Research on the antioxidant, wound healing, and anti-inflammatory activities and the phytochemical composition of maritime pine (Pinus pinaster Ait)

Journal of Ethnopharmacology Volume 211, 30 January 2018, Pages 235-246 Journal of Ethnopharmacology Author links open overlay panelİbrahimTümenabEsra KüpeliAkkolcHakkıTaştandIpekSüntarcMehmetKurtcab a Department of Forest Products Chemistry, Faculty of Forestry, Bartin University, 74100 Bartin, Turkey b Vocational School of Health Services, Bartin University, 74100 Bartin, Turkey c Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler, 06330 Ankara, Turkey d Department of Biology, Faculty of Science, Gazi University, Etiler 06330, Ankara, Turkey Received 21 March 2017, Revised 6 September 2017, Accepted 11 September 2017, Available online 14 September 2017. crossmark-logo https://doi.org/10.1016/j.jep.2017.09.009 Get rights and content Abstract Ethnopharmacological relevance Ethnobotanical investigations have shown that the Pinus species have been used against rheumatic pain and for wound healing in Turkish folk medicine. Material and methods In this study, phytochemical composition, antioxidant, anti-inflammatory, and wound healing activities of Maritime Pine (Pinus pinaster Ait.) that is collected in Turkey are investigated. Essential oil composition and the amount of extracts (lipophilic and hydrophilic) of maritime pine wood and fresh cone samples had been tested. Results The essential oil from cones of P. pinaster revealed the highest activities, whereas other parts of the plant did not display any appreciable wound healing, anti-inflammatory, or antioxidant effects. α-Pinene was the main constituent of the essential oil obtained from the cones of P. pinaster. Conclusion Experimental studies shown that P. pinaster's remarkable anti-inflammatory and wound healing activities support the traditional use of the plant, and suggest it could have a place in modern medicine. Graphical abstract fx1 Download high-res image (128KB)Download full-size image Keywords Essential oil Inflammation Martime pine Pinaceae Pinus pinaster Wound 1. Introduction Five Pinus species, Pinus brutia, P. nigra, P. sylvestris, P. pinea and P. halepensis, are found in Turkey; three of them, P. brutia, P. nigra and P. sylvestris, are used commercially. Earlier studies have shown that Pinus species growing in Turkey were principally used in refining the yield of turpentine manufacture. Pine oils are commonly used as fragrances in cosmetics, additive essences for food and beverages, and as intermediates in the synthesis of scented compounds. These compounds are likewise used in aromatherapy, as carminative, rubefacient, emmenagogue, and abortifacient agents. Because many studies contend that the causes for the chemical difference of pine oils come from the ecological, seasonal, genotypic, and conservational arguments used to assess them, these studies commence with the essential oils (EOs) of conifer species, and particularly those of Pinus (Bader et al., 2000; Velasquez et al., 2000; Barnola and Cedeno, 2000; Gomez da Silva et al., 2000; Koukos et al., 2000, 2001; Petrakis et al., 2001; Rezzi et al., 2001). Since Pinus pinaster Ait. is very fast growing and very resistant to drought, it has been cultivated as an exotic species in many places. In Portugal and France, plantations have existed for more than 600 years since being planted; they used in afforestation in South Africa, New Zealand, and Australia. The most prominent feature of P. pinaster is it can grow on poor soil that provides minimal nourishment (Saatçioğlu, 1969). It does not grow naturally in all regions of Turkey, but it grows in the coastal areas of Turkey, especially in the Black Sea, Marmara, and Aegean regions. In papers published to date, different extraction processes use various solvents that affect the composition of extracts and their biological activity. Bark extracts of P. pinaster that are used have a mixture of a large number of substances that are used to treat a wide range of dejenerative diseases through their antioxidative, anti-inflammatory, antitumor, antiatherogenic, antiviral, and antimicrobial properties. They have cardiovascular and cholesterol-lowering benefits and increase microcirculation via increasing capillary permeability (Gulati, 2005; Rohdewald, 2002). Furthermore, these extracts defend the nerve cells against beta-amyloids, or glutamate-induced toxicity, loss of histamine released from mast cells, and also inhibit pro-inflammatory cytokine actions (Blazso et al., 2004; Peng et al., 2002). Anti-inflammatory effects in asthma patients and reduction of attention-deficit illness and attention-deficit hyperactivity symptoms in children have been noted (Dvorakova et al., 2007; Lau et al., 2004). Essential oils from P. pinaster have generally been extracted from pines or needles and used as natural fragrance in cosmetics and flavoring additives in food and beverages (Maimoona et al., 2011). Studies on P. pinaster wood have determined the make-up as 44% cellulose, 26.6% lignin, 9.96% extractive material, and a density of 0.498 g/cm3 specific gravity (Pinto et al., 2004). The wood has abundant resin and is not very valuable in terms of its mechanical properties. The aim of the present study is to determine the volatile, lipophilic, and hydrophilic components of coastal pine wood, cones, and pine from coastal pine from different regions in Turkey, to examine the antioxidant effects of coastal pine, to determine its wound healing, anti-inflammatory and antioxidant properties, and its wound-healing mechanisms. 2. Material and methods 2.1. Plant material The needles, wood, and cones of Pinus pinaster Ait. were collected from the Karacaydere area in Bartin Province, Turkey. The samples were collected based on the conventional method for harvesting the plant at maturity and then stored at −24 °C pending the laboratory experiments (Tumen et al., 2010, 2012). Voucher specimens have been deposited in the Herbarium of the Faculty of Forestry, Bartin University, as BOF 516. The specimens were authenticated by Dr. Barbaros Yaman. 2.2. Hydrodistillation The EOs of the wood, cones, and needles of Pinus pinaster Ait. were obtained by hydrodistillation using a Clevenger apparatus (ILDAM CAM Ltd. Ankara-Turkey) on 1000 g each of the fresh samples. All oils were collected for 4–5 h. The samples were dried with anhydrous sodium sulphate in a sealed vial until used (Tumen and Reunanen, 2010; Tumen et al., 2010; Küpeli Akkol et al., 2015). 2.3. Preparation of the extracts Each sample (200 g) were extracted with hexane using a Soxhlet extractor for 10–12 h. After filtration of the hexane extract, the residue was extracted with acetone by the Soxhlet extractor for 10–12 h. After a final filtration, the organic phases were vaporized using a rotary evaporator (Buchi, Schweiz, Switzerland) at 40 °C in a vacuum to yield the crude extracts. 2.4. GC and GC-MS analysis A Shimadzu GCMS-QP2010 instrument equipped with a Teknokroma 5MS column (30 m × 0.25 mm, film thickness 0.25 µm) was used for the analysis of the EO samples. The carrier gas used was helium at a flow rate of 1.0 ml/min. The column oven temperature was started at from 60 °C and increased 3 °C/min at 5 min intervals until the temperature reached 280 °C. The temperatures of the split-injector and MS-transfer line were 260 °C and 280 °C, respectively. The MSD was operated in electron impact ionization mode at 70 eV electron energy. Samples were injected by splitting; the split ratio was 1:10 (Tumen et al., 2010). Compound identification was based on mass spectra, referring to NIST98 and WILEY275 mass spectral libraries. The measured retention index (RI) values of components were compared with data in the literature (Adams, 2007). The quantitative area-percent measurements were carried out based on peak-areas from the GC-MS data (Tumen and Reunanen, 2010). 2.5. Biological activity assessments 2.5.1. Animals Male Sprague-Dawley rats (160–180 g) and Swiss albino mice (25–30 g) procured from the Kobay animal breeding laboratory (Ankara, Turkey) were used in the experiments. The animals were left for 3 days to adapt to animal-room conditions and were fed a standard pellet diet and water ad libitum. The animals were kept in polysulfone cages at 21–24 °C, 40–45% wetness, and light-controlled (12 h light/12 h dark) conditions at the Laboratory Animals Breeding and Experimental Research Center, Gazi University, (Ankara, Turkey). For anti-inflammatory and wound healing activity tests, a minimum of six rats were used in each group. The present study was done with permission based on the universal guidelines on the animal experiments and biodiversity rights (Gazi University Ethical Council Project Number: G.U.ET- 08.037). 2.5.2. Preparation of test samples Ointments prepared from test samples using the Glycol sterilizer with Madecassol® pomate base, containing 1% extract / essential oil: 1,2-propylene glycol, using liquid paraffin (3: 6: 1). No product was applied to the negative control group; Madecassol® (Bayer, 00001199) (0.5 g) was used topically as the reference drug. In the anti-inflammatory activity assay model, test samples were suspended in 0.5% sodium carboxymethyl cellulose (CMC) solution, where necessary with an ultrasonic bath, and applied orally via stomach gavage specific to experimental animals. Control group animals received 0.5% CMC, which was used only for the preparation of test samples. Indomethacin (10 mg/kg) in 0.5% CMC was used as a reference drug. 2.5.3. In vivo wound healing activity models 2.5.3.1. Linear incision wound model In the linear incision wound model, the effect of the ointments applied during the experiment on the collagen production and wound tension enhancement effect was evaluated based on the method of Suguna et al. (2002). General anesthesia was performed using 0.01 cc Ketasol® (Richterpharma) injected intraperitoneally. Two 5 cm linear incision wounds were created with a bisturi 2 cm from the midline of the ridge sections. Three stitches were made at equal intervals using surgical silk thread. Formulations of ointments containing 500 mg of extract / essential oil were applied to external wounds once daily for 9 days. At the end of the 9th day, the stitches were removed. On the 10th day, the animals were sacrificed using ether anesthesia. The wound areas were cut with surgical scissors 2 cm from the wound edges. One of the wounds was reserved for histopathological examination. The tensile strength of the other wound was measured (Küpeli Akkol et al., 2011). The following formula was used in the linear incision wound model, while calculating the percent tensile force. BO: The tensile strength average of the group applied to the base ointment T: The tensile strength average of the group to which the test sample is applied 2.5.3.2. Circular excision wound model For determination of the size reduction in wound areas, that were measured daily in the circular excision wound model, the method by Sadaf et al. was applied to the mice by with some modifications (Sadaf et al., 2006). General anesthesia was performed with 0.01 cc Ketasol® (Richterpharma) injected intraperitoneally. A biopsy punch was used to create a circular excision wound with a diameter of 5 mm; ointment formulations containing 500 mg of extract / essential oil were applied externally for 12 days. The wound areas were photographed with a digital camera every day, and the reduction in wound area was calculated using the AutoCAD program (Küpeli Akkol et al., 2011). In the circular excision wound model, the following formula was used to calculate the percent contraction rates by which the decrease in wound areas were evaluated. BO: The average of the wound area of the group to which the base ointment is applied T: The average of the wound area of the group to which the test sample is applied 2.6. Histopathological examinations All skin tissues which contains normal ad experimental groups fixation made up in formaldehyde with %10. All tissues taken macroscope and detected by Thermo Scientific Excelsior (ES) machine. Later, all samples embedded parafine and all blocks were prepared by using Histocentre 2 machine. All sections which are 3.5 µm with marine glass was taken by parafine blocks by using Leica RM2255 microtome and than, the sections was stanined hematoxylin-eosin (HE) in Shandon Varistan machine. After examined under a light microscope (Nicon Eclipse Ci attached both polarizing attachment and Kameram Digital Image Analysis System). 2.7. L (-) Hydroxypyroline estimation A series of dilutions (0.5 μg/ml; 1 μg/ml; 1.5 μg/ml; 2 μg/ml and 2.5 μg/ml) were prepared from the stock solution by dissolving 5 mg of hydroxyproline in 50 ml of 0.001 N HCl for the measurement of the hydroxyproline standard. From each solution, 2 ml samples were taken in tubes. The tissues to be measured were weighed and placed in pyrex tubes, 5 ml of 6 N HCl was added over them, and the tubes were capped. The samples were hydrolyzed for 3 h at 130 °C. A few drops of 0.02% methyl red was added as an indicator. Next, 2.5 N NaOH was added until the pH of the solution was between pH 6 and 7 and it turned yellow. From both standard and test solutions, 2 ml samples were taken. Freshly prepared 1 ml chloramine T was added, and samples were kept at room temperature for 20 min; then 1 ml perchloric acid was added. Freshly prepared 1 ml 0.2 g/ml p-dimethylaminobenzaldehyde solution was added. It was shaken until the strata disappeared, held in a 60 °C water bath for 20 min and cooled in tap water for 5 min. The absorbance of the solutions was measured at 557 nm (Değim et al., 2002). 2.8. In vitro wound healing activity models 2.8.1. Hyaluronidase inhibitory activity assessment In our study, a method based on the measurement of the amount of N-acetylglucosamine released by sodium hyaluronate developed by Lee and Choi (1999) and Sahasrabudhe and Deodhar (2010) was used to determine anti-hyaluronidase activity. Accordingly, 50 μl of bovine hyaluronidase (7900 units / ml) was dissolved in 0.1 M acetate buffer (pH 3.6). This solution was dissolved at two different concentrations in 5% DMSO and embedded in 50 μl of the test sample solution. For the control group, 50 μl of 5% DMSO was used. Following the incubation at 37 °C for 20 min, 50 μl of calcium chloride (12.5 mM) was added to the mix and again incubated at 37 °C for 20 min. Then, 250 μl of sodium hyaluronate (1.2 mg/ml) was added and incubated at 37 °C for 40 min. The mixture was incubated for 3 min in boiling water bath after addition of 50 μl of 0.4 M NaOH and 100 μl of 0.2 M sodium borate. After addition of 1.5 ml of p-dimethylaminobenzaldehyde solution, the mixture was incubated at 37 °C for 20 min. The absorbance of the solution was measured at 585 nm using a Beckmann Due Spectrophotometer. 2.8.2. Collagenase inhibitory activity assessment Clostridium histolyticum collagenase (ChC) was dissolved in 50 mM Tris buffer (with 10 mM CaCl2 and 400 mM NaCl) as 0.8 units/ml. The substrate was prepared as 2 mM in the same buffer as N- [3- (2-furyl) acryloyl] -Leu-Gly-Pro-Ala (FALGPA). 25 μl of buffer, 25 μl test sample and 25 μl enzyme were added to each kettle. The samples were incubated for 15 min. Then, 50 μl substrate was added. The absorbance was measured at 340 nm. Three replicates were made for each sample (Barrantes and Guinea, 2003). 2.8.3. Elastase inhibitory activity assessment Test samples and human neutrophil elastase enzyme (HNE) (17 mU / ml) were incubated with 0.1 M Tris-HCl buffer (pH 7.5) for 5 min at 25 °C. Substrate N- (methoxysuccinyl) -ala-ara-pro-val 4-nitroanilide (MAAPVN) (500 μM) of the mixture HNE was added and left to incubate for 1 h at 37 °C. Subsequently, 1 mg/ml soybean trypsin inhibitor was added to the mixture. Absorption was measured at 405 nm due to formation of p-nitroaniline (Melzig et al., 2001). 2.9. Anti-inflammatory activity 2.9.1. Carrageenan-induced hind paw edema model One hour after oral administration of the test samples and indomethacin (10 mg/kg) used as a reference, to create edema, 25 μl of carrageenan suspension (Carrageenan, Sigma Co., No: C-1013) (50 mg was suspended in 2.5 ml of saline) was injected in the sub-plantar tissues of the right hind legs of the animals. In the sub-plantar tissue of the left hind legs of the animals, 25 μl of saline solution was injected for control purposes. The thickness of both feet was measured with a micrometric caliper (Ozaki Co., Tokyo, Japan) at 90-min intervals from the time of edema formation, and the swelling difference was recorded as edema amounts in the left and right hind legs. The results obtained from the test samples and control group animals were evaluated statistically (Kasahara et al., 1985; Küpeli, 2000). 2.9.2. Acetic-Acid-Induced Increase in Capillary Permeability After 30 min from the application of the flask test samples and the reference indomethacin, 0.1 ml of a 4% Evans Blue solution was injected into the marginal tail venous of each mouse. After 10 min, 0.4 ml 0.5% acetic acid solution was administered intraperitoneally. The animals were sacrificed by cervical dislocation 20 min later. The peritoneum was opened and the contents were rinsed with distilled water and transferred to 10 ml balloon jars containing 0.1 N NaOH and supplemented with distilled water to a volume of 10 ml. The absorbance of the dye was measured at 590 nm using a Beckman Due Spectrophotometer (Whittle, 1964; Yeşilada et al., 2007). The absorbance of the extruded dye was measured and the inhibition of inflammation was calculated using the following formula: Aa: The absorbance of the dye substance in the control group Ab: The absorbance of the dye in the group to which the test sample is applied 2.9.3. TPA-induced mouse-ear edema The administrations to the mice were 2.5 μg of TPA (12-O-tetradecanoylphorbol 13-acetate) dissolved in 20 μl of EtOH 70% (Yeşilada et al., 2007). An automatic pipette in 20 μl volume was used to apply the administrations from both anterior and posterior surfaces of the right ear. The same volume of solvent (EtOH 70%) as applied to the left ear (control) simultaneously with TPA. The reference drug was Indomethacin (10 mg/kg) in 0.5% CMC. The following procedures were adopted for the evaluation of the activity: 1. Following the induction of inflammation, at the 4th hour of the application, the thickness value of each ear was determined using a gauge calipers (Ozaki Co., Tokyo, Japan). The difference between the right and left ears due to TPA application was defined as the edema and consequently inhibition percentage was expressed as a reduction thickness with respect to the control group. 2. The animals were sacrificed four hours after the administration under deep ether anesthesia. Then, 6-mm diameter discs were removed from each ear, and their weights were measured. The difference in weight between the punches from right and left ears were accepted as the estimated swelling values and expressed as an increase in the ear thickness. 2.10. Antioxidant activity 2.10.1. DPPH photometric analysis Antioxidant activity (AA%) of each substance was analyzed by DPPH free radical assay. DPPH radical scavenging activity was measured based on the procedure of Brand-Williams et al. (1995). The samples were reacted with the stable DPPH radical in an ethanol solution. The reaction mixture consisted of adding 0.5 ml of sample, 3 ml of absolute ethanol, and 0.3 ml of 0.5 mM DPPH radical solution to ethanol. DPPH is reduced when it reacts with an antioxidant compound that can donate hydrogen. After 100 min from the initiation of the reaction, the changes in color (from deep violet to light yellow) were read [Absorbance (Abs)] at 517 nm using a UV–VIS spectrophotometer (DU 800; Beckman Coulter, Fullerton, CA, USA). The mixture of ethanol (3.3 ml) and sample (0.5 ml) were used as blanks. The control solution consisted of the mixture of ethanol (3.5 ml) and DPPH radical solution (0.3 ml). The scavenging activity percentage (AA%) was determined according to the method of Mensor et al. (2001): 2.10.2. ABTS radical scavenging assay ABTS assay was performed according to the procedure of Arnao et al. (2001) with some modifications. The stock solutions contained 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 14 h at room temperature in the dark. The dilution of the solution was prepared by mixing 1 ml ABTS solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.01 units at 734 nm using a spectrophotometer. In each assay, a fresh ABTS solution was used. Plant extracts (1 ml) were reacted with 1 ml of the ABTS solution. The absorbance value was then measured after 7 min at 734 nm using a spectrophotometer. The ABTS scavenging capacity of the extract was compared with that of BHT and ascorbic acid. The percentage inhibition was calculated as ABTS radical scavenging activity (%) = (Abscontrol -Abssample) / Abscontrol where Abscontrol is the absorbance of ABTS radical in methanol; Abssample is the absorbance of ABTS radical solution mixed with sample extract/standard. All the measurements were carried out in triplicate (n = 3) (Zheleva-Dimitrova et al., 2010). 2.10.3. Determination of reducing power The FRAP assay was carried out based on the Benzie and Strain (1996) procedure with some modifications. The stock solutions contained 300 mM acetate buffer (3.1 g C2H3NaO2 × 3H2O and 16 ml C2H4O2), pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3 × 6H2O solution. The freshly prepared working solution contained 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl3 × 6H2O solution. The solution was warmed to 37 °C before using. The extracts and essential oil (0.15 ml) reacted with 2.80 ml of the FRAP solution for 30 min in the dark. The absorbance value of the colored product (ferrous tripyridyltriazine complex) was measured at 593 nm. The standard curve was linear between 0.015 and 0.15 mM Trolox. The unit for the results were determined as mM TE/g dry mass. If the FRAP value measured was over the linear range of the standard curve, an additional dilution was carried out. All experiments were performed triplicate (n = 3). 2.10.4. Non-site-specific hydroxyl radical (•OH) scavenging activity assay Non-site-specific radical ·OH radical scavenging activity of melanoidins were measured based on the Gutteridge and Halliwell procedure (1988), with some minor modifications. The evaluation of the hydroxyl radical formation in a Fenton drive system in the absence or presence of melanoidins was carried out by both time-course to determine rate constants. The reaction mixture at a final volume of 1 ml contained 0.249 mM 2-deoxy-D-ribose, 1 mM H2O2, 100 μM FeCl3, 104 μM EDTA, 100 μM ascorbic acid, in 10 mM NaH2PO4–NaHPO4 buffer (pH 7.4). The solution of EDTA and FeCl3 was premixed one day before conducting the assay. All solutions were prepared before use in de-aerated water. Different concentrations of melanoidins or a reference antioxidant (trolox or chlorogenic acid) were tested in a final volume of 50 μl. The addition of H2O2 initiated the reaction. The reaction media were left to incubate in a water bath at 37 °C for up to 120 min. The reaction was stopped by adding 100 μl of 28% (w/v) cold TCA at the end of the incubation period. The chromogen development was maintained by heating the reaction vessel in a boiling bath for 30 min before addition of TBA solution (1% w/v in 0.05 M NaOH). Following the cooling step, chromogen development was measured spectrophotometrically at 532 nm against a blank containing phosphate buffer. The scavenger (melanoidin or reference antioxidant) rate constant (kS) was measured from the plot 1/A versus concentration of the scavenger (mg ml−1), where A is the absorbance at 532 nm. Gutteridge and Halliwell procedure (1988) proposed the reference value of 3.1 × 109 M−1 s−1 (0.0231 × 109 l g−1 s−1) for the second rate constant of the reaction of DR with ·OH (kDR); the same value is applied in this study. 3. Results and discussion The process begins with trauma and healing proceeds in three phases-inflammation, proliferation and maturation- that include cellular and biochemical events. The benefit of antioxidant compounds that exhibit free radical scavenging activity in wound healing is considerable. Phagocytosis, caused by monocytes, neutrophils, eosinophils, and macrophages in the inflammation phase of wound healing, leads to a situation known as oxidative burst, in which the consumption of oxygen dramatically increases. The resulting product consists of reactive oxygen and nitrogen derivatives. In addition to having the ability to kill microorganisms directly, these derivatives also act as triggering and messenger molecules. However, being produced at high rates increases damage in the inflamed area. Anti-inflammatory activity assays have been included in studies because it is a supportive observation for detecting wound healing activity, so that acute inflammation progresses with wound healing and continues in a controlled manner. The effect of antioxidant activity on wound healing is quite high. Compounds that have antioxidant effects inhibit lipid peroxidation and prevent cell damage and increase collagen fibrillary endurance. Assessment of the antioxidant effect of the plants tested is important in terms of providing information about wound healing effects (Getie et al., 2002; Shetty et al., 2008). Accordingly, in our study, free radical scavenging effect determination via DPPH and ABTS +, determination of reduction power and 2-deoxyribose degradation directed by non-specific hydroxyl radicals, were used to evaluate antioxidant activity (Kumarasamy et al., 2003). The in vivo wound healing potential of the n-hexane, acetone extracts, and essential oils prepared from some parts of P. pinaster was examined in the present study. As shown in Table 1, in the linear incision wound model, the highest wound tensile strength appeared in the volatile oil extracted from the cone. In a general evaluation of the extracts, it was observed that the best results were also in the cone samples. As seen in Tables 2, 3, the results obtained in the linear incision wound model were in line with those obtained in the circular excision wound model. At the end of the day, the volatile oil obtained from the cone produced 46.42% wound healing, and the hydroxyproline level was 34.1 μg/mg. Table 1. Effects of the test materials on linear incision wound model. Material Extract type Statistical mean ± SEM (Tensile strength %) Vehicle 8.42 ± 1.89 9.3 Negative Control 9.28 ± 2.01 – P. pinaster-Cone n-Hexane 10.90 ± 2.13 29.5* Acetone 9.90 ± 2.56 17.6 Essential oil 10.89 ± 1.87 29.3** P. pinaster-Needle n-Hexane 8.77 ± 2.19 4.2 Acetone 7.46 ± 2.38 – Essential oil 9.45 ± 2.22 12.2 P. pinaster-Wood n-Hexane 9.29 ± 2.31 10.3 Acetone 8.79 ± 2.43 4.4 Essential oil 9.56 ± 2.29 13.5 Madecassol® 13.48 ± 1.70 60.1*** SEM: Standard error of the mean. Percentage of tensile strength values: vehicle group was compared to negative control group; test materials and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001. Table 2. Effects of the test materials on circular excision wound model. Material Extract type Wound area ± SEM (% Contraction) 0 2 4 6 8 10 12 Vehicle 19.95 ± 2.36 18.05 ±1.92 (1.96) 16.91± 2.17 (0.99) 15.49 ± 2.06 (5.38) 12.43 ± 1.78 (8.47) 8.66± 1.30 (4.63) 4.31 ± 0.42 (7.71) Negative control 19.69 ± 2.45 18.41 ± 2.17 17.08 ± 1.79 16.37 ± 1.94 13.58 ± 1.86 9.08 ± 1.13 4.67 ± 0.75 P. pinaster-Cone n-Hexane 19.76± 2.54 18.17 ± 1.95 – 16.21 ± 2.04 (4.14) 14.01 ± 2.16 (9.55) 9.97 ± 1.36 (19.79) 6.92 ± 1.41 (20.09) 3.22 ± 0.29 (25.29) Acetone 19.92 ± 2.23 18.27 ± 2.14 – 16.96 ± 2.28 – 15.06 ± 2.18 (2.78) 10.23 ± 1.49 (17.69) 6.98 ± 1.53 (19.39) 3.63 ± 0.37 (15.78) Essential oil 19.49 ± 2.13 18.21 ± 1.99 – 15.32 ± 2.12 (9.40) 13.93 ± 2.03 (10.07) 9.80 ± 1.64 (21.16) 6.01 ± 1.20 (30.60)* 2.31 ± 0.11 (46.40)* P. pinaster-Needle n-Hexane 20.03 ± 2.30 19.01 ± 2.08 – 17.93 ± 2.11 – 15.16 ± 2.17 (2.13) 13.04 ± 1.94 – 7.88 ± 1.26 (9.01) 4.02 ± 0.61 (6.73) Acetone 19.92 ± 2.44 18.23 ± 2.09 – 17.30 ± 2.13 – 16.07 ± 2.29 – 12.99 ± 1.64 – 8.61 ± 1.29 (0.58) 4.94 ± 0.75 – Essential oil 19.78 ± 2.24 17.93 ± 1.78 (0.66) 16.98 ± 2.17 – 14.85 ± 2.15 (4.13) 10.94 ± 1.79 (11.99) 7.12 ± 1.50 (17.78) 3.42 ± 0.32 (20.65) P. pinaster-Wood n-Hexane 20.15 ± 2.32 18.36 ± 1.90 – 17.03 ± 1.97 – 14.99 ± 2.20 (3.23) 11.09 ± 1.66 (10.78) 7.08 ± 1.28 (18.24) 3.78 ± 0.39 (12.29) Acetone 19.99 ± 2.18 17.31 ± 1.79 (4.09) 16.38 ± 2.02 (3.13) 15.15 ± 2.16 (2.19) 11.75 ± 1.65 (5.47) 8.01 ± 1.72 (7.51) 3.97 ± 0.36 (7.88) Essential oil 19.37 ± 2.21 18.55 ± 1.86 – 16.14 ± 1.96 (4.55) 14.38 ± 2.02 (7.17) 10.82 ± 1.59 (12.95) 6.99 ± 1.21 (19.28) 3.20 ± 0.31 (25.75) Madecassol® 19.71 ± 2.10 15.10 ± 1.39 (16.34) 13.81 ± 1.78 (18.33) 10.23 ± 1.44 (33.96)* 6.08 ± 0.94 (51.09)** 1.36 ± 0.57 (84.30)*** 0.00±0.00 (100.00)*** SEM: Standard error of the mean. Percentage of tensile strength values: vehicle group was compared to negative control group; test materials and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001. Table 3. Effects of the test materials on hydroxyproline content. Material Extract type Hydroxyproline (µg/mg) ± SEM Vehicle 8.6 ± 1.45 Negative control 7.5 ± 1.19 P. pinaster-Cone n-Hexane 12.4 ± 1.31 Acetone 17.7 ± 1.58 Essential oil 34.1 ± 0.92** P. pinaster-Needle n-Hexane 6.3 ± 1.25 Acetone 8.8 ± 1.46 Essential oil 19.2 ± 1.18 P. pinaster-Wood n-Hexane 15.6 ± 1.83 Acetone 10.1 ± 1.52 Essential oil 14.3 ± 1.30 Madecassol® 47.6 ± 0.39*** *: p < 0,05; SEM: Standard error of the mean. ** : p < 0,01. *** : p < 0,001. In the histopathological analysis showed that the reference (Madecassol®) group which name is little damage skin have some little histopathological alternations, such as lower collagen fiber damaged in dermis. Moreover, normal epidermal layer, increasing collagen synthesis by fibroblast cells in dermis, mitotic division in the epitel tissue in epidermis was observed in this group (Fig. 1). Essential oil from cones of P. pinaster group which name is middle damage skin have also some histopathological alternations, such as epithelium degeneration, oil gland degeneration around hair follicles and vascularisation (Fig. 2). It is observed that increase in collagen synthesis in dermis tissue though some collagen fibers degeneration part of the dermis layer in the n-Hexane extract from cones of the plant group, (Fig. 3). The other tissues have more histopatholigal alternations on such as inflammatory cells in the dermis (Fig. 4; Acetone extract from cones group), increasing fat tissue and collagen fibers dejenerations in dermis (Fig. 5; Essential oil from needle group), much more polymorphonuclear and mononuclear inflammatory cells in dermis (Fig. 6; n-Hexane extract from needle group), epithelial dejenerations in epidermis and oil gland dejenerations arround the hair follicles (Fig. 7; Essential oil from wood group), seperation of epithelial tissue from the dermis layer and basal lamina destruction (Fig. 8; n-Hexane extract from needle group), intensive polymorphonuclear invasion in dermis layer basal lamina destruction (Fig. 9; Acetone extract from wood group), more epithelial tissue synthesis depending on more mitotic activity (Fig. 10; Acetone extract from needle group), collagen fibers damage in dermis layer, epithel layer dejeneration in epidermis (Fig. 11; Vehicle group), epithelial destruction, scab and ulcus formation in skin tissue (Fig. 12; Negative control group). Fig. 1 Download high-res image (328KB)Download full-size image Fig. 1. Histopathological view of wound healing and epidermal/dermal re-modeling in the reference ointment Madecassol® administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20X and the scale bars represent 120 µm for figure. mlc: Multi-layered ceratinous flat (Epidermis); c: Tight connective tissue with collagen fibers (Dermis); h: Hair follicle with oil gland (Dermis). Fig. 2 Download high-res image (340KB)Download full-size image Fig. 2. Histopathological view of wound healing and epidermal/dermal re-modeling in the essential oil from cones of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. mlc: Multi-layered ceratinous flat (Epidermis); v: Vascularisation and increasing vessel count (Dermis); g: Normal Oil Glands (Dermis). Fig. 3 Download high-res image (277KB)Download full-size image Fig. 3. Histopathological view of wound healing and epidermal/dermal re-modeling in the n-Hexane extract from cones of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 100× and the scale bars represent 120 µm for figure. m-mlc: Mitotic division in the multi-layered ceratinous flat (Epidermis); c: Increase in collagen fiber; f: fibroblast cell. Fig. 4 Download high-res image (281KB)Download full-size image Fig. 4. Histopathological view of wound healing and epidermal/dermal re-modeling in the acetone extract from cones of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. mlc: Multi-layered ceratinous flat (Epidermis); pmn: polymorphonuclear cells. Fig. 5 Download high-res image (403KB)Download full-size image Fig. 5. Histopathological view of wound healing and epidermal/dermal re-modeling in the essential oil from needle of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. Fat tissue increase and collagen fibers damage. Fig. 6 Download high-res image (313KB)Download full-size image Fig. 6. Histopathological view of wound healing and epidermal/dermal re-modeling in the n-Hexane extract from needle of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 40× and the scale bars represent 120 µm for figure. p: Microscopic papilla growth (Epidermis); pmn: polymorphonuclear cells; mnc: mononuclear cells (Dermis). Fig. 7 Download high-res image (301KB)Download full-size image Fig. 7. Histopathological view of wound healing and epidermal/dermal re-modeling in the essential oil from wood of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. h: Oil gland degeneration around hair follicle. Fig. 8 Download high-res image (400KB)Download full-size image Fig. 8. Histopathological view of wound healing and epidermal/dermal re-modeling in the n-Hexane extract from needle of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. s-mlc: Seperation multi-layered ceratinous flat from dermis and basal lamina dejeneration; pmn: polymorphonuclear cells (dermis); h: New oil glands occurrence arround the hair follicles. Fig. 9 Download high-res image (359KB)Download full-size image Fig. 9. Histopathological view of wound healing and epidermal/dermal re-modeling in the acetone extract from wood of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 40× and the scale bars represent 120 µm for figure. s-mlc: Seperation multi-layered ceratinous flat from dermis and basal lamina dejeneration; pmn: polymorphonuclear cells (dermis). Fig. 10 Download high-res image (255KB)Download full-size image Fig. 10. Histopathological view of wound healing and epidermal/dermal re-modeling in the acetone extract from needle of P. pinaster administered animals. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 100× and the scale bars represent 120 µm for figure. re: re-epithelization (epidermis). Fig. 11 Download high-res image (232KB)Download full-size image Fig. 11. Histopathological view of wound healing and epidermal/dermal re-modeling in the vehicle group. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 100× and the scale bars represent 120 µm for figure. e: Multi-layered ceratinous flat epithelium degeneration (Epidermis), cfd: Collagen fibers dejenaration; f: fibroblast cells. Fig. 12 Download high-res image (229KB)Download full-size image Fig. 12. Histopathological view of wound healing and epidermal/dermal re-modeling in the negative control group. Skin sections show the hematoxylin and eosin (HE) stained epidermis and dermis. The original magnification was 20× and the scale bars represent 120 µm for figure. e: Multi-layered ceratinous flat epithelium destruction; s: scab; u: ulcus. The extracellular matrix is composed of matrix metalloproteins in proteoglycan structures such as collagen, elastin, and fibronectin. In particular, collagen is a major structural protein that forms a supporting skeleton for the cells. Elastin provides the necessary flexibility for tissue, whereas hyaluronic acid ensures the continuity of structures by water retention. Hyaluronidase, collagenase, and elastase are metalloprotease enzymes that cause enzymatic degradation of extracellular matrix proteins. Under normal physiological conditions, these endogenous inhibitors maintain the healthy structure of the tissues. However, the shift of the equilibrium in favor of metalloproteases leads to uncontrolled destruction of connective tissue macromolecules, thus delaying wound healing and ultimately causing disturbances in the lungs and cardiovascular system. These enzymes play an important role in the pathophysiology of chronic wounds, which also has a role in the decomposition of TGF-β, PDGF, fibronectin, α−1 antiprotease, and α−2 macroglobin (Menke et al., 2007; Sahasrabudhe and Deodhar, 2010). Studies have shown that the level of metalloproteases in chronic wounds that do not heal is high. It is thought that keeping these enzymes at a minimal level may be necessary for proper wound healing (Edwards et al., 2004). Enzyme inhibition studies have shown that the volatile oil sample obtained from the cones of P. pinaster is more active than the other extracts, and that the highest enzyme inhibition activity is above the hyaluronidase inhibition (Table 4). Table 4. Collagenase, elastase, hyaluronidase enzyme inhibitory activities of test materials. Material Extract type Concentration (µg/ml) % Hyaluronidase Inhibition ± SEM % Collagenase Inhibition ± SEM % Elastase Inhibition ± SEM P. pinaster-Cone n-Hexane 100 18.27 ± 1.96 21.93 ± 1.78 19.40 ± 1.54 Acetone 100 20.13 ± 2.02 18.82 ± 1.15 21.18 ± 1.49 Essential oil 100 30.91 ± 0.86* 20.94 ± 1.51 20.07 ± 1.16 P. pinaster-Needle n-Hexane 100 16.13 ± 1.78 15.22 ± 1.25 17.16 ± 1.13 Acetone 100 15.78 ± 1.45 12.34 ± 1.38 15.08 ± 1.22 Essential oil 100 14.67 ± 1.49 17.11 ± 1.29 20.84 ± 1.30 P. pinaster-Wood n-Hexane 100 19.02 ± 1.62 16.39 ± 1.46 18.22 ± 2.03 Acetone 100 15.85 ± 1.92 20.34 ± 1.85 20.74 ± 1.98 Essential oil 100 21.34 ± 1.38 24.10 ± 1.52 17.43 ± 1.67 Tannic acid 100 76.23 ± 0.59*** – – Epigallocatechin gallate 100 – 40.54 ± 1.08** 72.14 ± 1.58*** SEM: Standard error of the mean. * p < 0.05. ** p < 0.01. *** p < 0.001. Arachidonic acid metabolites resulting from a long duration of acute inflammatory responses in the early phase of wound healing can cause tissue damage by endothelial injury. Therefore, the effects of extract and essential oils on the inflammatory response in the first phase of wound healing was investigated using the Whittle method, carrageenan-induced hind paw edema. Also used were TPA-induced ear edema models based on inhibition of acetic acid-induced capillary permeability increase, an acute anti-inflammatory activity evaluation method. None of the test samples showed an effect on carrageenan-induced hind paw edema (Table 5) and TPA-induced ear edema models (Table 6), whereas the volatile oil obtained from the cones showed an anti-inflammatory effect in the Whittle method with a 30.3% inhibition value at a 100 mg/kg dose (Table 7). Table 5. Preliminary anti-inflammatory activity assessment of test materials using carrageenan-induced paw edema model in mice. Material Extract type Dose (mg/kg) Swelling thickness (× 10−2mm) ± SEM (Inhibition %) 90 min 180 min 270 min 360 min Control 45.7 ± 3.2 48.5 ± 3.1 49.6 ± 3.5 52.7 ± 3.7 P. pinaster-Cone n-Hexane 100 46.1 ± 3.3 – 48.8 ± 2.9 – 50.1 ± 3.2 – 55.0 ± 3.1 – Acetone 100 42.1 ± 3.0 (7.9) 47.6 ± 3.8 (1.9) 50.5 ± 3.7 – 54.3 ± 4.1 – Essential oil 100 42.4 ± 3.1 (7.2) 45.8 ± 3.2 (5.6) 43.7 ± 3.5 (11.8) 45.3 ± 3.1 (14.0) P. pinaster-Needle n-Hexane 100 45.9 ± 3.3 – 50.6 ± 3.4 – 51.2 ± 3.6 – 53.7 ± 3.8 – Acetone 100 43.6 ± 2.7 (4.5) 46.2 ± 3.3 (4.7) 47.6 ± 3.4 (4.0) 54.8 ± 3.9 – Essential oil 100 40.9 ± 2.8 (10.5) 46.4 ± 3.2 (4.3) 48.1 ± 3.1 (3.0) 51.6 ± 3.5 (2.1) P. pinaster-Wood n-Hexane 100 48.5 ± 3.3 – 50.3 ± 3.7 – 52.7 ± 3.8 – 55.4 ± 3.6 – Acetone 100 44.0 ± 2.8 (3.7) 49.9 ± 3.9 – 51.7 ± 3.3 – 53.5 ± 3.1 – Essential oil 100 44.2 ± 3.3 (3.3) 44.7 ± 3.1 (7.8) 52.6 ± 3.7 – 54.2 ± 3.8 – Indomethacin 10 32.7 ± 2.6 (28.4)* 33.2 ± 2.9 (31.5)** 34.9 ± 2.5 (29.6)* 34.2 ± 2.1 (35.1)*** SEM: Standard error of the mean. * p < 0.05. ** p < 0,01. *** p < 0,001. Table 6. Effects of test materials on TPA-induced ear edema model. Material Extract type Dose (mg/ear) Swelling thickness (μm) ± SEM % Inhibition Weight edema (mg) ± SEM % Inhibition Control 194.3 ± 24.6 22.4 ± 3.7 P. pinaster-Cone n-Hexane 0.5 205.2 ± 21.7 – 25.7 ± 2.9 – Acetone 0.5 210.5 ±14.2 – 28.4 ± 3.1 – Essential oil 0.5 165.1 ± 17.8 15.0 19.9 ± 3.4 11.2 P. pinaster-Needle n-Hexane 0.5 198.9 ± 25.2 – 24.2 ± 2.8 – Acetone 0.5 206.1 ± 15.8 – 20.5 ± 2.5 8.5 Essential oil 0.5 214.7 ± 19.4 – 18.5 ± 2.6 17.4 P. pinaster-Wood n-Hexane 0.5 180.6 ± 19.2 7.1 29.3 ± 3.0 – Acetone 0.5 195.7± 20.1 – 20.3 ± 3.1 – Essential oil 0.5 209.5± 18.5 – 16.5 ± 2.2 – Indomethacin 0.5 100.6 ± 11.4 48.2*** 10.8 ± 1.9 51.8*** * p < 0.05; **: p < 0,01; SEM: Standard error of the mean. *** p < 0,001. Table 7. Effects of the extracts of test materials on increased vascular permeability induced by acetic acid in mice. Material Extract type Dose (mg/kg) Evans blue concentration (μg/ml) ± OSH Inhibition (%) Control 10.24 ± 1.97 P. pinaster-Cone n-Hexane 100 12.56 ± 1.86 – Acetone 100 10.92 ± 1.13 – Essential oil 100 7.13 ± 0.94 30.3** P. pinaster-Needle n-Hexane 100 10.75 ± 1.02 – Acetone 100 9.98 ± 0.75 2.6 Essential oil 100 8.74 ± 0.86 14.6 P. pinaster-Wood n-Hexane 100 9.59 ± 0.61 6.3 Acetone 100 9.25 ± 1.07 9.7 Essential oil 100 8.43 ± 0.57 17.7 Indomethacin 10 5.86 ± 0.38 42.8*** * p < 0.05; SEM: Standard error of the mean. ** p < 0.01. *** p < 0.001. Some in vitro studies have shown that P. pinaster bark extract has anti-inflammatory effects and inhibits the initiation of inflammation by preventing the release of pro-inflammatory mediators controlled in oxidative stress. The bark extract of P. pinaster inhibits the pro-inflammatory mediators in keratinocytes, leukocytes, and endothelial cells (Saliou et al., 2001; Bayeta and Lau, 2001; Peng et al., 2000). Also, an in vitro study has confirmed that bark extract of P. pinaster and its compounds inhibit the release of tissue-damaging matrix metalloproteinases enzymes elastase, collagenase, and gelatinase from inflammatory cells (Grimm et al., 2004). Similarly, later oral consumption of the extract, the enzymatic activity of COX-1 and COX-2, which are responsible for formation of prostaglandins, prostacyclin, and thromboxane in human serum samples of, was inhibited. That confirms that this enzyme can be responsible for relief from symptoms of pain and inflammation (Schäfer et al., 2006). One of the main pro-oxidant experiments, exposure to UV radiation, might possibly lead to the expression of many pro-inflammatory genes, together with TNF-α, IL-1 α, IL-1β, IL-6 and IL-8, (Gulati, 2005). Topical application of Pycnogenol®, a standardized extract of the bark of the French P. pinaster, might be used for important and dose-dependent safety from solar-simulated UV radiation-induced acute inflammation, photo-carcinogenesis, and immunosuppression by application after sunburn and day-to-day irradiation (Sime and Reeve, 2004). Saliou et al. demonstrated the protecting effect of Pycnogenol® against UV-light–induced skin damages (Saliou et al., 2001). It has been confirmed that the extract of P. pinaster inhibited the expression of pro-inflammatory cytokines and reduced the expression of mast cell-associated tryptase and stem cell factors (Matsumori et al., 2007). The anti-inflammatory effects of Pycnogenol® depend on the inhibition of NF-kappaB activation in lipopolysaccharide-stimulated monocytes, (Grimm et al., 2006). A double-blind, placebo-controlled study confirmed that Pycnogenol® suppressed pain and reduced the symptoms of knee osteoarthritis (Belcaro et al., 2007). Blazsó et al. reported that ingested in a controlled liquid diet, procyanidin-containing extracts from P. pinaster reduced the croton oil-induced ear edema in mice. Additionally, these extracts significantly inhibited the ultraviolet radiation-induced increased capillary permeability for the different polarity extracts applied topically on rats. In these experiments, normalization of capillary permeability was not related to the content of the extracts on sophisticated oligomeric procyanidins (Blazsó et al., 1997). Free radicals and oxidative reaction products are among the major causes of tissue damage. Large amounts of free radicals produced in the wound area cause connective tissue damage in the wound healing process. Various antioxidants are used to combat oxidative damage. In the present study, the best antioxidant activity was produced by acetone extracts, followed by hexane extracts and volatile oil samples (Table 8). Table 8. Antioxidant effects of test materials. Material Extract type DPPH IC50(µg/ml) ABTS IC50(µg/ml) Reducing capacity (%) OH-radical inhibition IC50(µg/ml) P. pinaster-Cone n-Hexane 192.54 115.82 25.13 184.26 Acetone 156.23 99.13 30.11 130.44 Essential oil 85.82 102.24 27.92 105.17 P. pinaster-Needle n-Hexane 203.28 170.92 16.28 158.26 Acetone 171.12 163.45 19.74 192.35 Essential oil 145.80 107.28 18.31 152.27 P. pinaster-Wood n-Hexane 138.56 156.20 20.03 165.24 Acetone 167.21 133.17 25.76 142.58 Essential oil 113.45 110.42 20.69 138.25 Ascorbic acid 6.25 10.04 60.12 4.13 In the Meullemiestre et al. study, P. pinaster sawdust waste essential oil was extracted using hydrodistillation, turbo-hydrodistillation, ultrasound-supported extraction hydrodistillation, microwave hydrodiffusion, and gravity and solvent-free microwave extraction; antioxidant activity, was assessed. The main components of the oil were identified as α-terpineol and β-caryophyllene. The highest antioxidant activity was found for both microwave techniques (Meullemiestre et al., 2014). Sonia et al. have produced fractions of different procyanidin composition from P. pinaster and the mixtures, without gallate esters, were active as free radical scavengers. Fractions obtained from pine bark were verified for antioxidant activity in hydrogen donation and electron transfer and inhibition of lipid peroxidation that was related with their galloylated complements. Though galloylation obviously decreases the free radical scavenging effectiveness in solution, it did not appear to play a major role in protection against lipid peroxidation in emulsion. Results showed that gallate esters seem to interfere with vital cell functions, thus gallate-free pine procyanidins might be the optimal and inoffensive chemopreventive agents for various applications in food and skin protection (Sonia Touriñ et al., 2005). Increased bacteria in the wound area responding to infection is another factor in delayed wound healing. Studies conducted on P. pinaster also suggest that the plant supports wound healing by showing antimicrobial action. The aqueous bark extract of P. pinaster was tested for antibacterial activity and its basic constituents against multidrug-resistant isolates of Acinetobacter baumannii by Ćurković-Perica. The high antibacterial activity was found in concentration of the extract lower than 200 mg/ml. Caffeic acid, catechin, epicatechin, gallic acid and vanillin, detected in the extract using high performance liquid chromatography, contributed to the antibacterial effect. Nevertheless, the antibacterial activity of the extract was higher than proanthocyanidins, which were present in a reasonably large amount in the extract, might have also funded to the activity of the extract (Ćurković-Perica et al., 2015). A similar study showed that the antibacterial potential of different polarity extracts of P. pinaster were tested on Staphylococcus aureus, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa. The results of phytochemical screening showed flavonoids, tannins, terpenes, sterols, coumarins, and saponins, which have already been characterized (Kahlouche-Riachi et al., 2015). The composition of the essential oil obtained from maritime pine wood, cones, and needles by hydrodistillation is shown in Table 9. Based on the experimental results, the main compounds were found to be α-pinene, β-pinene, β-myrcene, δ-3-carene, limonene, α-terpineol, junipene, trans-caryophyllene, α-amorphene, rimuene, cupressene, abietatriene, and abietadiene. Table 1 shows that α-pinene, which was one of the major compounds, existed most abundantly in the wood at 58.44%. Also in the wood, β-pinene (11.76%), limonene (4.09%), α-terpineol (5.32%), and junipene (6.10%) were present in their highest amounts. By comparison, α-pinene (32.57%), β-pinene (27.39%), δ-3-carene (7.32%), limonene (3.54%), junipene (9.45%), and trans-caryophyllene (1.49%) were found in cones. Major compounds in needles were identified as α-pinene (13.53%), β-pinene (9.81%), β-myrcene (4.14%), trans-caryophyllene (15.46%), α-amorphene (6.91%), rimuene (9.13%), cupressene (5.21%), abietatriene (8.36%), and abietadiene (10.91%). Table 9. Percent compositiona of essential oils of Pinus pinaster Ait. obtained by the hydrodistillation method. Nr RI Compoundsb Wood Cones Needles 1 774 Methyl benzene – 0.05 – 2 801 Hexanal 0.05 – – 3 918 Tricyclene 0.08 0.11 – 4 925 α-thujene – 0.10 – 5 933 α-pinene 58.44 32.57 13.53 6 944 Camphene 1.63 1.17 0.10 7 950 Verbenene 0.20 0.36 – 8 973 β-pinene 11.76 27.39 9.81 9 990 β-myrcene 1.75 3.20 4.14 10 1007 δ−3-carene – 7.32 0.37 11 1014 α-terpinene 0.02 0.06 – 12 1022 ρ-cymene 0.21 0.48 0.05 13 1025 β-phellandrene – – 0.85 14 1026 Limonene 4.09 3.54 – 15 1031 2-ethyl hexanol 0.06 – – 16 1048 β-ocimene – – 0.06 17 1057 γ-terpinene 0.02 0.08 0.02 18 1086 α-terpinolene 0.98 0.71 0.19 19 1089 Fencholenic aldehyde 0.04 0.05 – 20 1096 α-pinene oxide 0.02 – – 21 1099 Linalool – 0.10 0.09 22 1105 Pelargonaldehyde 0.07 – – 24 1111 Fenchyl alcohol 0.51 0.13 – 25 1125 α- campholene aldehyde 0.20 0.18 – 26 1136 Trans-pinocarveol 0.43 1.55 – 27 1140 Cis- verbanol – 0.03 – 28 1142 Camphor – 0.10 – 29 1144 Trans-verbenol 0.20 0.13 – 30 1146 Camphene hydrate 0.18 – – 31 1155 Isoborneol 0.02 – – 32 1159 Trans-pinocamphone 0.23 – – 33 1161 Pinocarvone 0.09 0.33 – 34 1164 Borneol 0.63 – – 35 1165 ρ-mentha-1,5-dien-8-ol – 0.89 – 36 1172 Cis-pinocamphone 0.40 – – 37 1176 Terpinen-4-ol 0.20 0.21 0.03 38 1184 ρ-Cymen-8-ol 0.16 0.27 – 39 1189 α-terpineol 5.32 1.20 0.31 40 1195 Myrtenol 0.64 1.24 – 41 1208 Verbenone 0.42 0.41 – 42 1218 Trans-carveol 0.11 0.12 – 43 1234 Methyl ether carvacrol – 0.02 – 44 1243 Carvone 0.06 0.05 – 45 1265 2-(E)-decenal 0.03 – – 46 1286 Bornyl acetate 0.02 0.25 – 47 1300 Tridecane – – 0.18 48 1350 α- longipinene 0.48 1.20 0.15 49 1364 Cis-undec-8-enal 0.04 – – 50 1368 Cyclosativene – 0.08 – 51 1371 Longicyclene 0.20 0.42 – 52 1372 α-ylangene – – 0.04 53 1376 α-copaene 0.09 0.19 0.61 54 1384 Geranyl acetate – 0.06 0.29 55 1390 Sativen 0.15 0.26 – 56 1392 β-elemene – – 0.05 57 1406 Junipene 6.10 9.45 0.64 58 1420 Trans-caryophyllene 1.64 1.49 15.46 59 1454 α-humulene 0.3 0.28 2.70 60 1457 β-farnesene 0.07 0.07 0.17 61 1474 Trans-cadina-1(6),4-diene – – 0.10 62 1477 Germacrene D – 0.01 0.40 63 1482 α-amorphene – 0.03 6.91 64 1487 Phenylethyl 2-methyl butyrate – – 0.33 65 1491 Phenethyl isovalerate – – 0.62 66 1501 α-muurolene 0.05 0.07 0.51 67 1515 γ-cadinene – 0.14 0.49 68 1525 δ-cadinene 0.06 – 1.52 69 1539 α-cadinene – – 0.05 70 1585 Caryophyllene oxide 0.08 0.04 0.47 71 1595 Diethyl phthalate 1.00 1.22 1.62 72 1599 Guaiol – – 0.56 73 1609 Geranyl isovalerate – – 0.14 74 1656 α-cadinol – – 0.35 75 1722 2 Cis-6-trans farnesol – – 0.21 76 1842 Farnesyl acetate – – 0.88 77 2000 Eicosane 0.06 – – 78 2019 Rimuen – – 9.13 79 2024 Cupressene – – 5.21 80 2064 Abietatriene – – 8.36 81 2087 Abietadien – – 10.81 82 2100 Heneicosane 0.11 – 0.07 83 2110 Sclareol – – 0.06 84 2153 Neoabietadien – – 0.87 85 2200 Docosane 0.09 – – 86 2239 Agathadiol – – 0.29 87 2300 Pentacosane 0.07 – – Total identified compounds 99.68 99.41 99.80 Total unidentified compounds 0.32 0.59 0.20 a peak area percents from total eluted components on GC-MS. b identified by MS and retention index (RI) data from literature (Adams, 2007). Earlier studies have revealed the essential oil composition of Pinus species. α-Pinene was originally thought to be the key constituent of the essential oils obtained from the cones of P. halepensis, P. nigra, and P. slyvestris. P. pinea has the higher amounts of limonene and β-pinene (Tumen et al., 2010). In our previous study on Coniferales, essential oils rich in limonene were found to have significant wound healing activity (Tumen et al., 2010). In earlier studies, α-pinene was described as a significant monoterpene with an anti-inflammatory effect (Rufino et al., 2014; Kim et al., 2015). Presence of α-pinene in P. pinaster might possibly contribute to the wound healing effect by providing an anti-inflammatory effect. Although an extensive delay in the inflammatory phase causes a delay in healing process, the anti-inflammatory effect is essential for wound healing activity. To shorten the healing period as well as to reduce pain and scarring, the anti-inflammatory effect is obligatory (Clark, 1991). Furthermore, monoterpene compounds were found to offer notable antioxidant activity (Emami et al., 2011). By way of its presence in many diseases, antioxidant activity similarly helps to support the wound-healing process. In this manner, the present study has contributed to scientific research and isolation studies on the effects of wound healing, anti-inflammatory and antioxidant effects of P. pinaster wood and cones; has shown the traditional use of these compounds to be supported by scientific evidence; and has provided the basis for the development of new and more effective compounds that can be offered for treatment. Conflict of interest The authors do not have any conflict of interest. Author contributions IT: GC-MS Analysis, Manuscript writing EKA: Project development, In vivo studies, Manuscript writing HK: Histopoathological Analysis, Manuscript writing IS:In vivo studies, Manuscript writing MK: GC-MS Analysis, Manuscript writing Acknowledgements A part of the study was presented orally at 5th International Conference on Science Culture and Sport, April 2016 (Kazakhstan) and this work was supported by the Scientific Research Projects Unit of Bartın University with the project number BAP-2012-1-40. References Adams, 2007 R.P. Adams Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry Allured Publishing, Carol Stream, IL, USA (2007) Arnao et al., 2001 M.B. Arnao, A. Cano, M. Acosta The hydrophilic and lipophilic contribution to total antioxidant activity Food Chem., 73 (2001), pp. 239-244 ArticlePDF (175KB) Bader et al., 2000 A. Bader, G. Flamini, P.L. Cioni, L. 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