Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats.

Objective: to compare the healing by second intention under the effects of topical application of honey, copaíba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) with a control group in rats. Methods: we carried out a skin resection, 1cm in diameter, on the back of 40 rats allocated to four groups of ten animals. All wounds were cleaned daily with 2ml of 0.9% NaCl solution. The first group (control - GC) was restricted to such procedure. In the wounds of the second (GM), third (GO) and fourth groups (GF), after cleaning, we respectively applied 1ml of honey, 1ml of copaíba oil-resin and 1ml of cream containing fibrinolysin, deoxyribonuclease and chloramphenicol. The wounds were occluded with sterile gauze. Immediately after the incision and on days three, seven and 14 of the experiment, the wounds were copied and contraction was analyzed using planimetry. After euthanasia, we histologically evaluated the inflammatory reaction and collagen in the scars. Results: the reduction of the wound area of GM (p=0.003), GO (p=0.011) and GF (p=0.002) were higher than the GC. The amount of type-I collagen present in GM and GO was higher than in GC and GF groups (p<0.05). There was a predominance of chronic inflammatory stage in GM (p=0.004), GO (p<0.001) and GF (p=0.003) when compared with GC. Conclusion: the topical use of honey and copaíba oil-resin increases wound contraction, the presence of type-I collagen and accelerates healing.


Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats.
Análise comparativa dos efeitos do mel, do óleo-serina de copaíba e de um produto comercial (fibrinolisina, desoxirribonuclease e cloranfenicol) na cicatrização por segunda intenção, em ratos. 2 Rev Col Bras Cir 46 (5):e20192245 Throughout history, natural compounds have been the basis of medicines, often starting from popular knowledge and coming to the market purified and improved. In this context, honey is the oldest material used to accelerate the healing process known to man 5 , presenting several properties that make it a potential element for dressing formulations. It can reduce edema, inflammation and exudation of a wound and increase the efficiency of the healing process 6 .
This viscous solution, produced by several bee species, is predominantly composed of sugars (75% to 79%) and water (20%) 6 . Other components are: proteins (invertase enzymes, catalase and glucose oxidase), vitamins (nicotinic acid, pyridoxine and thiamine), minerals (potassium, iron, magnesium, phosphorus, copper, zinc and calcium) and antioxidants. The quantities of these components vary according to species, seasonality, local flora and storage 7 .
Copaíba oil-resin is another compound that has shown potential in improving healing. It is an oil-resin extracted from trees of the genus Copaifera, which has more than 72 species. Among the highlights are its anti-inflammatory, analgesic, antitumor, antineoplastic and antinociceptive properties 8,9 . Because of these properties, honey and copaíba oil-resin are potential options to accelerate the healing process at a more affordable cost than current drugs, with easy access in the Brazilian territory.
In the formulation of medicines available on the market is fibrinolysin, a proteolytic enzyme that acts by favoring enzymatic debridement of injured tissues. This enzyme may have come associated with deoxyribonuclease, an enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA structure.
Many of these synthetic agents also come associated with an antibiotic, most commonly chloramphenicol and gentamicin. They are sold in the form of ointments, gel or adhesives to be applied to wounds.
The objective of this work is to evaluate the potential for secondary intention healing of topical application of honey and copaíba oil-resin by comparing them to the application of a commercial product containing fibrinolysin, deoxyribonuclease and chloramphenicol in rat skin wounds. Surgery, in species-specific polypropylene boxes with a white sawdust bed, with three animals per box. The room temperature was maintained at 22±2ºC, with a 12 hour light/dark cycle and the relative humidity and noise level of the environment. All the boxes were arranged on shelves at equal distance from the light source. The animals had free access to their own food and water. We randomly split the sample into four groups of ten animals.

Biondo-Simões
Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats. Once confirmed the death, we resected a square segment of skin, 3cm in the side that contained the scar in the central part. We extended the flap under filter paper and submerged it in formalin for fixation and further processing for histopathological study.
Paraffin blocks containing the material were cut 4µ thick and, after mounted on slides, were stained with hematoxylin-eosin (HE) and picrosirius (Sirius Supra Red F3BA).
In HE staining, we evaluated the inflammatory process by reading four 400x magnification fields. For classification and quantification, we assessed the type and quantity of predominant cells, poly and monomorphonuclear infiltrates. We used the following parameters: 0= absence, 1= one to ten cells per field, 2= 11 to 50 cells per field, and 3= 50 or more cells per field. With these data, it was possible to quantify the reaction in intense, moderate, discrete and absent. When there was a predominance of polymorphonuclear cells, we considered the reaction acute. When the monomorphonuclear cells were predominant, we called the reaction chronic, and when there was no predominance, we called it acute-chronic.
In the analysis of picrosirius-stained slides, the thicker and birefringent collagen fibers are orange-red (collagen I), and the thinner and more dispersed, weakly birefringent fibers are greenish (collagen III). The images were taken by a Sony CCD101 camera. We performed image analysis using the MediaCybernetics Image-Plus® 4.5 for Windows® application. We analyzed six fields located in the scar line with 100x magnification in each optical microscope with polarized light lens. In each one, we calculated the percentage of area occupied by the red and yellow (collagen I) and green (collagen III) fibers.

Biondo-Simões
Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats.

RESULTS
Two animals from the control group died due to anesthetic accidents on day zero.
The macroscopic evaluation of the wounds did not show the presence of hemorrhage or purulent discharge in the four groups throughout the study period.
The reduction in wound area, assessed by planimetry on days three, seven, and 14, showed no significant differences between groups (Table 1, Figure 1). However, when comparing wound contractions by paired groups, the wounds treated with copaíba oil were smaller than those in controls (p=0.008) between day zero and day seven.
The same was true for honey-treated (p=0.45) and for those treated with fibrinolysin with deoxyribonuclease and chloramphenicol compared with controls (p=0.001) ( Table 2).
Wound contraction from day zero to day 14 was more expressive in the honey-treated group (p=0.003) when compared with the control one.
We also observed a significant reduction in the group treated with copaíba oil (p=0.011). The group treated with fibrinolysin with deoxyribonuclease and chloramphenicol showed no significant difference compared with the control group (p=0.062) ( Table 2). In the quantitative, histological evaluation of collagen, although there was a higher average area in GC and GF, the difference was not significant in relation to the other groups (ANOVA p=0.612) ( Table 3, Figure 3).

Biondo-Simões
Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats. The pharmaceutical industry is always developing or improving products to aid healing.
As an example, there is the drug used in this study,    Copaíba oil-resin is also very old in use, being the first known citation in a letter from Petrus Martius to Pope Leo X, in 1534, that reported a drug used by the Indians, called "Copei" 10 . Like honey, the ability of copaíba oil-resin to improve the healing process is also known 8 . Considering that both honey and copaíba oil-resin are natural products, available practically all over the national territory, their use could be more accessible to the needy population.
The increase in contraction velocity in the first seven days of the study, observed in the three

Biondo-Simões
Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats. Both honey and copaíba oil-resin have the capacity to promote angiogenesis 11,16 , which facilitates oxygen delivery to the wound bed, the arrival of inflammatory cells and the early establishment of granulation tissue. In addition, both compounds exhibit direct, already determined anti-inflammatory activities 5,9 . In this study, the inflammatory reaction was minimal. In GM, GO and GF, however, it was in the chronic phase (predominance of monomorphonuclear cells), suggesting that they were able to modulate the inflammatory response pattern in the scar bed, reaching more advanced stages in a shorter period of time, when compared with GC. The same inflammatory pattern has been shown in other studies 17,18 . By increasing the contraction capacity of the wound bed while improving the inflammatory pattern, the compounds provide a more conducive environment for the maturation of scar elements such as collagen.
There was therefore a greater amount of type-I collagen fibers in GM and GO compared with the other groups, and these data are already present in the literature 7,8 . Thus, it can be inferred that honey and copaíba oil-resin induce fibroblast proliferation and extracellular matrix formation.
As the type-I collagen is more resistant, a higher concentration of it indicates a better quality wound when compared with GF and GC.
In addition, for the group treated with copaíba oil-resin, we observed a smaller total amount of collagen in the wound bed. Since copaíba oil-resin is able to increase the activity of metalloproteinase type 2 18 , an enzyme responsible for the collagen remodeling and homeostasis phase, it is believed that the topical application of the oil-resin leads to the earlier occurrence of the later healing phases.
A major advantage of honey and copaíba oil-resin over the commercial product is their easy access. In this study, we used commercialized products, sold in common retail. The commercial product is sold only in drugstores and requires a prescription, which makes it difficult to use for populations with low access to medical services.
In addition, both honey and copaíba oil-resin can cost 60 times less than the commercial product, depending on where in the country they are purchased. The use of these natural products makes treatment more affordable and more accessible, increasing patient compliance, especially for minor injuries that would not receive any kind of medical care but are subject to the same complications as others.
Therefore, there is evidence to say that honey and copaíba oil-resin are as effective as the commercial wound care product, with the advantage of forming a better quality scar, having greater accessibility and reduced treatment cost.
However, there was no significant difference in the action of honey and copaíba oil-resin compounds when compared with each other. Other studies are still needed to compare the performance of the different types of honey and copaíba oil-resin and to show their mechanisms of action, thus providing the evidence for their clinical recommendation.

Biondo-Simões
Comparative analysis of the effects of honey, copaiba oil-resin and a commercial product (fibrinolysin, deoxyribonuclease and chloramphenicol) on second intention healing, in rats.