Rosemary is an evergreen, fragrant, decorative, perennial plant that may grow up to 2 m in many parts of the world. It is mainly native to the Mediterranean Region^4,5,6. The Rosemary extracts, ursolic acid, oleanolic acid, and micromeric acid are considered to have the most effective anti-inflammatory^7,8 and anti-tumor activities⁸. Rosemary has an antibacterial⁹ and antioxidant effect in vivo^10,11. It increases the synthesis of prostaglandin E2, decreases production of leucotrien B4 in the polymorph leucocytes and prevents complement system in humans¹⁰. It decreases plasma glucose level¹² and has antitumorigenic activity^13,14. Sprouts of the plant have been known to have hepatoprotective and increasing bile secretion activity in rats^13,15 Rosemary powder is used as anti-inflammatory agent in horses, pigs and cattle while rosemary ointment can be used for breast disinfection and its solution is used locally for wound treatment¹⁶.

The aim of this study was to investigate the effects of Rosemarinus officinalis extract on wound healing compared to povidone-iodine and isotonic saline solution.

Animals
Twelve healty, adult New Zealand rabbits (6 male and 6 female), weighing about 2500±200 g were used for the study. They were individually housed and fed in normal diet and water ad libitum in a room with natural light cycle and constant temperature (24±2 ºC). The Harran University Animal Care and Use Committee has approved the study protocol.

Instrumentation and surgical procedure
All rabbits were anaesthetized with intramusculary administrations of 10 mg/kg xylazine hydrochloride and 50 mg/kg ketamine hydrochloride. The dorsal aspects of 12 rabbits were clipped and prepared for aseptic surgery. On dorsal aspect of each animal, one cranially and two caudally located full-thickness skin wounds in 3.14 cm diameter were created using a template prepared from a used X-ray film (Figure 1.A).

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Figure 1: A. Representative open wounds of rabbits on day 0: a. Rosmarinus officinalis group, b. Iodine group, c.Isotonic saline group. B. Representative open wounds of rabbits on day 14: a. Rosmarinus officinalis group, b. Iodine group c. Isotonic saline group.

Treatment protocol
Each rabbit was treated once daily, before bandaging, with topical 5% Rosemarinus officinalis (rosemary) extract and 10% povidone-iodine and 0,9% isotonic saline solution respectively.

Evaluations of wound healing
Observations during daily wound care: Each wound was evaluated at the time of the daily bandage changes for the presence of exudate and wound healing until day 14.

Measurement of wound diameter: The wound diameters was measured by caliper both horizontally and vertically and the average was calculated postoperative on the 4^th, 7^th, 10^th and 14^th days.

Histopathological evaluation: Biopsy specimens, which were collected with clockwise rotation on the 4^th, 7^th, 10^th and 14^th postoperative days (POD), wounds' diameters were measured with calipers in milimeters. Skin specimens were fixed in 10% neutral buffered formalin and processed routinely for histopathological examination. Five micrometers sections were stained with haematoxylin and eosin (H&E). Progressive decrease in neutrophil numbers, progressive increase in angiogenesis, density of macrophages, the ratio of fibroblast, fibrocyte and collagen deposition etc. are the parameters used to assess the progression of healing from the inflammatory into the repair stage. In every skin section, an area just beneath the epidermis or crust formation was randomly selected. Thereafter, four consecutive areas moving towards the deep dermis were selected. The five selected areas were examined under ×400 magnification. The number of neutrophils, fibroblast, fibrocyte was scored as 0–25 = 1, 26–50 = 2, 51–75 = 3, and > 75 = 4¹⁸. The same areas were also examined for the number of vessels and the actual count was noted. Masson's trichrom staining was made for the purpose of find out connective tissue proliferation in wound areas¹⁹.

Statistical analysis
Statistical analyses were carried out using statistics software (SPSS). Kruskal Wallis and Jonkheere-Terpstra methods were used to test the significances of intergroup differences in quantitative (wound size) and qualitative variables, respectively.

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Figure 2: A. General histological aspect of the wounds on day 4th Rosmarinus officinalis group. Crust (bold arrow), infiltration of numerous inflammatory cells (mainly neutrophils) in the dermis (head arrow), haemorrhage (thin arrows). H&E., Bar:100 μm. B. General histological aspect of the wounds on day 14th Rosmarinus officinalis group. Completed re-epithelialization (bold arrow), neovascularization in the dermis (head arrows). H&E., Bar: 50 μm.

Day 7: Reduction in the sizes of wounds treated with rosemary extract and povidone-iodine solution, compared to isotonic saline solution, was statistically significant. Even though reduction in wound sizes of rosemary extract group smaller than the povidone-iodine solution group, the difference was not statistically significant. Microscopically, as opposed to other groups, re-epithelialization has been found to have started in the rosemary extract group. The number of neutrophils was lower than that on day 4 in the povidone-iodine solution and rosemary extract groups while neutrophils were still abundant in the isotonic saline group. The differences in wound size measured on day 7 between groups were statistically significant (P<0.01) while those measured on other days were not (P>0.05) (Table 1).

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Table 1: Measurements of the wound diameter in the groups.

Day 10: Though scarce, moderate macrophage infiltrations were still present in the inflamed regions of the rosemary extract and povidone-iodine solution groups on day 10 of healing. Furthermore, fibroblasts were abundant and formation of numerous vessels and increase in the amount of collagen were noted. Re-epithelialization was ongoing. The moderate neutrophil and macrophage infiltrations and infrequent fibroblasts were still present in the isotonic saline group.

Day 14: On day 14 of healing process, microscopical examination showed that the number of fibroblasts was lower, the number of fibrocytes was higher and the amount of collagen was increased in povidone-iodine solution and rosemary extract groups (Figure 2.B). Re-epithelialization process was found to be complete (Figure 1.B).

Presence of infrequent macrophages and neutrophils, as well as fibroblasts and fibrocytes were observed with isotonic saline solution dressing. This was statistically significant. Numbers of neutrophils counted on day 14 were significantly different between groups (P<0.01) (Table 2) but the differences in other days were not significant (P>0.05).

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Table 2: Neutrophil count in the groups.

Measurements of wound size revealed that wounds healed faster with rosemary extract than povidone-iodine or isotonic saline solution. Neutrophil infiltration significantly regressed on day 7 with rosemary extract and on day 10 with povidone-iodine solution. On the other hand, neutrophil infiltration in the wound region was still present on day 14 with isotonic saline solution. In conclusion, rosemary extract was found to be more effective on wound healing that povidone-iodine or isotonic saline solution. In the wound area, the connective tissue proliferation was confirmed by Masson's trichrom staining. The connective tissue proliferation was defined as the least on day 4th, getting increased on days 7^th, 10^th and 14^th in the wound area.

The wound healing process involves many complex factors. These may be classified as local and systemic factors, organ and species variability in response to injury. In this study, we adopted an experimental design including daily observation, macroscopical and histological evaluations of the wound healing process and statistical methods.

Re-epithelialization, collagen deposition and neovascularization are all among the parameters used for the analysis of healing wounds^20-22. The immature vessels are originated from mature vessels of deeper tissues. Anastomoses also occur among these vessels to provide the wound area with sufficient blood supply²³. In the present study, increase in neovascularization was observed in the rosemary extract and povidone-iodine solution groups on day 10 significantly.

Rosemary extract has long been used in medicine and food industry, thanks to its antibacterial^7,8, antiinflammatory and antioxidant^10,24,25 activities. Many studies have been carried out on Rosemary's bactericidal and bacteriostatic effects⁹. The present study is important since it demonstrated the antiseptic, antimicrobial and epithelializing effects of rosemary extract on wound healing.

As a result, we have noted that open wounds in rabbits treated with rosemary extract tend to heal faster than similar wounds treated with povidone-iodine and isotonic saline solution. These results indicate that rosemary extract provide a good alternative for the treatment of open wounds in rabbits.

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