Evaluation of lipid profile and antioxidant activities of an herbal combination therapy on formalin-induced inflammation in albino rats

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Introduction
Herbal medicine is gaining popularity both in developing and developed countries because of its natural constituents 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. 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, which 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). This study is aimed at evaluating lipid profile and antioxidant activities of an herbal combination therapy on formalininduced inflammation in Albino rats.

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 N5 0 23'33.6876" longitude and E6 0 59'10.5504 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.

Preparation of extract
Three hundred grams (300 g) powdered sample 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 evaporated and dripped into the extraction chamber where it was collected. The active component left behind in the flask was 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.

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℃) (25 ℃) 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 were done in compliance with the National Institute of Health Guide for care and use of laboratory animals.

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 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 their mean body weight according to OECD'S guideline on volume selection; (Barret, et al., 1991). All the groups received their treatments accordingly, while groups 7 and 9 received 5mls normal saline and group 8 received 5mg/kg 5 mg/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.

Biochemical Analysis
The blood samples were collected into EDTA and heparinized bottles by ocular puncture then sent to the laboratory for analysis. Blood samples were centrifuged for 10mins at 3000rpm and the serum was use to examine malondialdehyde (MDA) according to the method of Ohkawa & Ohishi (1979), Catalase was determined using Clairborne method (1985), and Superoxide Dismutase (SOD) was assessed according to misra and fridovich method (1972).

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.

Lipid Profile Results
The There was a marked significant decrease in the high density lipoprotein (HDL) level of the standard control compared to all other groups and normal control group. Apart from the group treated with 600mg/kg 600 mg/kg MSB+PL which is similar to the normal control, all other groups decreased significantly and are comparable to the negative controls as shown in figure 3 The low density lipoprotein (LDL) of all the test groups revealed a significant increase compared to the normal control group as shown in figure 4.

Antioxidant Results
The Catalase Activity (CAT) (U/G) 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 a significant decrease in the catalase activity of the negative control compared to the normal control and other treated groups as shown in Figure 5 The superoxide dismutase (SOD) (µ/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 showed that there were clear significant decrease in the SOD level of the negative control, standard control and all the rats fed with low doses of the extract compared to the normal control. However, the groups fed with higher doses of the extract were comparable to the normal control as shown in Figure 6.

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 the untreated inflamed group (group 9), this significant increase was also seen in the lowest dose of MSB+EU group (200mg/kg 200 mg/kg body weight) and all doses of the MSB+PL groups except the highest (600mg/kg 600 mg/kg body weight animals). There was no significant difference observed between the rest of the groups and the normal control as shown in Figure 7

Discussion
The The enzymatic antioxidant systems such as catalase and superoxide dismutase play a corresponding role in the avoidance of oxidative damage by reactive oxygen species. SOD is one of the chief cellular defense enzymes that dismutate superoxide radicals to water and oxygen. Catalases (CAT) on the other hand are heme-containing proteins that defend the cells from toxic effects of reactive oxygen species by converting hydrogen peroxide to water and molecular oxygen. The increase in the activities of antioxidant enzymes is comparable to that reported by udegbunam, et al., (2014) who reported increase in the SOD levels, and Ruma et al., 2015) who also reported increase in the antioxidant activity. This may suggest the fact that upon injection of inflammatory agent, excess reactive oxygen species are generated causing oxidative stress, the production of ROS could overwhelm the capacity of endogenous antioxidant enzymes system resulting to significant decrease. However, administration of the extracts increases the antioxidant enzyme activity, thereby neutralizing the free radicals generated. It may also be suggested that the standard drug had no significant effect on the SOD enzyme activity.

Conclusion
It is now widely acknowledged that early diagnosis of inflammation and aggressive treatment to control disease activity offer the highest likelihood of preserving function and preventing disability.
The result of the present study reveals the effects of combined ethanol extracts of mango stem bark + pawpaw leaves and mango stem bark + eucalyptus leaves respectively on lipid profile and antioxidant activities.
The result on antioxidant revealed a significantly increased level of Malondialdehyde (MDA), decreased SOD and CAT in the negative control. This indicates production of free radicals due to the presence of formaldehyde in the tissues.
The lipid profile assessment revealed significant changes in the LDL, HDL, cholesterol, and triglycerides; which indicate possible disposition of the animals to dyslipidemia.

Contribution to knowledge
The study revealed that the combined extracts of mango stem bark + pawpaw leaves and mango stem bark + eucalyptus leaves is observed to reverse the significant changes on the lipids caused by the inflammation and piroxicam (the standard drug) may cause significant metabolic disorder in prolong usage . The extracts reduced the level of MDA in the groups treated with the extracts, and increase the antioxidant activity of SOD and CAT. This justifies the antiinflammatory use of both extract combinations in ameliorating the inflammatory process by mopping up the free radicals produced.

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.