Biomedical and Biotechnology Research Journal (BBRJ)

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 2  |  Issue : 4  |  Page : 254--259

Evaluation of tumor necrosis factor alpha (TNFα), interleukin 4, interleukin 6, aspartate aminotransferase, and alanine aminotransferase in rabbits overdosed with ibuprofen and supplemented with guava leaf (Psidium guajava) extract


Mathew Folaranmi Olaniyan1, Ruth Avedoya Atibor2, Temitayo Afolabi2,  
1 Department of Medical Laboratory Science, Edo University, Iyamho, Nigeria
2 Department of Medical Laboratory Science, Achievers University, Owo, Nigeria

Correspondence Address:
Prof. Mathew Folaranmi Olaniyan
Department of Medical Laboratory Science, Edo University, Iyamho
Nigeria

Abstract

Introduction: Guava leaf contains low calorie, vitamins, minerals antioxidants, polyphenolic, and flavonoid compounds which may play important role in prevention of cancers, aging, cell differentiation, apoptosis and may also have immune enhancing properties while ibuprofen is a nonsteroidal anti inflammatory drug commonly used to relief pain. It reduces hormones that cause inflammation and pain in the body.This project work was therefore designed to evaluate the plasma level of pro (tumor necrosis factor alpha (TNFα)) and anti inflammatory (interleukin 4 [IL 4] and 6) cytokines, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in rabbits overdosed with ibuprofen and supplemented with guava leaf extract. Methods: Fifteen rabbits grouped into three experimental groups labeled A–C (with subgroups B1, B2, C1, and C2) with A as control were investigated. An overdose of ibuprofen of 1600 mg/kgBW was administered to Group B and C rabbits to induce toxicity, while 400 ml/kgBW guava leaf extract was used to reduce and treat ibuprofen toxicity. Plasma tumor necrosis factor alpha (TNFα), IL-4 and 6, AST, and ALT were analyzed biochemically by spectrophotometry and enzyme-linked immunosorbent assay. Results: The result obtained showed a significant increase in the plasma value of TNF-α, IL-4, IL-6, ALT, and AST in rabbits induced with 1600 mg/kgBW of ibuprofen per oral fed with normal meal for 7 days (Group B1 and C1) compared with normal rabbits fed with normal meal and water only for 7 days (Group A) (P < 0.05). There was a significant decrease in the plasma value of TNF-α, IL-4, IL-6, and AST in rabbits given 400 ml/KgBW of guava leaf extract daily for 7 days after 7 days of postibuprofen administration (Group B2 and C2) compared to when the rabbits were given 1600 mg/kgBW of ibuprofen per oral with normal meal and water observed for 7 days (B1 and C1) (P < 0.05). Conclusion: Guava aqueous extract has been demonstrated to reduce the biochemical alterations in the plasma values of (TNF-α, IL-4, IL-6, AST, and ALT) following ibuprofen overdose as a result of the phytochemical contents of the guava leaf.



How to cite this article:
Olaniyan MF, Atibor RA, Afolabi T. Evaluation of tumor necrosis factor alpha (TNFα), interleukin 4, interleukin 6, aspartate aminotransferase, and alanine aminotransferase in rabbits overdosed with ibuprofen and supplemented with guava leaf (Psidium guajava) extract.Biomed Biotechnol Res J 2018;2:254-259


How to cite this URL:
Olaniyan MF, Atibor RA, Afolabi T. Evaluation of tumor necrosis factor alpha (TNFα), interleukin 4, interleukin 6, aspartate aminotransferase, and alanine aminotransferase in rabbits overdosed with ibuprofen and supplemented with guava leaf (Psidium guajava) extract. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 Mar 23 ];2:254-259
Available from: http://www.bmbtrj.org/text.asp?2018/2/4/254/247243


Full Text



 Introduction



Guava (Psidium guajava) is a fruit-bearing tree commonly known as guava of the family Myrtaceae is a native of tropical America.[1] Guava which is used as a traditional medicine is found in countries with hot climates such as South America, Europe, Africa, and Asia (Gutierrez et al., 2008). Psidium guava popularly known as guava has traditional medicine values for a number of ailments.[2] Guava (P. guajava) is low in calories and fats but contains vitamins, minerals, antioxidants, polyphenolic, and flavonoid compounds with properties for prevention of cancers, aging, cell differentiation, apoptosis, and immune-enhancing properties[3],[4] It is used traditionally to alleviate of diarrhea, dehydration, treatment of gastroenteritis, dysentery, stomach pain, diabetes mellitus, and wounds. It has antioxidant and anti-inflammatory properties.[5],[6],[7]

Guava leaves contain phenolic compounds and flavonoids with high antioxidant activity. Main active substances in guava leaves are garlic acid, caffeic acid, guaijaverin, tannins, carotenoids, and triterpenoids.[7]

According to Mittal et al.,[8] phytochemicals in leaves of guava include α-pinene, β-pinene, limonene, menthol, terpinyl acetate, isopropyl alcohol, longicyclene, caryophyllene, β-bisabolene, caryophyllene oxide, β-copanene, farnesene, humulene, selinene, and guayavolicacids, cineol, quercetin, 3-L-4-4-arabinofuranoside (avicularin) and its 3-L-4-pyranoside (Essential oil), resin, tannin, and eugenol.

Ibuprofen is a nonsteroidal anti-inflammatory drug. It acts by reducing hormones that cause inflammation and pain in the body. Ibuprofen is used to reduce fever and treat pain or inflammation caused by many conditions such as headache, toothache, back pain, arthritis, menstrual cramps, or minor injury because the tablet possesses analgesic and antipyretic activities. The adverse reactions include peptic ulcer or perforation, bleeding, vision disorders, nausea, epigastric pain, heartburn, dizziness, rash (discontinue if occurs), edema, renal papillary necrosis, jaundice, and hepatitis.[9]

This work was therefore designed to evaluate the tumor necrosis factor alpha (TNFα), IL-4, IL-6, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in rabbits overdosed with ibuprofen and supplemented with guava leaf (Psidium guajava) extract.

 Methods



Study area

This study was carried out at Achievers University animal house, Owo Local Government Area of Ondo State in Nigeria. Achievers University is in Owo, Ondo State, Nigeria. The university is a private-sector initiative, established in 2007 and accredited by the National Universities Commission. It is located on land in the Idasen community of Owo, consisting of Ulale 1, Ulale 11, Ulema, Ijegunma, Isijogun, and Amurin Elegba (formerly Amurin and Ogain). The university sprang from the Achievers Group of Education and Training Organization, located in Ibadan Oyo State of Nigeria owned and run by Mr. Bode Ayorinde and other educationalists. The university commenced academic activities during the 2007/2008 academic session. In the Nigerian National University Commission annual university rankings for 2013, it was rated 53rd.[10] The proposal was reviewed and approved by Research and Ethical committee of the Department of Biological Sciences, Achievers University, Owo, Nigeria before the commencement of the work. (AUOBS/2018/31/112; 13th March 2018).

Study population

Fifteen rabbits were purchased from Owo through the Department of Biological Sciences, Achievers University, Owo, Nigeria. Rabbits purchased were identified and confirmed having the same sex (male). Before performing the experiment, ethical clearance was obtained from the biomedical research and ethical committee of the Department of Biological Sciences, Achievers University, Owo, Nigeria, and was carried out in strict compliance with the guidelines for the care and use of animals for research which is in line with that said by the World Health Organization.

Sample size

Fifteen apparently healthy rabbits were bought and classified into three groups of 5 rabbits each. Body weight of animals before and after the experiment was measured using weighing balance.

Study design

Experimental and observational study involves 15 rabbits divided into three groups:

Control group

Group A: Five rabbits fed with normal meal and water throughout the period of the research (normal control).

Experimental groups

Group B: Five rabbits fed with normal meal and overdosed with 1600 mg/kgBW of ibuprofen for 7 days and were treated with 400 mg/KgBW of methanolic extracts of guava leaf for 14 days.

Group C: Five rabbits fed with normal meal and overdosed with 1600 mg/kgBW of ibuprofen for 7 days and were treated with 400 mg/KgBW of aqueous extracts of guava leaf for 14 days.

Blood sampling

Group A: Five milliliters of blood sample was collected from the vein lining the ear of all the five rabbits into lithium heparinized bottles feeding with normal meal and water (normal meal) after 14 days. Each of their weight was documented.

Group B: Five milliliters of blood sample was collected from the vein lining the ear of each rabbit into lithium heparinized bottle after 7 days following the administration of 1600 mg/kgBW of ibuprofen. Five milliliters of blood sample was collected into lithium heparinized bottle from each of the five rabbits after 14 days of treatment with 400 ml/KgBW methanolic extracts of guava leaf. Each of their weight was documented.

Group C: Five milliliters of blood sample was collected from the vein lining the ear of each rabbit into lithium heparinized bottle after 7 days following the administration of 1600 mg/kgBW of ibuprofen. Five milliliters of blood sample was collected into lithium heparinized bottle from each of the rabbits after 14 days of treatment with 400 ml/KgBW aqueous extracts of guava leaf. Each of their weight was documented.

Treatment of normal control and experimental rabbits

Control group

Group A: Five rabbits fed with normal meal and water throughout the period of the research (normal control).

Experimental group

Group B: Five rabbits fed with normal meal and overdosed with 1600 mg/kgBW of ibuprofen for 7 days and were treated with 400 mg/KgBW of methanolic extracts of guava leaf for 14 days.

Group C: Five rabbits fed with normal meal and overdosed with 1600 mg/kgBW of ibuprofen for 7 days and were treated with 400 mg/KgBW of aqueous extracts of guava leaf for 14 days.

Preparation of guava leaf extract

The fresh and tender local guava leaf was collected and presented to the Department of Biological Sciences, Achievers University, Owo, for confirmation and certification.

The leaves were then dried in a shade under room temperature for 14 days and then crushed into coarse powdery substance using electric grinder. The coarse powdery substance was then dried again and sieved to get fine powder using fine plastic sieve.

Extract preparation

Fifty grams of the sieved powder was weighed accurately and subjected to extraction at room temperature using 350 ml methanol and distilled water separately. The extracts obtained were filtered, concentrated after dryness in an evaporator maintained at 45°C.

Preparation of ibuprofen powder

Ibuprofen tablets were purchased and grinded into powder using laboratory pestle and mortal.

Blood sample preparation

Whole blood samples collected from the veins lining the ear of each of the rabbit were collected in lithium-heparinized tubes. The blood sample was spun using bench/macrocentrifuge for the extraction of the plasma.

Estimation of blood parameters

Pro inflammatory (tumor necrosis factor alpha (TNFa) and anti-inflammatory (interleukin-4 and interleukin-6) cytokines

Tissue necrotic factor-alpha enzyme-linked immunosorbent assay

Plasma TNF-alpha was analyzed using Abcam's kit. Abcam's TNF-alpha human in vitro enzyme-linked immunosorbent assay (ELISA) kit is designed for the quantitative measurement of TNF-alpha in supernatants, buffered solutions, serum, and plasma samples.

A monoclonal antibody-specific TNF-alpha has been coated onto the wells of the microtiter strips provided. Samples, including standards of known TNF-alpha concentrations, control specimens, or unknowns are pipetted into these wells. During the first incubation, the standards or samples and a biotinylated monoclonal antibody specific for TNF-alpha are simultaneously incubated. After washing, the enzyme streptavidin- Horseradish Peroxidase (HRP) that binds the biotinylated antibody is added, incubated, and washed. A TMB substrate solution is added which acts on the bound enzyme to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of TNF-alpha present in the samples.

Interleukin-4 and 6 enzyme-linked immunosorbent assay

Abcam's IL-4 Human ELISA kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human IL-4 in serum, plasma, and cell culture supernatants.

This assay employs an antibody specific for human IL-4 coated on a 96-well plate. Standards and samples are pipette into the wells and IL-4 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human IL-4 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IL-4 bound. The stop solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm.

Aspartate aminotransferase assay

The aminotransferase are a group of enzymes that catalyze the interconversions of amino acids by transfer of amino groups. AST or glutamate oxaloacetate transaminase has been found in the cytoplasm and the mitochondria of cells. In cases of mild tissue damage, for example, liver the predominant form of serum AST is that from the cytoplasm, with a smaller amount coming from the mitochondria. Severe tissue damage will result in more mitochondria enzyme being released. Elevated levels of AST can signal myocardial infarction, hepatic disease, muscular dystrophy, and organ damage.[11]

Method: Colorimetric method (Reitman and Frankel)Principle: AST is measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4-dinitrophenyl-hydrazine.

Alanine aminotransferase assay (Berger, 2006)

ALT belongs to a group of enzymes called aminotransferases. The enzyme ALT been found to be in highest concentrations in the liver, with decreasing concentrations found in the kidneys, heart, skeletal muscle, pancreas, spleen, and lung tissue, respectively. ALT measurements are used in the diagnosis and treatment of certain liver diseases (e.g., viral hepatitis and cirrhosis) and heart diseases.

It is often tested in combinations with AST as part of a liver panel with ALT levels being higher in most types of liver disease.

Principle

ALT catalyzes the transamination of L-alanine to α-ketoglutarate, forming L-glutamate and pyruvate. The pyruvate formed is reduced to lactate by lactate dehydrogenase with simultaneous oxidation of reduced nicotinamide-adenine dinucleotide. The change in absorbance is directly proportional to ALT activity and is measured using a bichromatic (340,700 nm) rate technique.

Method of data analysis

A Statistical Package For the Social Sciences (International Business Machines Corporation (IBM) SPSS, Armonk, New York, United States) 18.0 was used for the analysis of the data appropriately. Continuous variables were displayed as means and standard deviation and categorical variables were displayed as percentage. The level of significance was taken at 95% confidence interval and P ≤ 0.05 was considered statistically significant.

 Results



The result obtained showed a significant increase in the plasma value of TNF-α, IL-4, IL-6, ALT, and AST in rabbits induced with 1600 mg/kgBW of ibuprofen per oral fed with normal meal for 7 days (Group B1 and C1) compared with normal rabbits fed with normal meal and water only (Group A) (P < 0.05) [Table 1], [Table 2] and [Figure 1].{Table 1}{Table 2}{Figure 1}

There was a significant decrease in the plasma value of TNF-α, IL-4, IL-6, and AST in rabbits given 400 ml/KgBW of guava leaf extract daily for 14 days after 7 days of postibuprofen administration (Group B2 and C2) compared to when the rabbits were given 1600 mg/kgBW of ibuprofen per oral with normal meal and water observed for 7 days (B1 and C1) (P < 0.05) [Table 1], [Table 2] and [Figure 1].

However, there was no significant difference in the plasma value of ALT in rabbits given 400 ml/KgBW of guava leaf extract daily for 14 days after 7 days of postibuprofen administration (Group B2 and C2) compared to when the rabbits were given 1600 mg/kgBW of ibuprofen per oral with normal meal and water for 7 days (B1 and C1) (P > 0.05) [Table 1], [Table 2] and [Figure 1].

Furthermore, there was no significant difference in the plasma value of TNF-α, IL-4, IL-6, ALT, and AST in rabbits given 400 ml/KgBW of guava leaf methanolic extract daily for 14 days (B2) compared with groups given 400 ml/KgBW of guava leaf aqueous extract daily for 7 days after 7 days (C2) of postibuprofen administration (P > 0.05) [Table 1], [Table 2] and [Figure 1].

 Discussion



This work has been used to reveal possible increase in plasma TNF-α, IL-4, IL-6, AST, and ALT in ibuprofen overdose and a decrease in the plasma value of the parameters following guava leaf extract supplementation.

The findings of this work showed the effectiveness of ibuprofen overdose at inducing toxicity as reflected by a significant increase in the plasma value of TNF-α, IL-4, IL-6, AST, and ALT.[11]

Increase in plasma TNF-α could be associated with its physiological properties as TNF-alpha is produced by macrophages, broad variety of cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts, and neurons. Large amounts of TNF-alpha are released upon inflammation due to toxicity.[12] It is involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction.[12],[13],[14],[15] It has a number of physiological activities on different organ systems, generally together with IL-1 and IL-6. On the liver, it stimulates the acute phase response.[12],[13],[14],[15] Significantly higher TNF-α, IL-4, and IL-6 in Group B1 following overdose of ibuprofen could also be associated with pro-inflammatory activities of TNF-α which act to make disease worst by stimulating inflammatory process.[12],[13],[14],[15]

Significant increase in plasma IL-4 and IL-6 could be associated with the anti-inflammatory activities of these cytokines to act against pro-inflammatory cytokines to reduce inflammation and promote healing.

There was a significant decrease in plasma TNF-α, IL-4, and IL-6 when the rabbits were supplemented with guava leaf extract after the overdose of ibuprofen. This could be associated with the excessive utilization of anti-inflammatory activities of IL-4 and IL-6 to stop inflammatory process including their activities against pro-inflammatory cytokine (TNF-α) thereby reducing the production of pro-inflammatory cytokines. In addition, significant decrease in plasma anti-inflammatory cytokine IL-4 and IL-6) could be associated with the immune regulatory process possibly aimed at reducing production of pro-inflammatory cytokine (TNF-α) to reduce inflammatory process and promote healing.

Reduction in plasma TNF-α after guava leaf extract supplementation could be due to possible pro-inflammatory reduction effect of guava leaf extract due to its phytochemical constituent such as the tannins, polyphenolic compounds, flavonoids, ellagic acid, triterpenoids, guiajaverin, quercetin, and other chemical compounds present in the P. guajava which are speculated to account for the observed anti-inflammatory of the plant's leaf extract.[16],[17],[18]

Significant increase in plasma AST and ALT when the rabbits were given ibuprofen overdose could be attributed to hepatotoxicity effect of ibuprofen overdose which could cause liver dysfunction and damage. Raised AST and ALT were however reduced when the rabbits were supplemented with 400 mg/BKW of aqueous and methanolic leaf extract of guava leaf for 14 days. This is consistent with the report of Roy and Das[19] that in the acute liver damage induced by different hepatotoxins, P. guajava leaf methanolic and ethyl acetate extracts significantly reduced the elevated serum levels of AST, ALT, alkaline phosphatase, and bilirubin in carbon tetrachloride and paracetamol-induced hepatotoxicity. Meanwhile, P. guajava leaf aqueous extract significantly reduced the elevated serum levels of alkaline phosphatase, ALT, and bilirubin in carbon tetrachloride-induced hepatotoxicity. Histological examination of the liver tissues supported the hepatoprotection. Thus, the methanolic extract of leaves of P. guajava plant possesses better hepatoprotective activity compared to other extracts[19] which was also revealed in this work by the significant alterations in plasma levels of cytokines and liver enzymes as they are some of the indices of liver health and toxicity.[19],[20],[21],[22],[23],[24]

 Conclusion



The aqueous and methanolic extract of guava (P. guajava) leaf has been demonstrated to reduce ibuprofen-induced toxicity/inflammation as revealed by the biochemical alterations in the plasma values of TNF-α, IL-4, IL-6, AST, and ALT following ibuprofen overdose and supplementation with the guava leaf extract.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Barbalho SM, Farinazzi-Machado FM, Goulart RA, Brunnati AC, Ottoboni AM, Nicolau CC. Review on Psidium guajava (Guava): A plant of multipurpose medicinal applications. Med Aromat Plants 2012;1:1-6.
2Berger MM. Ibuprofen toxicity on liver enzymes. J Clin Invest 2006;77:152-201.
3Chen HC, Sheu MJ, Lin LY, Wu CM. Volatile constituents of six cultivars of mature guava (Psidium guajava L.) fruits from Taiwan. Acta Hortic 2008;765:273-7.
4Chen HY, Yen GC. Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava L.) leaves. Food Chem 2007;101:686-94.
5Chen KC, Hsieh CL, Huang KD, Ker YB, Chyau CC, Peng RY, et al. Anticancer activity of rhamnoallosan against DU-145 cells is kinetically complementary to coexisting polyphenolics in Psidium guajava budding leaves. J Agric Food Chem 2009;57:6114-22.
6Cheng FC, Shen SC, Wu JS. Effect of guava (Psidium guajava L.) leaf extract on glucose uptake in rat hepatocytes. J Food Sci 2009;74:H132-8.
7Dowlati Y, Herrmann N, Swardfager W, Liu H, Sham L, Reim EK, et al. A meta-analysis of cytokines in major depression. Biol Psychiatry 2010;67:446-57.
8Gutiérrez RM, Mitchell S, Solis RV. Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol 2008;117:1-27.
9Han EH, Hwang YP, Kim HG, Park JH, Choi JH, Im JH, et al. Ethyl acetate extract of Psidium guajava inhibits IgE-mediated allergic responses by blocking fcεRI signaling. Food Chem Toxicol 2011;49:100-8.
10Han EH, Hwang YP, Choi JH, Yang JH, Seo JK, Chung YC, et al. Psidium guajava extract inhibits thymus and activation-regulated chemokine (TARC/CCL17) production in human keratinocytes by inducing heme oxygenase-1 and blocking NF-κB and STAT1 activation. Environ Toxicol Pharmacol 2011;32:136-45.
11Kamath JV, Rahul N, Ashok-Kumar CK, Lakshmi SM. Psidium guajava L: A review. Int J Green Pharm 2008;2:9-12.
12Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: Integrating mammalian biology. Cell 2001;104:487-501.
13Macleod AJ, Troconis NG. Volatile flavor components of guava. Phytochemistry 1975;21:1339-42.
14Mittal P, Gupta V, Kaur G, Garg A, Singh A. Photochemistry and pharmacological activities of Psidium guajava. Int J Pharm Sci Res 2010;1:9-19.
15Morton JF. Guava: Fruits of Warm Climates. Published by Julia F. Morton 20534 SW 92 Ct. Miami, FL.. Web-publications by Purdue University, West Lafayette, Indiana p.356-63. 33189ISBN: 0-9610184-1-01987.
16Ojewole JA. Hypoglycemic and hypotensive effects of Psidium guajava linn. (Myrtaceae) leaf aqueous extract. Methods Find Exp Clin Pharmacol 2005;27:689-95.
17Ojewole JA. Antiinflammatory and analgesic effects of Psidium guajava linn. (Myrtaceae) leaf aqueous extract in rats and mice. Methods Find Exp Clin Pharmacol 2006;28:441-6.
18Olszewski MB, Groot AJ, Dastych J, Knol EF. TNF trafficking to human mast cell granules: Mature chain-dependent endocytosis. J Immunol 2007;178:5701-9.
19Roy CK, Das AK. Comparative evaluation of different extracts of leaves of Psidium guajava linn. For hepatoprotective activity. Pak J Pharm Sci 2010;23:15-20.
20Shao M, Wang Y, Liu Z, Zhang DM, Cao HH, Jiang RW, et al. Psiguadials A and B, two novel meroterpenoids with unusual skeletons from the leaves of Psidium guajava. Org Lett 2010;12:5040-3.
21Shao M, Wang Y, Huang XJ, Fan CL, Zhang QW, Zhang XQ, et al. Four new triterpenoids from the leaves of Psidium guajava. J Asian Nat Prod Res 2012;14:348-54.
22Swardfager W, Lanctôt K, Rothenburg L, Wong A, Cappell J, Herrmann N, et al. A meta-analysis of cytokines in Alzheimer's disease. Biol Psychiatry 2010;68:930-41.
23Victor FC, Gottlieb AB. TNF-alpha and apoptosis: Implications for the pathogenesis and treatment of psoriasis. J Drugs Dermatol 2002;1:264-75.
24Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin 2007;45:27-37.