A Therapeutic Approach for Wound Healing by Using Essential Oils of Cupressus and Juniperus Species Growing in Turkey

Juniperus and Cupressus genera are mainly used as diuretic, stimulant, and antiseptic, for common cold and wound healing in Turkish folk medicine. In the present study, essential oils obtained from cones of Cupressus and berries of Juniperus were evaluated for their wound healing and anti-inflammatory effects. In vivo wound healing activity was evaluated by linear incision and circular excision experimental wound models, assessment of hydroxyproline content, and subsequently histopathological analysis. The healing potential was comparatively assessed with a reference ointment Madecassol. Additionally acetic-acid-induced capillary permeability test was used for the oils' anti-inflammatory activity. The essential oils of J. oxycedrus subsp. oxycedrus and J. phoenicea demonstrated the highest activities, while the rest of the species did not show any significant wound healing effect. The experimental study revealed that J. oxycedrus subsp. oxycedrus and J. phoenicea display remarkable wound healing and anti-inflammatory activities, which support the folkloric use of the plants.


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Evidence-Based Complementary and Alternative Medicine  .87 ± 1.41 (50.1) * * * * P < 0.05; * * P < 0.01; * * * P < 0.001; SEM: Standard error of the mean. Percentage of tensile strength values: vehicle group was compared to negative control group; the oils and the reference material were compared to vehicle group. and berries of J. communis, J. excelsa, J. foetidissima, J. oxycedrus, and J. phoenicea by means of in vivo circular excision and linear incision wound models and anti-inflammatory activity.

Plant
Material. Berries and cones of 5 different Juniper species and 2 different varieties of Cypress belonging to Cupressaceae family natively growing in Turkey were chosen as the study material. Specimens were taken from each species as 1 kg weight from their growth sites and stored at −24 • C until analysis [10]. Species names, sampling site, local name, collection date, and altitude of all specimens are listed in Table 1.

Hydrodistillation.
The essential oils of each sample were obtained by hydrodistillation with a Clevenger apparatus using 250 g (partially crushed) of fresh berries and cones. The essential oils were collected for 3 h and dried over anhydrous sodium sulphate and under refrigeration in a sealed vial until required [11,12].
The animals were left for 3 days at room conditions for acclimatization. They were maintained on standard pellet diet and water ad libitum throughout the experiment. A minimum of six animals were used in each group. Throughout the experiments, animals were processed according to the suggested European ethical guidelines for the care of laboratory animals.

Preparation of Test Samples for Bioassay.
Incision and excision wound models were used to evaluate the wound healing activity. For the in vivo wound models, test samples were prepared in an ointment base (vehicle) consisting of glycol stearate, 1, 2 propylene glycol, liquid paraffin (3 : 6 : 1) in 1% concentration. 0.5 g of each test ointment was applied topically on the wounded site immediately after wound was created by a surgical blade.
The animals of the vehicle group were treated with the ointment base only, whereas the animals of the reference drug group were treated with 0.5 g of Madecassol (Bayer, 00001199). Madecassol contains 1% extract of Centella asiatica.
For the assessment of anti-inflammatory activity, test samples were given orally to test animals after suspending in a mixture of distilled H 2 O and 1% Tween 80. The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of dosing vehicle. Indomethacin (10 mg/kg) in 1% Tween 80 was used as reference drug. 0.00 ± 0.00 (100.00) * * * * P < 0.05; * * P < 0.01; * * * P < 0.001; SEM: standard error of the mean. Percentage of contraction values: vehicle group was compared to negative control group; the oils and the reference material were compared to vehicle group.

Wound Healing Activity
Linear Incision Wound Model. Animals, seven rats in each group, were anaesthetized with 0.15 cc Ketalar [13], and the hairs on the dorsal part of the rats were shaved and cleaned with 70% alcohol. Two 5 cm length linear-paravertebral incisions were made with a sterile blade through the shaved skin at the distance of 1.5 cm from the dorsal midline on each side. Three surgical sutures were placed each 1 cm apart.
The ointments prepared with test samples, the reference drug (Madecassol) or ointment base (glycol stearate: propylene glycol: liquid paraffin (3 : 6 : 1)) were topically applied on the dorsal wounds in each group of animals once daily throughout 9 days. All the sutures were removed on the last day, and tensile strength of previously wounded and treated skin was measured by using a tensiometer (Zwick/Roell Z0.5, Germany) [14,15].
Circular Excision Wound Model. This model was used to monitor wound contraction and wound closure time. Each group of animals (seven animals in each) was anaesthetized by 0.01 cc Ketalar. The back hairs of the mice were depilated by shaving. The circular wound was created on the dorsal interscapular region of each animal by excising the skin with a 5 mm biopsy punch; wounds were left open (Tramontina, Machado, Nogueira Filho Gda, Kim, Vizzioli & Toledo, 2002). Test samples, the reference drug (Madecassol, Bayer) and the vehicle ointments were applied topically once a day till the wound was completely healed. The progressive changes in wound area were monitored by a camera (Fuji, S20 Pro, Japan) every other day. Later on, wound area was evaluated by using AutoCAD program. Wound contraction was calculated as percentage of the reduction in wounded area. A specimen sample of tissue was isolated from the treated skin of each group of mice for the histopathological examination [16].

Hydroxyproline Estimation.
Tissues were dried in hot air oven at 60-70 • C till consistent weight was achieved. Afterwards, samples were hydrolyzed with 6 N HCl for 4 hours at 130 • C. The hydrolyzed samples were adjusted to pH 7 and subjected to chloramine T oxidation. The coloured adduct formed with Ehrlich reagent at 60 • C was read at 557 nm [17].  5.28 ± 0.26 50.6 * * * * P < 0.05; * * P < 0.01; * * * P < 0.001 significant from the control; SEM: standard error of the mean.
Standard hydroxyproline was also run and values reported as mg/g dry weight of tissue [18].

2.3.5.
Histopathology. The skin specimens from each group were collected at the end of the experiment. Samples were fixed in 10% buffered formalin, processed, and blocked with paraffin and then sectioned into 5 micrometer sections and stained with hematoxylin & eosin (HE) and Van Gieson (VG) stains. The tissues were examined by light microscope (Olympus CX41 attached Kameram Digital Image Analyze System) and graded as mild (+), moderate (++), and severe (+++) for epidermal or dermal remodeling. Re-epithelization or ulcer in epidermis, fibroblast proliferation, mononuclear and/or polymorphonuclear cells, neovascularization, and collagen depositions in dermis were analyzed to score the epidermal or dermal remodeling. At the end of the examination, wound healing phases-inflammation, proliferation, and remodeling-were determined for all groups.

Anti-Inflammatory Activity
Acetic-Acid-Induced Increase in Capillary Permeability. Effect of the test samples on the increased vascular permeability induced by acetic acid in mice was determined according to Whittle method [19] with some modifications [20]. Each test sample was administered orally to a group of 10 mice in 0.2 mL/20 g body weight. Thirty minutes after the administration, the tail of each animal was injected with 0.1 mL of 4% Evans blue in saline solution (intravenous) and then we waited for 10 min. Then, 0.4 mL of 0.5% (v/v) AcOH was injected intraperitoneally. After 20 min incubation, the mice were killed by dislocation of the neck, and the viscera were exposed and irrigated with distilled water, which was then poured into 10 mL volumetric flasks through glass wool. Each flask was made up to 10 mL with distilled water, 0.1 mL of 0.1 N NaOH solution was added to the flask, and the absorption of the final solution was measured at 590 nm (Beckmann Dual Spectrometer; Beckman, Fullerton, Calif, USA). A mixture of distilled water and 0.5% CMC was given orally to control animals, and they were treated in the same manner as described above.

Statistical
Analysis of the Data. The data on percentage anti-inflammatory and wound healing was statistically analyzed using one-way analysis of variance (ANOVA). The values of P ≤ 0.05 were considered statistically significant. Histopathologic data were considered to be nonparametric; therefore, no statistical tests were performed.

Results and Discussion
In the present study, wound healing and anti-inflammatory effects of the oils obtained from Cupressus sempervirens var. horizontalis, C. sempervirens var. pyramidalis J. communis, J. excelsa, J. foetidissima, J. oxycedrus, and J. phoenicea were evaluated. The oils were mixed with glycol stearate, 1,2 propylene glycol, liquid paraffin (3 : 6 : 1) in 1% concentration for the assessment of the wound healing activity. Linear incision and circular excision experimental wound models were employed. Moreover, skin samples were evaluated histopathologically. The experimental results are given in Tables 2-6.
As shown in Table 2, topical application of the ointment prepared with the oils of aerial parts of J. oxycedrus subsp. oxycedrus and J. phoenicea onto the incised wounds demonstrated the best wound healing activity. On day 10 the tensile strength values were 31.5% and 36.3%, respectively. The rest of the oils did not show any remarkable wound healing activity.
The contraction values of vehicle, negative control, oils and reference drug treated groups were shown in Table 3. The oils obtained from the berries of J. oxycedrus subsp. oxycedrus and J. phoenicea were found to have wound healing potential, while the vehicle and negative control groups and the other oil ointments showed no statistically significant wound healing activity. The wound contractions were determined as For the assessment of collagen synthesis hydroxyproline levels of treated tissues were evaluated. As shown in Table 4 significant increase in hydroxyproline content was observed for the tissues treated with the essential oils of J. oxycedrus ssp. oxycedrus and J. phoenicea.
Phases in wound healing processes (inflammation, pro liferation, and remodeling) were observed and within the experimental groups with different degree (Table 5, Figures 1 and 2). The reference drug, essential oils of J. oxycedrus subsp. oxycedrus and J. phoenicea treated groups demonstrated faster remodeling respectively compared to the other groups which showed delayed wound healing processes. As an evidence of delay, wound-associated tissue debris still remained in the dermal tissues.
The effect of the extracts on the inflammatory phase of wound healing was examined by using the method of Whittle, based on the inhibition of acetic-acid-induced capi-llary permeability. As shown in Table 6, the inhibitory activity was observed for the oils from Juniperus oxycedrus ssp. oxycedrus and J. phoenicea at the dose of 200 mg/kg with the highest inhibitory values of 33.1% and 27.3%, respectively, while the essential oils from both species showed no significant effect at the dose of 100 mg/kg. On the other hand the rest of the oils did not show any inhibitory activity.
Previous studies revealed that the essential oils of Juniperus and Cupressus are rich in α-pinene. Particularly J. phoenicea possesses the highest α-pinene content compared to the other species in comparative phytochemical analysis; β-Pinene, sabinene, and limonene are the constituents which are also present in the essential oils of both genera [8,10]. In a previous study, α-pinene was found to have moderate anti-inflammatory effect at 500 mg/kg dose on carrageenaninduced hind paw edema test [21]. Anti-inflammatory activity is essential for wound healing, since a long duration in the inflammatory phase causes a delay in healing process. In order to shorten the healing period as well as for minimal pain and scar, anti-inflammatory activity is required [22]. Presence of α-pinene in Juniperus species could probably contribute to wound healing activity by providing antiinflammatory effect. Moreover, limonene is an important monoterpene which has a role in wound healing [23]. In a model of chronic skin inflammation, the researchers have also shown a significant effect of limonene and perillyl alcohol on skin repair and proinflammatory cytokines levels [24]. Furthermore, J. oxycedrus ssp. oxycedrus and J. phoenicea were found to have high flavonoid and phenolic acid content which provide them to show remarkable antioxidant activity [5]. As in many of the diseases, antioxidant activity also helps to promote wound healing process [25].
Collagen is an important extracellular matrix protein which provides strength and integrity to the tissue and plays an important role in haemostasis and epithelisation. High levels of hydroxyproline in regenerated tissue suggest enhanced collagen synthesis. Hence enhanced collagen synthesis by J. oxycedrus ssp. oxycedrus and J. phoenicea may significantly contribute to healing and provide strength to repaired tissue [18].

Conclusion
According to the experimental results, the oils from berries of J. oxycedrus ssp. oxycedrus and J. phoenicea were found to have better activity on the wound healing compared to the other essential oils and control groups. This might be due to the synergistic effect of the constituents present in the oils. This study provides a scientific evidence for the folkloric utilization of Juniperus for wound healing activity.