Anti-inflammatory efficacy of Mangifera indica stem-bark, carica papaya and eucalyptus leaf extracts on formalin-induced inflammation in rats

*Corresponding author: Onyedikachi Joannah Akporhono Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Anti-inflammatory efficacy of Mangifera indica stem-bark, carica papaya and eucalyptus leaf extracts on formalin-induced inflammation in rats A.A. Emejulu 1, C.P. Nzebude 1, A.V. Iwueke 2 and Onyedikachi Joannah Akporhono 1, * 1 Department of Biochemistry, Federal University of Technology, Owerri. 2 Department of Science, Imo State University of Agriculture and Environmental Sciences, Umuagwo. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 Publication history: Received on 27 September 2025; revised on 03 November 2025; accepted on 06 November 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.33.2.0392 Abstract Inflammation is part of the body's defense mechanism and can be acute or chronic. Inflammatory disease prevalence is on the increase in Africa and anti-inflammatory efficacious alternatives are continually sought for. Effects of herbal remedies consisting of ethanol extracts of Mangifera indica Stem-bark (MSB), Carica papaya (PL) and Eucalyptus (EU) leaf extracts were evaluated on formalin-induced inflammation in albino rats using standard procedures. Forty-five (45) rats were divided into nine (9) groups of five (5) rats each. Induction was by subcutaneous injection of 0.1ml (2.5% v/v in normal saline) formaldehyde solution (formalin) via the right hind paw of all groups except normal control, group seven (7), on 1st & 3rd day of experiment. Groups 1-3 were treated with 200mg/kg, 400mg/kg, and 600mg/kg combined ethanol extracts MSB+PL respectively. Groups 4-6 received 200mg/kg, 400mg/kg, and 600mg/kg combined extracts of MSB+EU respectively. Group 8(Standard) received 5mg/kg piroxicam and group 9(negative control) received 5ml/kg normal saline for 10 days. Paw thickness and body weights taken on days 3, 7&10. MSB+EU combination was most efficacious and effect was dose-dependent. Tumor necrosis factor alpha (TNFα) and ESR (erythrocyte sedimentation rate) did not significantly differ between the groups. White blood cells (WBC), Malondialdehyde (MDA) were highest in untreated group and platelet count lowest. There were no significant changes in liver marker enzymes except in MSB+EU 600mg/kg group, which also corroborated histological findings. The study therefore justifies the antiinflammatory use of both extract combinations and suggests MSB+EU maybe most effective, however, questions are raised on its effect on liver integrity. Further studies on human clinical parameters and anti-inflammatory properties of these extract over an extended period is suggested to further confirm their potential role in anti-inflammation as well as determination of anti-inflammatory effect of these extracts on immunological pro-inflammatory molecules (IL-6, ILIB, TNF-a). Keywords: Mangifera indica; Carica papaya; Eucalyptus; Inflammation; Hematology; Antioxidant; TNF1. Introduction Inflammation is part of the body's defense mechanism and can be acute or chronic. Acute inflammation is the initial response and is characterized by the increased movement of plasma and innate immune system cells, such as neutrophils and macrophages, from the blood into the injured tissues. Cardinal signs of inflammation—edema, hyperalgesia, and erythema—develop immediately following subcutaneous injection of inflammatory agent, resulting from action of proinflammatory agents—bradykinin, histamine, tachykinins, complement and reactive oxygen and nitrogen species. Upon the presence of the inflammatory agent, cell membranes induce the activation of phospholipase A2 followed by release of arachidonic acid and inflammatory mediators such as cytokines, serotonin, histamine, prostaglandin and leukotrienes that increase vascular permeability, thus facilitating the migration of leukocytes to the site of inflammation (Sarkhel, 2016). GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 179 Herbal medicine are gaining popularity both in developing and developed countries because of their natural origin and less side effects, many traditional medicines in use are derived from medicinal plants, minerals and organic matter (Grover, et al., 2002). Plants are rich sources of many different bioactive phyto-compounds, including phenolic components, anthocyanins, carotenoids, vitamin E, and vitamin C, which exhibit good antioxidant and health enhancement properties (Liu, 2003, Usman, et al., 2001). This study is aimed at assessing the anti-inflamatory efficacy and hepatotoxicity of mangifera indica stem-bark (msb), carica papaya (pl) and eucalyptus (EU) leaf extracts on formalin-induced inflammation in rats. 2. Materials and methods 2.1. Sample collection and preparation The leaves of Carica papaya, and Eucalyptus were gotten from Ihube town in Okigwe L.G.A of Imo State and Magnifera indica stem bark was gotten from Federal University of Technology Owerri Imo State at N5023’33.6876” longitude and E6059’10.5504 latitude. These samples were washed thoroughly with clean tap water, and then air-dried under room temperature, the dried samples were grinded to fine powder and then stored in a clean bottle. 2.2. Preparation of extract Three hundred grams (300g) powdered samples was placed in a stoppered container and 1200mls of the solvent (ethanol) was added. They were allowed to stand at room temperature for 48 hours, with frequent agitation. The extract was filtered with a fine cloth and then re-filtered using Whatman filter. The filtrate was poured in to a clean round bottom flash at a volume that will not allow the filtrate to siphon into the extraction chamber. The temperature is adjusted in accordance with the boiling point of ethanol (78.4℃). The solvent evaporates and drips into the extraction chamber where is collected. The active component left behind in the flask is dried in water bath to completely evaporate the remaining solvent and then preserved in tightly corked labeled bottles and stored in a refrigerator until required. 2.3. Experimental animals Fifty male adult albino rats (110-120g) were obtained from Awka. The animals were housed in plastic cages and allowed to acclimatize for 7days, under normal room temperature (25 ± 20C) and natural light cycle and maintained on standard pellets (Vital Feeds, Jos, Nigeria) and water ad libitum throughout the study period. Animals handling and use was done in compliance with the National Institute of Health Guide for care and use of laboratory Animals. 2.4. Induction of Animals Non-immunological (Udegbunam, et al., 2014) inflammation was induced according to the method described by (Amraoui et al., 2019). Inflammation was induced in rats by subcutaneous injection of 0.1ml (2.5% v/v in normal saline) formaldehyde solution (formalin) into the sub-planter region of right hind paw of albino rats on first and third day of the experiment. Prior to induction, the mean body weights were calculated and different concentrations of the extract were prepared based on the mean body weight according to OECD’S guideline on volume selection; (Barret, et al., 1991). 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑑𝑜𝑠𝑒 = weight of animal (g) 1000 (g) × 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑜𝑠𝑒 (𝑚𝑔) 𝑠𝑡𝑜𝑐𝑘 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = required dose (mg) 1000 (g) × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛t All the groups receives their treatments respectively, while groups 7 and 9 receives 5mls normal saline and group 8 receives 5mg/kg piroxicam (standard drug), one (1) hour before induction. After induction the rats were observed for 1 hour and their paw size were measured after one hour (day1). The paw thickness and body weight of the rats were taken before induction and on day 3, 7 and 10 using vernier caliper and the animals were observed daily for signs/symptoms of inflammation. All the rats had free access to food and water throughout the time of the experiment and treatments were administered orally by intubation. 2.5. Biochemical analysis The blood samples were collected into EDTA and heparinized bottles by ocular puncture, the animals killed by cervical dislocation and the liver organs were collected in plain bottles, preserved using 10% formalin and then sent to the laboratory for analysis, blood samples were centrifuged for 10mins at 3000rpm and the serum was use to examine GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 180 aspartate amino transferase (AST) and alanine amino transferase (ALT) according to the method of Reitman and Frankel method, (1957), Alkaline phosphates (ALP) was assessed according to Kochmar, J.F and moss, D.W method, (1986) and Total Protein using Biuret method by Tietz, (1995). Randox kit was used. Heamatological parameters were carried out using microheamatocrit centrifuge (UNICO, CMH30, USA), Determination of Lipid profile was carried out using enzymatic end-point method (Unit mmol/L) (Y Kayamori, et al., 1979), Malondialdehyde (MDA) using Ohkawa & Ohishi method (1979). Erythrocyte Sedimentation Rate (ESR) was carried out using westergren technique (Bharathi, et al., 2011), Rat Tumor Necrosis Factor α (TNF-α) was carried out using enzyme-linked immunosorbent assay method (ELISA) (Engvail, 1972). 2.6. Histopathology examination (Slaoul and Fiette, 2011) The liver of rats were fixed in 10% neutral buffered formalin solution for 24 hours and cleared in xylene solution followed by embedding on the paraffin block. The tissues were then cut using rotary microtome into 4µm thick, mounted on glass slides, stained with hematoxylin and eosin, and examined under a high powered microscope at magnification ×400. 2.7. Statistical analysis The data obtained were analyzed using using students package for social sciences (SPSS) version 20 computer software Analysis of Variance (ANOVA). Values for p≤0.05 were considered statistically significant. 3. Results 3.1. Hematological Results The packed cell volume (PCV) of inflammation-induced rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed that there were no significant difference in PCV levels between most of the various groups and the normal control animals except in the lower -dose (200mg/kg MSB+PL and 200mg/kg MSB+EU) groups. The lower-dose test groups (200mg/kg MSB + PL and 200mg/kg MSB + EU) were significantly lower than that of the normal control group, but very similar to that of the negative control group. The result of the hemoglobin revealed no significant difference in the level of Hb concentration of the test groups compared to the normal control group, except in the lower dose groups (200mg/kg MSB/PL and MSB/EU) which were significantly lower than the normal control group but similar to the negative control as shown in figure.1a below. The result of the red blood cell (RBC) revealed no significant differences between the high dose (600mg/kg) concentrations of the extracts compared to the normal control. However, the low doses 200mg/kg MSB+EU and both 200 & 400mg/kg MSB+PL significantly decreased compared to the normal control. This level of decrease is very similar to that of the standard but significantly different from that of the negative control which was most decreased. The WBC was significantly elevated in all the groups compared to the normal control, except in the high dose (600mg/kg body wt.) of both combinations. The result seen in the high dose combinations is very similar to that of the standard. The negative control and the 200mg/kg body wt. extract combinations have the highest levels of elevation. Results are shown in Figure 1b below The platelet result showed significant increase in all the groups compared to the normal control. The groups fed with higher doses are similar to the standard control, while the groups treated with low doses are comparable to the negative control result as shown in Figure1c below The erythrocyte sedimentation rate (ESR) of the inflamed rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves showed that there was clearly no significant difference seen in the ESR of all the animals treated with various concentrations of the combined extracts between groups. Hence the extract did not produce any significant change in the ESR of the test groups even at higher doses as shown in Figure 1d. Legend: GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 181 • MSB= mango stem bark • PL= pawpaw leaves • EU= eucalyptus • NS= normal saline Figure 1a The Packed Cell Volume (PCV) And Hemoglobin (Hb) Level of Inflamed Rats Treated With Different Concentrations of Combined Ethanol Extracts Of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet are statistically significant (p<0.05) Figure 1b The Red Blood Cell (RBC) And White Blood Cell (WBC) Level Of Inflamed Rats Treated With Different Concentrations of Combined Ethanol Extracts Of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 182 Figure 1c The Platelet Level of Inflamed Rats Treated With Different Concentrations Of Combined Ethanol Extracts of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) Figure 1d The Erythrocyte Sedimentation Rate (ESR) of inflamed Rats Treated With Different Concentrations of Combined Ethanol Extracts of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) 3.2. Liver Function Test Result The aspartate amino transferase (AST) activity (IU/L) of inflammation-induced rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed no significant change in the serum activity of AST in all the treated groups compared to normal control. However, there was a significant change/decrease in the AST activity of standard control as shown in 2a The alkaline phosphatase (ALP) activity (IU/L) of inflamed rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed elevated activities of ALP in the groups treated with MSB+PL and 600mg/kg MSB+EU compared to the normal control as shown in Figure 2b. Alanine amino transferase (ALT) activity (IU/L) of inflamed rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed that apart from the group fed with 200mg/kg MSB+PL, all other groups are significantly lower in ALT activity compared to the normal control as shown in Figure 2c. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 183 The albumin (ALB) concentration (g/l) of inflamed rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed that all the groups showed no significant difference in the ALB concentration compared to the normal control group as shown in Figure 2d Figure 2a The Aspartate Amino Transferase (AST) Activity (IU/L) of Inflamed Rats Treated With Different Concentrations of Combined Ethanol Extracts of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) Figure 2b The Alkaline Phosphatase (ALP) Activity (IU/L) of Inflamed Rats Treated With Different Concentrations of Combined Ethanol Extracts Of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 184 Figure 2c The Alanine Amino Transferase (ALT) Activity (IU/L) of inflamed Rats Treated With Different Concentrations Of Combined Ethanol Extracts Of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) 3.3. Antioxidant Results The malondialdehyde (MDA) (µmol/Ml) of inflamed rats treated with different concentrations of combined ethanol extracts of mango stem bark with pawpaw leaves and mango stem bark with eucalyptus leaves revealed that Malondialdehyde concentration significantly increased in all the groups, but highest in the untreated inflamed group (group 9) compared to the normal control. The group treated with higher doses is comparable to the standard control, while the group treated with lower doses is similar to the negative control as shown in Figure 3a Figure 3a The Malondialdehyde (MDA) (µmol/Ml) Of inflamed Rats Treated With Different Concentrations Of Combined Ethanol Extracts Of Mango Stem Bark With Pawpaw Leaves And Mango Stem Bark With Eucalyptus Leaves Respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) 3.4. Tumor necrosis factor Tumor necrosis factor-alpha result of combined ethanol extract of mango stem bark with eucalyptus leaves and mango stem bark with pawpaw leaves result showed that there was no significant difference observed in the TNF-α of all the test groups compared to the test controls as shown In Figure 4.a. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 185 3.5. Weight and paw size Results on weight (g) of the rats on combined ethanol extract of mango stem bark with eucalyptus leaves and mango stem bark with pawpaw leaves, showed that the weights of the animals in the different treated groups did not significantly differ from that of the control before induction. However after treatment, the weights significantly increased in all the groups compared to the normal control, except in the higher dose-treated groups, which were similar to that of normal control animals. This is shown in figure 3.5a. Results on paw size of the rats on combined ethanol extract of mango stem bark with eucalyptus leaves and mango stem bark with pawpaw leaves showed that there were no significant difference in the paw size of all the test groups and controls one (1) hour after induction. On the 3rd day, there were significant increase in the paw size of the test groups compared to normal control groups, this increase is comparable to standard and negative controls. On the 7th and 10th day, the paw sizes of all the test groups were significantly higher than normal control. However, the test groups with high doses were comparable to standard control while the low dose test groups were similar to negative control as shown in table 1 One hour after induction, it was observed that d rats were licking/ chewing the injected paw, partial elevation of the injected paw (edema). On the 4th and 5th day, it was observed that the rats always cluster together (reduced mobility), could not stand firmly on the injected paw, walking one-sidedly and increased size of the injected paw and was lifted off the floor. On day 7and 10, it was observed that elevated paw was slightly reduced in some groups. Figure 4a The TNF-α of inflamed rats treated with different concentrations of combined ethanol extracts of Mango stem bark with pawpaw leaves and mango stem bark with Eucalyptus leaves respectively. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 186 Figure 5a The weight of rats before induction and 10th day before sacrifice. Bars are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) Table 2 Effect of treatment on paw size of inflamed rats treated with different doses of combined ethanol extract of mango stem bark with Eucalyptus leaves and mango stem bark with pawpaw leaves respectively: Values are mean ± standard deviation. Bars bearing different alphabet letter(s) are statistically significant (p<0.05) Group Paw before induction 1hour after induction 3rd day 1hr after induction (3rd day) 7th day 10th day 200mg/kg MSB + PL 4.37± 0.15a 6.36± 0.20a 5.65±0.17b 7.55±0.08ab 7.26±0.2a 6.75±0.16a 400mg/kg MSB + PL 4.34± 0.12a 6.57± 0.10a 6.11±0.10b 7.52±0.11ab 6.46±0.11ab 6.29±0.09b 600mg/kg MSB + PL 4.33± 0.07a 6.31± 0.16a 5.72±0.18b 7.48±0.13ab 6.44±0.15b 6.20±0.18b 200mg/kg MSB + EU 4.39± 0.11a 6.27± 0.13a 5.45±0.12b 7.52±0.23ab 6.48±0.19ab 6.45±0.17ab 400mg/kg MSB + EU 4.28± 0.05a 6.65± 0.06a 6.07±0.19b 7.49±0.15ab 6.35±0.13b 6.18±0.22b 600mg/kg MSB + EU 4.37± 0.05a 6.39± 0.13a 5.87±0.18b 7.51±0.18ab 6.38±0.21b 5.95±0.17b 5ml NS (Normal Control) 4.27± 0.21b 4.31± 0.18a 4.44± 0.08e 4.44±0.08e 4.66±0.08e 4.68±0.08e 5mg/kg Std drug (+ive control) 4.45± 0.12a 6.53± 0.13a 6.48± 0.19b 7.31±0.09b 6.30±0.21b 6.02±0.23b 5ml NS (Negative control) 4.24± 0.06a 6.33± 0.18a 7.34±0.10a 8.44±0.18a 7.49±0.08a 7.42±0.11a Histological Results Mag. X 400 Hematoxyline & Eosine The histological section of the liver tissue treated with 200mg/kg, 400mg/kg and 600mg/kg Combined Ethanol Extracts of Mango Stem Bark With Pawpaw Leaves (MSB+PL) respectively are shown in plate 3.6.1a to 3.6.1 c) below. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 178-195 193 The present study established that combined ethanol extracts of mango stem bark + pawpaw leaves did not show any toxic effect on the liver of the rats, hence may possibly be safe for consumption. However, mango stem bark + eucalyptus leaves at high doses may be toxic. The antioxidant examination revealed increased level of Malondialdehyde (MDA) in the negative control. This indicates production of free radicals due to the presence of formaldehyde in the tissues and prevention of it in the treated groups. The tumor necrosis alpha examination (TNF-a) revealed no significant change, hence the acute inflammation induced by formalin is non-immunologic. The result also showed that combined ethanol extracts of mango stem bark + pawpaw leaves and mango stem bark + eucalyptus leaves, have significant anti-inflammatory effect due to the marked reduction in paw size volume of the rats. Hence these extracts may be used in the management of painful arthritic inflammatory condition. Histological examination carried out on the liver of the rats revealed no alteration in the liver cell integrity of the rats treated with the mango stem bark + pawpaw leaves extracts. However, questions are raised on its effect on liver integrity with mango stem bark + eucalyptus leaves extract due to the distortion of the liver and inflammatory cells observed in the sinusoids. Contribution to knowledge The study justifies the anti-inflammatory use of both extract combinations and suggests MSB+EU maybe most effective, however, questions are raised on its effect on liver integrity. Recommendation for further study Based on the results, Further studies are suggested on effects on other body organs and to determine the antiinflammatory effect of these extracts on immunological pro-inflammatory molecules (such as cytokines, IL-6, IL-1β, TNF-α, ESR and C-reactive proteins) on arthritic rats induced by complete Fraud’s Adjuvant. Further studies are also required for isolation of active constituents and cellular characterization so as to exclusively establish these plant parts (mango stem bark, eucalyptus leaves and pawpaw leaves) as a potential safer disease modifying agent in the management/treatment of arthritic inflammation. Compliance with ethical standards Acknowledgments The authors firstly thank the Almighty God (The Lord Jesus Christ) for His Grace throughout the duration of this work. 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