ANTI-INFLAMMATORY POTENTIAL OF MICROEMULSION AND PURE BULLFROG OIL IN MUSCLE INJURY

Rev Bras Med Esporte – Vol. 23, No 3 – Mai/Jun, 2017 ABSTRACT Introduction: Every day science seeks new ways to treat various diseases through drugs that are efficient and viable. Thus, therapeutic alternatives that meet such demand are targets of study. Microemulsions are one of these new alternatives due to their peculiar pharmacodynamic and pharmacokinetic characteristics. Objective: The aim of this study was to analyze the anti-inflammatory potential of microemulsion and pure bullfrog oil using an experimental model of muscle injury. Methods: Male Swiss mice were divided into three groups: control, microemulsion and pure bullfrog oil. After the pre-treatment, a muscle injury was induced in the animals’ leg and subsequently evaluations were carried out in the horizontal extent of edema and compared between the groups at predetermined times. Following evaluation of muscle injury, dissection of the right gastrocnemius muscles was performed for histological analysis. Results: The microemulsion and pure bullfrog oil showed good anti-inflammatory activity, acting similarly in reducing edema during the first two hours, but without statistical significance from the 3rd to the 24th hour after induction. The histological analysis revealed that the muscle tissue of the animals treated with the microemulsion presented mild cellular infiltrate and little wear of muscle fibers when compared with the muscular tissue of animals treated with the pure bullfrog oil. The histological analysis of the hepatic tissue showed signs of injury in the liver lobes of the pure bullfrog oil group, not observed in the microemulsion group. Conclusion: The microemulsion showed good anti-inflammatory potential in the acute phase of the inflammatory response, reducing the formation of edema and preserving muscle tissue against the occurrence of lesions and without inducing injury in hepatic tissue.


INTRODUCTION
Current drugs are delivered through conventional pharmaceutical formulas that typically cannot reach the adequate concentrations at the target tissue, due to biological barriers that ultimately expose other tissues to the toxic effects of the medication.For their peculiar characteristics, such as viscosity, ease of preparation, high pharmaceutical solubility and high bio availability, beside low toxicity levels, microemulsions are presented as new alternatives 1 .Microemulsions are thermodynamically stable and isotropically clear of two immiscible liquids (oil/water) 1 .The term microemulsion (ME) has been used to define systems of micro heterogenic phases of up to five constituents: water, oil phase, tensoactive and cotensoactive.From the structural point of view, microemulsions can be of the following types: oil in water (O/W), water in oil (W/O) or bi-continuous 2 .In this study, water in oil (W/O) microemulsion was used, composed by the aqueous phase (ethanol at 70%), soy lecithin as the tensoactive and bullfrog oil (FO) as the oily phase.The lecithin is used as a tensoactive on the preparation of microemulsions for their lipophilic and hydrophilic properties and for the balance between these properties and a strong tendency to form liquid crystals 1 .The active ingredient tested in this study and transported in a microemulsion was the bull-frog oil (FO).This oil has already been broadly utilized by the population -sometimes indiscriminately -for treatment of several ailments.The FO is obtained from the bull-frog' s (Rana catesbeiana) fat tissue, a frog species native to eastern North America, but currently cultivated in several regions of the world.There is presently a growing investigation of possible therapeutic properties for FO, due to the presence of polyunsaturated fatty acids (PUFA), omega-3 and omega-6, in its composition 3 .The oil proportions of polyunsaturated fatty acids on bull-frog oil are 31.7% of oleic acid (omega-6) and 12.9% of linolenic acid (omega-3) 4 .Some studies have been conducted to evaluate their power on anti-inflammatory processes [5][6][7][8] ; however, the results are still not very conclusive for proving this potential.The increased consumption of PUFA by the human being brings substantial changes to the organism, such as increased membrane fluidity, an influence on the expression of membrane receptors, nutrient transport and signs of transduction, acting on cellular growth 9 .PUFA and the products derived from its cellular metabolism may modulate the kinases activities involved on the NF-kB activities (Kappa B nuclear factor), which is an inflammation factor activated by the phosphorylation of IkB protein.
As a consequence of this activation, the NF-kB migrates to the nucleus of the cell, connecting to the DNA sequences (kB sites) which are located on the regions promoting genes related to apoptosis, cellular adhesion, immune response, inflammation, cellular stress and tissue remodeling.It is believed that PUFA can be relevant to the inflammatory process for being connected to the suspension of factors like NF-kB, modulating its activity and diminishing the tissue wear due to the liberation of cytokines 10 .As it' s a substance rich in fatty acids, the possibility that the administration of pure FO and in microemulsion may cause hepatic overload must be analyzed.
As previously said, microemulsion is a new system for drug release, which raises the hypothesis that it may be less metabolically offensive to the liver when compared with the pure bullfrog oil.The changes on the composition of fatty acids in liver tissues may influence the lipid metabolism and regulate inflammatory processes in mammals 11 .However, when it comes to the ingestion of fatty acids, attention is required for the possibility of fat accumulation in the organ, which may predispose the individual to a situation of hepatic steatosis, first event of non-alcoholic disease on fat liver.It becomes important to analyze hepatic lipids not only as hepatotoxic factors, but as markers of the hepatic exposure to free fatty acids, possible inducers of hepatotoxicity.The objective of this study was to investigate if the microemulsion of bullfrog oil may act as a modulator of the activities triggered by the muscular inflammation, in an effort to avoid the exacerbated wear of the tissue (injury), providing it a better regeneration, as well as if it may contribute for the preservation of the liver, avoiding its overburden.

MATERIALS AND METHODS
To obtain and determine the microemulsion, the study of ternary diagrams was required to determine the regions (points) of the microemulsion.The diagram was obtained from the proportions of oil phase and tensoactive, titrated with alcohol solution at 70%, used as the aqueous phase, as represented on Figure 1.
A microemulsion composed of 90% of oil phase, 5% tensoactive (soy lecithin) and 5% aqueous phase (water/ethanol 70%) was used.18 male mice from the Swiss lineage, Mus musculus species were used and randomly selected, separated in three groups (n=6): one control group (saline) and two experimental groups (microemulsion and pure bull-frog oil).100 µl of the respective solutions was administered to each group for 4 consecutive days using the gavage technique.With the pre-treatment completed, the animals were anesthetized by inhalation for subsequent induction of the muscular lesion, with the inoculation of 20 µl of formalin at 10% on the animals' gastrocnemius venter muscle.The lesion was evaluated from the muscle's horizontal extension (edema), using a digital metallic caliper ruler, positioned on the medium third of the animal's leg.The extension of the lesions was verified in the intervals of 5 minutes, 1, 2, 3, 4, 5 and 24 hours after the induction.For histological analysis, dissections of the gastrocnemius muscle were performed, to evaluate the presence of tissue injury.In parallel, samples of the animal livers were taken for evaluation of the parenchyma and analysis of signs of hepatotoxicity.As shown in Figure 2, when the microemulsion and the negative control (saline) groups are compared, a statistically significant difference (p<0.001) can be observed.When the micro-emulsion and the FO (pure frog oil) groups are compared, no statistically significant difference is observed (p=0.9027),showing that the micro-emulsion with oil concentration of 90% and the pure frog oil provide the same efficiency on the edema reduction.
When the efficiency of the microemulsion is compared to the pure frog oil on the measurement intervals, both demonstrated to be effective up to the second hour, where greater statistical difference from the negative control group is observed, indicating lesser extension of the edema in both groups.No statistical difference was observed from the third to the twenty-fourth hour after the induction of the lesion.Significant morphologic differences were observed on the analysis of the tissue samples from the microemulsion and pure frog oil groups.Discrete edema presence and small signs of tissue injury were observed on the microemulsion group (Figure 3A).
The analysis of the muscular tissue on the pure frog oil group showed relevant presence of edema and fragmentation of muscular fibers, denoting the installation of more evident muscle injury (Figure 3B).
Considering that the pure frog oil is rich in polyunsaturated fatty acids, which are metabolized by the liver, samples of the animals' livers in the microemulsion and pure frog oil groups were taken for histopathological analysis, with the intention to verify the existence of signs of hepatotoxicity.Undamaged hepatic lobes were observed on the hepatic tissue samples from the microemulsion group, with hepatocyte cords and capillary sinusoids positioned radially from the perimeter to the center of the lobe (Figure 4A).
The blood vessels appear congested with limpid light and preserved without any structural or morphological damage Signs of injury on the lobes were observed on the hepatic tissue samples from the pure bullfrog oil group, as well as hepatocytes growth and signs of subscapular necrosis, injuries associated with the increased fat absorption by the animals, which denotes the hepatotoxic potential of the pure bullfrog oil (Figure 4B).The study was approved by the institution's ethics committee UNI-RN under n.protocol 001/2015.

Figure 1 .
Figure 1.Graphic representation of the construction on the phase diagram.

Figure 2 .
Figure 2. Evaluation of the edema through the measurement of the animal leg diameter on the microemulsion, saline and pure frog-oil groups.

Figure 3A .
Figure 3A.Histologic section of muscle tissue on animal from the microemulsion group (10x magnification).

Figure 3B .
Figure 3B.Histologic section of muscle tissue on animal from the FO group (10x magnification).

Figure 4A .
Figure 4A.Histologic section of hepatic tissue on animal from the microemulsion group (100x magnification).

Figure 4B .
Figure 4B.Histologic section of hepatic tissue on animal from the FO group (100x magnification).