. Flavonoids are a group of plant metabolites thought to provide health benefits through cell signaling pathways and antioxidant effects. These molecules are found in a variety of fruits and vegetables14. These group compounds are found in nature, especially in plants, and can capture free radicals. Antioxidants are found in many foods, including vitamin E, vitamin C, and carotenoids15.
Antioxidant activity or inhibition of generation of free radicals plays a crucial role in protection against sodium nitrite induced-nephrotoxicity16. It has been claimed that protective agents against free radicals, such as antioxidants, may be useful therapeutics for sodium nitrite toxicity in the kidneys. The availability of ethnomedical evidence for the application of okra for kidney problems coupled to the free radicals scavenging abilities of the methanol extracts of the plant16 prompted us to evaluate its nephroprotective effect against sodium nitrite-induced nephrotoxicity in mice.
MATERIAL AND METHODS:
Sodium nitrite was purchased from Sigma-Aldrich (USA; Cat No 6080-56-4 and 458-37-7). SOD assay kit was obtained from NWLSS (USA; Cat. No. E-BC-K022). CAT assay kit was obtained from (EnzyChromTM Catalase Assay Kit ECAT-100). BUN and Cre assay kit were obtained from Diasys.
Fresh okra pods were purchased from okra farm in Jember, East Java, Indonesia. The okra pods were cleaned and air-dried at a temperature of 30±5ºC. The okra pods were blended and got 150g of coarse powder stored in an airtight container. Then, the maceration process was carried out in methanol (three times). The extract was filtered and the concentrate was evaporated at 30ºC by rotary evaporator, the result of evaporation obtained was ±75mL, then it was freeze-dried.
Animal model and experimental design:
All procedures involving animal care were approved by the Animal Care and Use Committee (ACUC) of Veterinary Faculty, Airlangga University, Surabaya, Indonesia (number 2.KE.138.06.2019). The mice used as animal model were placed in ventilated cages given condition of 12 h light and 12 h dark. Mice were acclimated for a week before divided into 6 groups: normal control (no treatment/KN), negative control (exposed only sodium nitrite/K-) at dose of 50mg/kg BW, and P1, P2, P3, P4 with OPM administration at doses 50, 100, 200, 400mg/kg BW and sodium nitrite, respectively via gavage for 19 days. The 20th, mice were sacrificed and blood was taken from mice through the heart. Blood was left to coagulate at room temperature for 2 hours before centrifuged at 3000rpm for 10 minutes. Serum was isolated and used to measure the activity of SOD and CAT, the level of BUN and Cre. The kidneys were removed and rinsed with PBS to analyze the histopathological studies.
The total SOD activity was determined using a reaction system consisting of xanthine and xanthine oxidase that produces O2-. The O2- oxidizes hydroxylamine forming nitrite which appears purplish-red after chromogenic reaction. 20μL sample was added to the tube and add reagent 1-7, mix thoroughly by vortex mixer, incubate for 40 min at 37℃. After 40 min, mix fully and stand for 10 min at room temperature. Set to zero with distilled water and measure the OD values of each tube at 550nm with a 1cm diameter cuvette. Calculate the SOD activity of sample used the formula:
OD Control – OD Sample
---------------------------------------- X Dilution Factor
OD Control
The protocol relies on the reaction of the enzyme in the presence of an optimal concentration of H2O2. Add 10 μL sample into wells of the 96-well plate. Also, for each assay run, prepare one sample blank well that contains only 10μL assay buffer. Add 400μL assay buffer to positive control tube and mix well. Add 10μL of the reconstituted positive control into separate wells. Mix 5 μL 3% H2O2 and 914μL dH2O (final 4.8 mM). Prepare 50μM H2O2 substrate for sample, positive control, and sample blank by mixing for each well. Add 1μL of the 4.8mM H2O2 with 95μL assay buffer. Add 90μL of the 50μM substrate to these wells to initiate the catalase reaction. Tap plate quick to mix and incubate 30 min at room temperature. Calculate the CAT activity of sample used the formula:
R sample blank - R sample
Catalase (U/mL) ------------------------------------------ × n
Slope (μM-1) ×30 min
Measurement of BUN and Cre Levels:
Serum BUN levels are commonly used to assess glomerular filtration rate, concentrating and diluting capacity of tubular function of kidneys. An increase in the values of these markers may indicate development and extent of renal tubular damage16. Preparation of blank and standard solutions, and sample. Add 10μL samples and R1 solution 1000μL and incubated for 5 min. Add a solution of R2 250μL, incubated for 1 min. Read optical density at 340nm. Calculate the BUN levels of sample used the formula:
∆A sample
Blood urea nitrogen = --------------- × Standard concentration (mg/dL)
(BUN) (mg/dL) ∆A standard
Serum Cre are commonly used to assess kidney function. Add samples of 50μL and R1 of 1000μL and incubated for 5 min. Add a solution of R2 as much as 250μL, incubated for 1 min. Calculate the Cre activity of sample used the formula:
∆A sample
Creatinine (mg/dL) = ---------------- × Standard concentration (mg/dL)
∆A standard
Histopathological Kidney Study:
Sodium nitrite enhances the intracellular formation of ROS causing kidney damage. In the present study, we analyzed the kidney levels of two antioxidants (SOD and CAT activity). Negative control (sodium nitrite exposed) showed significant (p<0.05) decrease in the levels of SOD and CAT compared with the normal control. OPM administration groups with (50, 100, 200, and 400 mg/kg BW) showed a significant (p<0.05) increase in the activity of SOD and CAT compared with the sodium nitrite exposed. The results showed in Fig. 1.
Fig 1. The effect of OPM on the activity of SOD and CAT induced by sodium nitrite. KN (normal control); K- (negative control); P1, P2, P3, P4 with doses of 50, 100, 200, and 400 mg/kg BW. * Significantly different from negative control (α=0.05).
An increase in the BUN and Cre serum indicates kidney damage. The negative control group (exposed with sodium nitrite) showed significant (p<0.05) increase in BUN level, but non-significant (p˃0.05) in Cre level compared with the negative control group. In contrast, the groups administered with OPM (50, 100, 200, and 400mg/kg BW) showed significantly (p<0.05) decreased serum BUN level, but not significant (p˃0.05) in Cre level. The results showed in Fig. 2.
Fig 2. The effect of extract OPM on the levels of BUN and Cre induced by sodium nitrite. KN (normal control); K- (negative control); P1, P2, P3, P4 with doses of 50, 100, 200, and 400 mg/kg BW. * Significantly different from negative control (α=0.05).
Histopathological study was performed using light microscopy. Microscopic examination of normal kidney showed intact tubules and glomerulus. In the sodium nitrite exposed group, kidney tissues showed tubular necrosis indicated cell damage. Administration with extracts OPM 50, 100, 200, and 400 mg/kg BW significantly prevented histopathological changes towards normal (Fig. 3).
Fig 3. Kidney histology with magnification of 400x (50μm). KN (normal control); K- (negative control); P1, P2, P3, P4 with doses of 50, 100, 200, and 400mg/kg BW. Black arrow (cell necrosis), green arrow (cell inflammation), blue arrow (proximal tubules constriction)
The selection of cells necrosis was based on criteria: karyolysis, the cell nucleus was shrinking, and cell lysis. Fig 4 showed that the percentage of cell necrosis was significantly increased in negative control (exposed to sodium nitrite/K-) compared with another group. Administration with OPM showed that the percentage of cell necrosis was significantly decreased.
Fig 4. The percentage of cell necrosis in the renal proximal tubule. KN (normal control); K- (negative control); P1, P2, P3, P4 with doses of 50, 100, 200, and 400 mg/kg BW. * Significantly different from negative control (α=0.05).
DISCUSSION:
Nitrite salts are methemoglobin (metHb)-forming agents, one of environmental and industrial pollutants, producing severe organ damage in animals and humans. Studies have shown that the kidney is one of the primary targets in sodium nitrite associated toxicity3,17. Sodium nitrite can react quickly with superoxide to form peroxynitrites, peroxynitrites are known to increase lipid peroxidation, increased lipid peroxidation can be harmful to organs and can increase reactive oxygen species and produces oxidative damage in the kidney18. Antioxidant enzyme levels are used as markers of oxidative stress in sodium nitrite toxicity. Based on the present study sodium nitrite-induced toxicity might inhibit tissue antioxidants activities of SOD and CAT. The examined serum in mice indicated that the decrease of SOD and CAT activities might result from oxidative stress because these enzymes catalyze the decomposition of ROS19. The levels of these antioxidants might provide a clear indication of the extent of cytotoxic damage that occurs in kidney tissues. Therefore, some studies have said that antioxidants should be one of the important components of effective treatment of sodium nitrite exposure20.
Recently, many natural products and herbs are used for research, due to their high therapeutic potential and low price with little side effects compared with synthetic drugs, the present study investigated the nephroprotective effect of OPM. The purpose of this study was to observe the protective effects of extract OPM on kidney damage of mice that were given sodium nitrite exposure. Sodium nitrite significantly decreased SOD and CAT activities. SOD and CAT are important antioxidant enzymes. They have an important role against ROS. SOD is a metalloenzyme that catalyzes superoxide radicals (O2-) to oxygen and nonreactive compounds such as H2O2. CAT is an intracellular antioxidant enzyme that enzymatically catalyzes H2O2 into water and oxygen. Furthermore, this enzyme can be used to inhibit oxidative stress in cells. It has been reported that sodium nitrite can cause tissue hypoxia.
Tissue hypoxia can increase the reactive oxygen of species, causing decreased endogenous antioxidant activity such as superoxide dismutase (SOD) and catalase (CAT)7. The present study showed that the activity of SOD and CAT in mice kidneys was inhibited by sodium nitrite exposure. These results are following previous research21. This suggested that sodium nitrite toxicity can inhibit the activity of the antioxidant enzymes (SOD and CAT) that can cause oxidative stress. The administration of OPM decreased the accumulation of free radicals, which might increase the activities of SOD and CAT in the serum of sodium nitrite treated mice. OPM acts as a scavenger for the oxygen-derived free radicals, thus protecting against cellular damage. The mechanism of OPM as an antioxidant is by donating H atoms, it will reduce the amount of ROS22. Besides, OPM can also work by activating the Nrf2 gene. Nrf2 is a leucine zipper protein (bZIP) which regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation22.
In regards to BUN and Cre, the present experiment indicated that sodium nitrite exposure induced a significant increase in these parameters, suggesting an impairment of kidney functions. These effects could also be attributed to the changes in the threshold of tubular re-absorption, renal blood flow and glomerular filtration rate (GFR)23. The BUN and Cre serum are recommended for the assessment of kidney injury in preclinical studies as it is considered a more specific and sensitive indicator of kidney damage. Low levels of BUN and Cre serum are normally found in the blood, but when the kidney is damaged or diseased, BUN and Cre levels go up. Most increases in serum BUN and Cre levels are caused by kidney damage24. The present study showed increased BUN and Cre levels in the kidney damaged that were given sodium nitrite in comparison to the negative controls. A similar observation was reported by (8), who reported that sodium nitrite induced significant elevation of serum creatinine and BUN levels. Our results indicated that OPM had a nephroprotective activity against sodium nitrite-induced nephrotoxicity, where the pretreated groups with OPM at doses 200 and 400mg/kg BW, showed an increase in activity of SOD, CAT, and decreased in the BUN and Cre levels. This might be through its direct action on free radicals of sodium nitrite, protecting the kidney from cellular damage by maintaining its membrane integrity.
Histopathological results showed structural changes in the kidney tissue of sodium nitrite toxins were reported by some researchers25. Histopathological view of kidney sections in the sodium nitrite treated group showed necrosis in tubules, as compared to the normal control group. Necrosis tubular epithelial changes caused by organ damage due to oxidative stress and Histopathology analysis of kidneys tissue demonstrated increased inflammation in tubular epithelial cells and cell swollen. Damage to the proximal tubular epithelial cells decreases its function in the reabsorption of components that are still useful by the body, such as glucose, amino acids, bicarbonate, potassium ions, calcium, phosphate ions, uric acid, and water26.
Kidneys damage were considered mild in the groups treated with OPM 100mg/kg while mice treated with 200 and 400mg/kg OPM showed regeneration in tubular epithelial cells. We think that morphological changes in kidneys tended to be considered mild in sodium nitrite and OPM treatment.
It could be concluded that extracts OPM through its antioxidant mechanisms as a scavenger for the oxygen-derived free radicals protects from sodium nitrite-induced kidney damage in mice. These results showed that extracts OPM has a potential nephroprotective effect in a dose-dependent manner that minimizes or diminishes compounds with nephrotoxic effect induced by sodium nitrite toxicity.
ACKNOWLEDGMENTS:
We are thankful to Master Research Funding by the Ministry of Research and Higher Education in 2019, Indonesia.
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Received on 26.09.2019 Modified on 28.11.2019
Accepted on 23.01.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(8):3648-3652.
DOI: 10.5958/0974-360X.2020.00645.9