Journal of Ethnopharmacology 134 (2011) 89–96 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm A novel wound healing ointment: A formulation of Hypericum perforatum oil and sage and oregano essential oils based on traditional Turkish knowledge Ipek Süntar a , Esra Küpeli Akkol a , Hikmet Keleş b , Alper Oktem c , Kemal Hüsnü Can Başer d , Erdem Yeşilada e,∗ a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03030 Afyonkarahisar, Turkey Langenhagen 49-33617 Bielefeld, Germany d Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey e Faculty of Pharmacy, Yeditepe University, Atasehir 34755, Istanbul, Turkey b c a r t i c l e i n f o Article history: Received 31 August 2010 Received in revised form 22 November 2010 Accepted 25 November 2010 Available online 3 December 2010 Keywords: Excision wound Hypericum perforatum Incision wound Medicinal plants Origanum majorana essential oil Origanum minutiflorum essential oil Olive oil Salvia triloba essential oil Tensiometer Wound healing a b s t r a c t Aim of the study: Hypericum perforatum L. (Hypericaceae), olive oil (Oleaceae), Origanum Tourn ex L. and Salvia L. species (Lamiaceae) are used against inflammatory disorders and for healing of skin wounds in traditional Turkish medicine. A new ointment formulation was developed to provide more efficient wound healing activity. The content of the formulation was as follows; olive oil extract of flowering aerial parts of Hypericum perforatum L., olive oil, an equivalent mixture of Origanum majorana L. and Origanum minutiflorum Schwrd. et Davis essential oils (Origani aetheroleum), Salvia triloba L. essential oil. The aim of the present study is to assess the wound healing potential of this new formulation by using in vivo and in vitro models as well as histopathological methods. Materials and methods: For the evaluation of wound healing potential of this formulation (HPP crème mit Rotöl ointment) in vivo wound healing experimental models were employed on rats and mice and the efficiency was comparatively assessed against a reference ointment Madecassol® . Tissue sections were also evaluated histopathologically. Furthermore, the wound healing activity of each component was also investigated individually to determine the improvement in the healing capacity of the formulation. Results: The ointments of HPP crème mit Rotöl and Hypericum perforatum L. demonstrated the highest activities on both wound models when compared to reference ointment Madecassol® , while the other ingredients did not show any remarkable wound healing effect. However, the efficacy of the formulation was remarkably higher than the Hypericum L. ointment alone which was also confirmed by histopathological evaluation. On the other hand, the formulation did not reduce elastase activity in vitro, but inhibited the collagenase activity. HPP crème mit Rotöl also exerted bactericidal and candicidal activities. Conclusions: The experimental studies revealed that HPP crème mit Rotöl formulation displays remarkable wound healing activity. To be acting on the different stages of wound healing process could be considered as a beneficial effect of the formulation for the treatment of wounds. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Wound healing progression comprised systematical processes of events which repair the damaged tissue partially or completely. This complex cascade of event starts from the moment of injury and continues for varying periods of time depending on the severity of wounding. The process can be broadly categorized into three stages; inflammatory phase (consisting of the establishment of homeostasis and inflammation); proliferative phase (consisting ∗ Corresponding author. Tel.: +90 216 5780000x3021; fax: +90 216 5780068. E-mail address: yesilada@yeditepe.edu.tr (E. Yeşilada). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.061 of granulation, contraction and epithelialization) and finally the remodeling phase which ultimately determines the strength and appearance of the healed tissue (Kondo, 2007). Wound healing process is promoted efficiently by the use of traditional remedies which are mainly based on plant sources. These remedies have been shown to affect one or more stages of the healing process. In this context, traditional medicines provide a vast source for the discovery of original drug leads. Among these traditional wound healing remedies the olive oil extract of flowering aerial part of Hypericum perforatum possesses an exceptional place in Turkish folk medicine. This oleate is a popular home-remedy for the rapid recovery of cuts and burns externally as well as peptic ulcers internally (Yesilada et al., 1993, 1995). 90 I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 Olive oil is well-known for its taste and health benefits particularly in the Mediterranean societies. Olive oil is also known to improve the wound healing process in traditional medicines. A mixture of olive oil and white wax is applied onto wounds twice a day in Northwest part of Anatolia (Yesilada et al., 1999). The oil is also pounded with salt and ground soap and applied onto cuts and wounds for rapid healing (Yesilada et al., 1993). In Bulgaria, Italy and Portugal, olive oil is also used to sooth and to heal burns (Leporatti and Ivancheva, 2003; Neves et al., 2009). Essential oils from several plants – such as lavender oil – have been used for the first aid treatment of wounds, abscess and burns (Cuttle et al., 2009; Steflictsch, 2009). In Turkish folk medicine thyme or oregano oils obtained from various “kekik” species (mainly Origanum sp., Thymus sp., Thymbra sp. or Satureija sp.) have been used as a remedy against wounds and burns (Dursun et al., 2003; Altiok et al., 2010). The members of Origanum genus are known and used as oregano in Anatolia (Turkish names; Yalancı kekik, kekik or keklik otu, etc.). Due to their rich carvacrol or thymol content, the essential oils of various oregano species have been shown to possess potent antibacterial, antifungal and anticandidal activities (Tabanca et al., 2001; Altiok et al., 2010). Turkey is a rich repository of sage species, i.e., over 90 species and eventually Salvia species have been used for the treatment of various complaints in Turkish folk medicine. Among these, various records were documented that Salvia species have been used to treat inflammatory skin diseases (Baytop, 1999), to stop bleeding or as antiseptic for wounds (Fujita et al., 1995; Honda et al., 1996; Sezik et al., 1997). In particular, the essential oil of Salvia triloba is popular in southwest Anatolia as “almıya yağı, acı elma yağı” to treat foot infections, especially caused by dermatophytes and fungi (Honda et al., 1996). Based on the traditional documents on Turkish folk medicine, a new wound healing formulation was framed to produce an efficient wound healing medicine. In a previous study, Hypericum perforatum oleate was found to possess potent wound healing activity (Peşin Süntar et al., 2010a) and therefore this oleate was selected as one of the main components of the formulation after diluted with equal amount of olive oil. In order to warrant antimicrobial efficiency, oregano oil (particularly against bacterial and Candida infections) and sage oil (against dermatophytes and fungal infections) were added. Aim of the present study was to evaluate the wound healing potential of this new formulation by using several in vivo and in vitro methods. For the assessment of the wound healing activity linear incision and circular excision wound models were employed. In vitro antimicrobial activity was assessed against bacteria and yeast strains. Moreover, the effects on elastase and collagenase enzymes were also evaluated to reveal the activity mechanism partially. 2. Materials and methods 2.1. Plant materials Olive oil, essential oils of Origanum majorana L., Origanum minutiflorum O. Schwarz et. P.H. Davis and Salvia triloba L. (syn. Salvia fruticosa Mill.) were purchased from producers (Doğtab Co., Antalya, Turkey). Reference essential oils are stored by the company. The compositions of the product and suppliers are as follows: essential oils of two Origanum species were mixed in equivalent ratio (1:1) and named “Origani aetheroleum” (12.5%, w/w), essential oil of Salvia triloba (7.5%, w/w), olive oil macerate of Hypericum perforatum L. (Johanniskrautöl; charge no. 124979, Primavera; 30:70 ratios) (10%, w/w), Shea Butter (Butyrospermum parkii) (Care Chemicals) (55%, w/w) and silica (5%, w/w) (Cab-O-Sil Co.). 2.2. GC–MS analysis The GC–MS analysis was carried out with an Agilent 5975 GC–MSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 mm film thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was kept at 60 ◦ C for 10 min and programmed to 220 ◦ C at a rate of 4 ◦ C/min, and kept constant at 220 ◦ C for 10 min and then programmed to 240 ◦ C at a rate of 1 ◦ C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 ◦ C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. 2.3. GC analysis The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 ◦ C. To obtain the same elution order with GC–MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The compositions were as follows: for Origanum aetheroleum; carvacrol (70.0%), linalool (9.7%), p-cymene (6.5%) and for Salvia triloba essential oil; 1,8-cineol (65.3%), beta-pinene (7.9%), camphor (3.7%), alpha-pinene (3.5%) and beta-tujen (3.5%). 2.4. Identification of components in essential oils Identification of the essential oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, Adams Library, MassFinder 3 Library) (McLafferty and Stauffer, 1989; Koenig et al., 2004) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data (Joulain and Koenig, 1998; ESO 2000) was used for the identification. 2.5. Biological activity tests 2.5.1. Animals Male, Sprague–Dawley rats (160–180 g) and Swiss albino mice (20–25 g) were purchased from the animal breeding laboratory of Saki Yenilli (Ankara, Turkey). 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 was used in each group. Throughout the experiments, animals were processed according to the suggested international ethical guidelines for the care of laboratory animals under the audit of Gazi University Commission of Animal Ethics. 2.5.2. 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 95% Shea Butter and 5% silica. Firstly, each test material was individually prepared as ointment by mixing in the following proportions with ointment base: (1) Hypericum perforatum ointment: 10% Hypericum perforatum olive oil macerate in ointment base; (2) olive oil ointment: 10% olive oil in ointment base; (3) Oregano ointment: 12.5% Origani aetheroleum in ointment base and (4) sage ointment: 7.5% Salviae trilobae aetheroleum in ointment base. On the other hand, these test materials were combined in ointment base in the same proportions and this formulation was named “HPP crème mit Rotöl”. 0.5 g of each test ointment was applied topically on the wounded site once a day throughout the experiment. I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 Animals of the vehicle group were treated with the ointment base only, whereas animals of the reference drug group were treated with 0.5 g of Madecassol® (Bayer, 00001199). Madecassol® contains 1% extract of Centella asiatica. 2.5.3. Wound healing activity 2.5.3.1. Linear incision wound model. All the animals were anaesthetized with 0.15 cm3 Ketalar® and the back hairs of the rats were shaved by using a shaving machine. Five cm long, two linearparavertebral incisions were made with a sterile surgical blade through the full thickness of the skin at the distance of 1.5 cm from the midline of each side of the vertebral column (Ehrlich and Hunt, 1968). The wounds were closed with three surgical interrupted sutures of 1 cm apart. The extracts, the reference drug (Madecassol® ) and the vehicle were topically applied once in a day throughout 9 days. Animals of the negative control group were not treated with any material. All the sutures were removed on the 9th post wound day. On day ten all the animals were killed under anesthesia. One linear-paravertebral incised skin was measured using tensiometer (Zwick/Roell Z0.5, Germany) for its tensile strength, the other incised skin was sent for histopathological examination (Suguna et al., 2002; Lodhi et al., 2006). Tensiometer measures the breaking strength in N (Newton), which is called tensile strength (Küpeli Akkol et al., 2009; Peşin Süntar et al., 2010b). Tensile strength (TS) of extract (%) = Tensile strength of reference (%) = Tensile strength of vehicle (%) = TSextract − TSvehicle × 100 TSvehicle TSreference − TSvehicle × 100 TSvehicle TSvehicle − TSnegative TSnegative × 100 2.5.3.2. Circular excision wound model. This model was used to monitor wound contraction and wound closure time. Each group of animals (six animals in each) was anaesthetized by 0.01 cm3 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 et al., 2002). The extracts, 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 healed skin of each group of mice for the histopathological examination (Sadaf et al., 2006). 2.5.4. Histopathology The cross-sectional full-thickness skin specimens from each group were collected at the end of the experiment to evaluate for the histopathological alterations. Samples were fixed in 10% buffered formalin, processed and blocked with paraffin and then sectioned into 5 ␮m sections and stained with hematoxylin and eosin (HE), Van Gieson (VG) and toluidine blue (TB) 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-epithelialization or ulcus in epidermis; fibroblast proliferation, mononuclear and/or polymorphonuclear cells, neovascularization and collagen depositions in dermis were analyzed 91 to score the epidermal or dermal re-modeling. Van Gieson stained sections were analyzed for collagen deposition and toluidine blue stained sections checked for metachromatic staining of mast cells. At the end of the examination, obtained results were combined and staged for wound healing phases as inflammation, proliferation, and re-modeling in all groups. 2.5.5. Inhibition of collagenase and elastase by HPP crème mit Rotöl 1 g of the HPP crème mit Rotöl was dissolved in 10 ml ethanol and n-hexane, respectively. These stock solutions at the concentrations of 0.1 g/ml were further diluted 1:1000, 1:500, 1:200, 1:100, 1:50, 1:20, 1:10, 1:5 und 1:2 in phosphate buffered saline (PBS) for testing elastase inhibition (n-hexane stock solution) and for repression of collagenase activity (ethanol stock solution) by using Enzyme Immunoassay (ELISA). Tests were performed in duplicate, and each sample was measured in four replicas. Elastase and collagenase enzyme inhibitory effects were tested in Klinik für Dermatologie und Dermatologische Allergologie, Universitatklinikum Jena, Germany (Authors thank C. Wiegand and U.-C. Hipler). 2.5.6. Antimicrobial activity Testing was based on the Standard methods of the German Society of Hygiene and Microbiology (DGHM) for testing of chemical disinfection processes (issue: 2001-09-01). Validation and control procedures were therefore carried out in accordance with this standard processing. Due to the screening character of this study, the test was only performed using Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10231) as test-organisms. For the quantitative suspension test, a suspension of test organisms was added to the test sample (an aliquot of the product HPP crème mit Rotöl was diluted with 1% Tween in hard water) and the mixture of interfering substance (0.3% albumin + 0.3% sheep erythrocytes). The mixture was maintained at 21 ± 1 ◦ C for the required contact times (60 and 360 min). At the end of the contact time, an aliquot of 1 ml was taken; the microbiocidal activity in this portion was immediately neutralized with a mixture (3% Tween 80 + 0.3% lechitine + 0.1% histidine + 0.5% sodium-thiosulfate). A 0.1 ml sample (per dilution step) of this suspension was spread on a nutrient plate using the pour-plate technique. Tests were performed at room temperature (21 ± 1 ◦ C), the test organisms were incubated at 36 ± 1 ◦ C (for Candida albicans 30 ± 1 ◦ C). The number of surviving test organisms in the test mixture was calculated for each sample and reduction was determined with respect to a corresponding test suspension without test product (water control/DGHM control Co1). The experimental conditions (control Co 1), the dilution–neutralization method and (control Co 2) and the non-toxicity of the neutralizer (control Co 3) were validated according to the DGHM standard methods. 2.5.7. Statistical analysis of the data The data on percentage 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. In vitro inhibition of collagenase and elastase values was expressed as means ± S.E.M. The statistical significance of differences between mean results for wound dressing samples compared to the controls was assessed using unpaired Student’s t-test (Microsoft® Excel). Results with p < 0.05 were considered statistically significant and expressed as follows: *p < 0.05, **p < 0.01 and ***p < 0.001. ± ± ± ± ± ± ± 20.86 14.18 12.14 7.49 3.07 1.17 0.00 2.36 1.81 (7.9) 1.24 (12.1) 1.76 (15.8) 1.95 (45.9)** 0.13 (74.8)*** 0.86 (91.8)*** ± ± ± ± ± ± ± Percentage of contraction values: Vehicle group was compared to negative control group; ointments were compared to vehicle group. S.E.M., standard error of the mean. * p < 0.05. ** p < 0.01. *** p < 0.001. 19.93 15.83 13.07 8.02 2.90 1.01 0.17 2.82 1.98 (5.6) 1.29 (6.7) 1.35 (14.5) 1.19 (19.0) 0.10 (20.9) 0.44 (26.1) ± ± ± ± ± ± ± 21.49 16.23 13.85 8.14 4.34 3.17 1.53 1.72 2.38 (4.1) 1.92 (4.3) 1.30 (8.2) 1.04 (7.1) 0.85 (17.7) 0.19 (25.1) ± ± ± ± ± ± ± 19.09 17.59 15.43 10.71 5.62 4.48 2.81 ± ± ± ± ± ± ± 2.43 2.90 1.13 1.96 1.68 0.55 0.04 19.07 15.98 13.26 8.14 3.34 1.55 0.35 ± ± ± ± ± ± ± 2.68 2.03 (7.0) 2.26 (10.8) 1.22 (14.5) 2.02 (37.7)* 0.93 (61.3)*** 0.31 (83.1)*** 19.55 16.73 13.94 8.76 4.73 3.37 1.58 ± ± ± ± ± ± ± 2.63 2.16 (2.7) 1.52 (6.3) 1.02 (7.9) 0.23 (11.8) 0.18 (15.9) 0.21 (23.7) 20.46 16.84 14.23 8.74 4.98 3.30 1.55 Madecassol® HPP crème mit Rotöl 2.26 2.61 (2.3) 2.32 (3.6) 1.58 (11.1) 1.79 (4.6) 1.22 (10.5) 0.14 (26.3) ± ± ± ± ± ± ± 21.45 17.19 14.87 9.52 5.36 4.01 2.07 The wound healing potential of a formulation “HPP crème mit Rotöl” which was set up on the basis of traditional medicine knowledge was investigated in the present study. In order to reveal combined interaction of the ingredients in the formulation, each material was also investigated individually for its wound healing potential. The experimental results were listed in Tables 1 and 2. Linear incision and circular excision wound models were employed on rats and mice for the wound healing activity assessment. As shown in Table 1, topical application of the HPP crème mit Rotöl and Hypericum perforatum ointment onto the incised wounds demonstrated the best wound tensile strengths by the highest value of 45.2% and 33.3% on day 10, respectively. However, the rest of the test ointments did not show any significant activity on tensile strength in this model. Wound contraction values in the healing progression are shown in Table 2. The HPP crème mit Rotöl and Hypericum perforatum ointment were found to have wound healing potential, while the vehicle and negative control groups and rest of the test ointments showed no statistically significant wound healing activity on excision wound model. The wound contractions were 45.9%, 74.8% and 91.8% for HPP crème mit Rotöl treated group, while 37.7%, 61.3% and 83.1% for Hypericum perforatum ointment treated group, on days 8, 10 and 12, which were comparable to reference drug Madecassol® (42.7%, 70.8% and 100%). Stages in wound healing processes (inflammation, proliferation, and remodeling) were observed and recorded within the experimental groups (Table 3). Wound healing processes delayed in the negative control and vehicle treated groups, while faster re-modeling in different degrees were observed with the test groups. In experimental groups, best re-modeling, particularly, reepithelialization was observed in the reference drug-treated group and with decreasing order in HPP crème mit Rotöl, Hypericum perforatum ointment, Salvia trilobae aetheroleum ointment, Origani aetheroleum ointment and olive oil ointment groups, respectively. Weak foreign body reaction, superfluous process in wound healing, characterized with a few foreign body giant cells, which generally localized in peripheral sides of some hair follicles were detected in linear incision model groups. Histopathological results are supported with the photographs of the tissues, which stained with HE, VG and TB (Fig. 1). The HPP-Creme mit Rotöl was not able to reduce elastase activity in vitro. However, significantly inhibited the collagenase enzyme activity in concentration dependent manner (Fig. 2). The IC50 was determined at a concentration of 7.4 ± 1.1 mg/ml (Fig. 3). 0 2 4 6 8 10 12 3. Results and discussion Salviae trilobae aetheroleum Percentage of tensile strength values: vehicle group was compared to negative control group; ointments were compared to vehicle group. S.E.M., standard error of the mean. ** p < 0.01. *** p < 0.001. Origani aetheroleum 1.38 1.21 1.06 2.14 2.43 2.27 0.97 1.63 Olive oil 7.7 – 33.3** 10.8 13.5 15.5 45.2*** 51.3*** ± ± ± ± ± ± ± ± Hypericum perforatum 14.93 13.86 19.90 16.54 16.94 17.25 21.68 22.59 Negative control Vehicle Negative control Hypericum perforatum Olive oil Origani aetheroleum Salvia trilobae aetheroleum HPP crème mit Rotöl Madecassol® Vehicle Tensile strength (%) Wound area ± S.E.M. (contraction %) Statistical mean ± S.E.M. (Newton) Day Material Table 2 Effects of the test ointments prepared from Hypericum perforatum, olive oil, Origani aetheroleum, Salviae trilobae aetheroleum, and the combined formulation HPP crème mit Rotöl on circular excision wound model. Table 1 Effects of the test ointments prepared from Hypericum perforatum, olive oil, Origani aetheroleum, Salviae trilobae aetheroleum, and the combined formulation HPP crème mit Rotöl on linear incision wound model. 2.11 1.95 (17.5) 1.16 (18.4) 1.57 (21.3) 0.70 (42.7)** 0.88 (70.8)*** 0.00 (100)*** I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 92 I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 93 Table 3 Histopathological evaluation of the wound sections treated with test ointments on the wound healing processes and healing phases.a Groups Vehicle Negative control Hypericum perforatum Olive oil Origani aetheroleum Salviae trilobae aetheroleum HPP crème mit Rotöl Madecassol® Wound healing processes Healing phases S U RE FP CD MNC PMN NV I P R ++/+++ ++/+++ ++ ++/+++ ++ ++ ++ +/++ +++ +++ −/+ ++/+++ ++/+++ ++ −/+ −/+ − − ++ −/+ + + ++ ++/+++ ++/+++ ++/+++ ++ ++ ++ ++ +/++ +/++ ++ ++ ++ ++ ++ ++ +/++ +/++ ++/+++ ++/+++ +/++ ++ ++ +/++ +/++ + ++/+++ ++/+++ + ++ ++ +/++ + + ++/+++ +++ ++ ++/+++ ++ ++ ++ ++ +++ +++ +/++ ++/+++ ++ ++ + + ++ ++ ++ ++ ++ ++ + + − − ++ −/+ + + ++ ++/+++ S, scab; U, ulcus; RE, re-epithelialization; FP, fibroblast proliferation; CD, collagen depositions; MNC, mononuclear cells; PMN, polymorphonuclear cells; NV, neovascularization; I, inflammation phase; P, proliferation phase; R, re-modeling phase. a HE and VG stained sections were scored as mild (+), moderate (++) and severe (+++) for epidermal and/or dermal re-modeling. Collagen is the main structural protein component of connective tissue. Chvapil et al. (1986) demonstrated that collagen sponge enhances the connective tissue formation and increases the vascularization of the repaired tissue. Therefore it is known that collagen effectively increases the process of healing. Motta et al. (1983) revealed that collagen also enhances suitably modulated collagen lysis and favours a more orderly fibroblast proliferation, probably by acting as a recruiting factor for these cells and taking part in their metabolism directly. Following injury, basal keratinocytes move from the basement membrane and interact with new connective tissue proteins in the dermis and wound bed. Contact with native type I collagen that Fig. 1. Histopathological view of wound healing and epidermal/dermal re-modeling in the vehicle, negative control, Hypericum perforatum, olive oil, Origani aetheroleum, Salviae trilobae aetheroleum ointments, HPP crème mit Rotöl and Madecassol® treated animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis in A, and the dermis stained with Masson’s trichrome (MT) and toluidine blue (TB) in B and C, respectively. The original magnification was x100 and the scale bars represent 75 ␮m for figures in A, and the original magnification was 400× and the scale bars represent 25 ␮m for both B and C. Data is representative of 6 animals per group. (1) Vehicle group, 10 day old wound tissue treated with only vehicle, (2) negative control group, 10 day old wound tissue, untreated group, (3) Hypericum perforatum group, 10 day old wound tissue treated with Hypericum perforatum ointment (10%), (4) olive oil group, 10 day old wound tissue treated with olive oil ointment (10%), (5) Origani aetheroleum group, 10 day old wound tissue treated with Origani aetheroleum ointment (12.5%), (6) Salviae trilobae aetheroleum group, 10 day old wound tissue treated with Salviae trilobae aetheroleum ointment (7.5%), (7) HPP crème mit Rotöl group, 10 day old wound tissue treated with HPP crème mit Rotöl, (8) reference drug group, 10 day old wound tissue treated with Madecassol® . Arrows pointing events during wound healing; s, scab; re, re-epithelialization; f, fibroblast; c, collagen; mnc, mononuclear cells; pmn, polymorphonuclear cells; m, mast cells; nv, neovascularization. I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 94 Fig. 1. Continued. induces expression of collagenase. Collagenase is a member of the matrix metalloproteinase family of enzymes, which are capable of degrading triple-helical fibrillar collagens into fragments, thereby altering its structure and, hence, the affinities to which cells bind it. Thus, collagenase serves a beneficial role in wound healing during re-epithelialization by facilitating the movement of keratinocytes over the collagen rich dermis (Pilcher et al., 1998). In fact, controlled proteolysis is needed. In a previous study, it was revealed that, tissue inhibitor of metalloproteinase was not detected in the epidermis of any chronic wound sample while it was expressed in keratinocytes bordering normally healing wounds. Tissue inhibitor of metalloproteinase expression near the basement membrane in acute, but not in chronic wounds suggests that the balance between metalloproteinases and their inhibitors may be altered in poorly healing wounds. Thus, the collagenase inhibitory effect of HPP crème mit Rotöl might contribute a better healing in especially chronic wounds. According to the “Catalogue of requirements for including chemical disinfectants processes” in the DGHM list of disinfec120 120 collagenase activity % collagenase activity [%] 100 100 * ** *** 80 60 *** 40 20 60 40 20 *** 0 *** 0 80 control 0,1 0,2 0,5 1 2 5 10 20 50 weighted sample of HPP-Creme [mg/mL] Fig. 2. HPP-Creme mit Rotöl had a significant inhibitory effect on collagenase activity. 0,1 1 10 weighted sample of HPP-Creme mit Rotöl mg/mL Fig. 3. A half maximal inhibition of collagenase activity (IC50 ) under test conditions was achieved at 7.4 ± 1.1 mg/ml concentration. I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 95 Table 4 Results of quantitative suspension test of HPP crème mit Rotöl against Pseudomonas aeruginosa. Concentration Dilution 48.85% 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−3 0.1 ml × 10−4 Time: 60 min cfu/plate 44.5% WSH (co1) 0 0 0 0 0 0 0 0 >300 >300 >300 31 Time: 360 min log10 (x) RF 0.00 ≥6.49 0.00 ≥6.49 6.49 cfu/plate 0 0 0 0 0 0 0 0 >300 >300 >300 46 log10 (x) RF 0.00 ≥6.66 0.00 ≥6.66 6.66 WSH (co1), water control; x, average value; RF, reduction factor. Table 5 Results of quantitative suspension test of HPP crème mit Rotöl against Staphylococcus aureus. Concentration Dilution 48.85% 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−3 0.1 ml × 10−4 Time: 60 min cfu/plate 44.5% WSH (co1) 12 2 0 0 18 1 0 0 >300 >300 >300 131 Time: 360 min log10 (x) RF 1.08 6.04 1.26 5.86 7.12 cfu/plate 0 0 0 0 0 0 0 0 >300 >300 >300 124 log10 (x) RF 0.00 ≥7.09 0.00 ≥7.09 7.09 WSH (co1), water control; x, avarage value; RF, reduction factor. tants (issue: 2002-02-04) the provided batch of the product HPP crème mit Rotöl, applied at a product concentration of 44.5%, possesses bactericidal activity (log RF≥5) in 60 min, and candicidal activity (log RF≥4) in 360 min at 21 ◦ C under dirty conditions (0.3% albumin + 0.3% sheep erythrocytes) against reference strains of Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, respectively (Tables 4–6). Antimicrobial agents also provide a better and rapid healing by forming a barrier against microbial contamination. The essential oils in HPP crème mit Rotöl formulation might possibly promote the healing process at the initial steps of wound healing by acting as antimicrobial components. In our previous study, the wound healing and anti-inflammatory activities of the Hypericum perforatum L. extracts and the fractions were investigated. The olive oil extract of Hypericum perforatum exerted a significant wound healing effect on excision and incision wound models. For the determination of the active wound healing ingredient(s), the ethanolic extract of the plant was then subjected to successive bioassay-guided fractionation processes. The ethyl acetate extract and its fractions composed of flavonoids [hyperoside, isoquercitrin, rutin and (−)-epicatechin] (Fr. A) and naphthoquinones (hypericins) (Fr. B) showed potent wound healing activity. Moreover, these extracts and fractions exerted a dose-dependent anti-inflammatory activity. These results suggest that anti-inflammatory activity of the active fractions might have a contributory role in the wound healing effect of the plant (Peşin Süntar et al., 2010a). Results of the present study have clearly demonstrated that HPP crème mit Rotöl, which was set up on the basis of traditional Table 6 Results of quantitative suspension test of HPP crème mit Rotöl against Candida albicans. Concentration Dilution Time: 60 min cfu/plate 48.85% 44.5% WSH (co1) 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 1 ml × 100 0.1 ml × 100 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−1 0.1 ml × 10−2 0.1 ml × 10−3 0.1 ml × 10−4 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 125 n.d. WSH (co1), water control; x, avarage value; RF, reduction factor; n.d., not done. Time: 360 min log10 (x) >5.48 >5.48 6.10 RF <0.62 <0.62 cfu/plate 6 0 0 0 24 3 0 0 >300 >300 92 n.d. log10 (x) RF 0.78 5.19 1.38 4.58 5.96 96 I. Süntar et al. / Journal of Ethnopharmacology 134 (2011) 89–96 medicine knowledge, possesses a potent wound healing activity. Principally, the potent wound healing effect of the formulation might be attributed to Hypericum perforatum oleate in the formulation, however, the degree of potency was much higher in the developed formulation than Hypericum perforatum ointment itself. In fact, it was higher than that of the reference drug Madecassol® in the 8th and 10th days on excision wound model. This might be due to the combined interaction of essential oil components (sage and oregano) in the formulation. 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