Saturday, 21 October 2017

A new clerodane furano diterpene glycoside from Tinospora cordifolia triggers autophagy and apoptosis in HCT-116 colon cancer cells

Journal of Ethnopharmacology Volume 211, 30 January 2018, Pages 295–310 Cover image Neha Sharmaa, b, Ashok Kumarc, f, P.R. Sharmac, f, Arem Qayumc, f, Shashank K. Singhc, f, Prabhu Dutta, Satya Pauld, Vivek Guptae, M.K. Vermab, N.K. Sattia, , , , R. Vishwakarmaa a Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India b Analytical Chemistry Division (Instrumentation), CSIR‐ Indian Institute of Integrative Medicine, Jammu 180001, India c Cancer Pharmacology Division, CSIR‐ Indian Institute of Integrative Medicine, Jammu 180001, India d Department of Chemistry, University of Jammu, Jammu 180006, India e Post- Graduate Department of Physics, University of Jammu, Jammu 180006, India f AcSIR: Academy of Scientific and Innovative Research, Jammu- Campus, Jammu, India Received 5 July 2017, Revised 15 September 2017, Accepted 24 September 2017, Available online 27 September 2017 Show less https://doi.org/10.1016/j.jep.2017.09.034 Get rights and content Abstract Ethnopharmacological relevance Tinospora cordifolia is a miraculous ayurvedic herb used in the treatment of innumerable diseases such as diabetes, gonorrhea, secondary syphilis, anaemia, rheumatoid arthritis, dermatological diseases, cancer, gout, jaundice, asthma, leprosy, in the treatment of bone fractures, liver & intestinal disorders, purifies the blood, gives new life to the whole body; (rejuvenating herb) and many more. Recent studies have revealed the anticancer potential of this plant but not much work has been done on the anticancer chemical constituents actually responsible for its amazing anticancer effects. This prompted us to investigate this plant further for new potent anticancer molecules. Aim of the study The present study was designed to isolate and identify new promising anticancer candidates from the aqueous alcoholic extract of T. cordifolia using bioassay-guided fractionation. Materials and methods The structures of the isolated compounds were determined on the basis of spectroscopic data interpretation and that of new potent anticancer molecule, TC-2 was confirmed by a single-crystal X-ray crystallographic analysis of its corresponding acetate. The in vitro anti-cancer activity of TC-2 was evaluated by SRB assay and the autophagic activity was investigated by immunofluorescence microscopy. Annexin-V FITC and PI dual staining was applied for the detection of apoptosis. The studies on Mitochondrial Membrane potential and ROS (Reactive oxygen species) production were also done. Results Bioassay guided fractionation and purification of the aqueous alcoholic stem extract of Tinospora cordifolia led to the isolation of a new clerodane furano diterpene glycoside (TC-2) along with five known compounds i.e. cordifolioside A (β-D-Glucopyranoside,4-(3-hydroxy-1-propenyl)- 2,6-dimethoxyphenyl 3-O-D-apio-β-D-furanosyl) (TC-1), β-Sitosterol(TC-3), 2β,3β:15,16-Diepoxy- 4α, 6β-dihydroxy-13(16),14-clerodadiene-17,12:18,1-diolide (TC-4), ecdysterone(TC-5) and tinosporoside(TC-6). TC-2 emerged as a potential candidate for the treatment of colon cancer. Conclusion The overall study on the bioassay guided isolation of T.cordifolia identified and isolated a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. We have reported a promising future candidate for treating colon cancer. Graphical abstract fx1 Figure options Abbreviations DAPI, 4′-6-Diamidino-2-phenylindole; MMP, Mitochondrial membrane potential; ROS, Reactive oxygen species; PTPC, permeability transition pore complex; PCD, Programmed cell death; MDC, Monodansylcadaverine; UV visspec, Ultra violet-visible spectrophotometer; IR, Infra-red; FTIR, Fourier Transform Infra-Red; LCMS, Liquid Chromatography-Mass Spectrometry; MeOH, Methanol; EtOAc, Ethyl acetate; CAN, Acetonitrile; TLC, Thin Layer Chromatography; CHCl3, Chloroform; DEPT, Distortionless enhancement by polarization transfer; HMBC, Heteronuclear Multiple Bond Correlation; NOESY, Nuclear Overhauser Effect SpectroscopY; TCA, Trichloroacetic acid; SRB, Sulforhodamine B; OD, Optical density; PBS, Phosphate buffer saline; DCFH-DA, dichlorodihydro-fluorescein diacetate) Keywords Tinospora cordifolia; DAPI (4′-6-Diamidino-2-phenylindole); Apoptosis; HCT116; MMP potential; ROS 1. Introduction Research on herbal drugs has stimulated multifold during the last few decades with special emphasis being on investigating novel candidates to combat existing life threatening diseases. Tinospora cordifolia is a miraculous ayurvedic herb used in “Rasayanas” for boosting immunity and body's strife against various infecting organisms ( Tirtha, 2005). It is globally distributed in Bangladesh, South Asia, Indonesia, Myanmar, Thailand, Sri Lanka, Pakistan, Nepal, in certain parts of China and throughout India ascending up to an altitude of 1200 m above sea level (Khare, 2007). Apart from more than 100 vernacular names such as Guduchi, Amrit (Sanskrit) and Abb-e-Hyat (Urdu) meaning water of life, it is also known as Giloya, a traditional term that alludes to the heavenly panacea which gives new life to the whole body, increases the human life span and keeps them perpetually young ( Sinha et al., 2004; Singh et al., 2003a ; Singh et al., 2003b). Giloya is used for curing innumerable health conditions such as diabetes, cancer, gonorrhea, secondary syphilis, irregular fever, skin diseases, anaemia, gout, rheumatoid arthritis, general and cardiac debility, cough, vomiting, chronic diarrhoea, asthma, in the treatment of bone fractures, liver & intestinal disorders, quite effective against lethal conditions like Swine Flu, Dengue and Malaria. It also purifies the blood and gives new life to the whole body; hence called as the rejuvenating herb (Singh, 1983; Shah, 1984; Bhatt, 1987; Shah et al., 1983; Kritikar, 1975; Zhao et al., 1991; Mhaiskar et al., 1980). The plant is used in numerous ayurvedic preparations such as Amritashtakachurana, Dashmoolarishta, Sanjivanivati, Kanta-kariavleha, GuduchyadichurnaChyavanprasha, Guduchisaatva, BrihatGuduchitaila, Guduchitaila, Stanyashodhanakashayachurana, Punehnimbachurna, Guduchighrita, Amrita guggulu, etc (Sinha et al., 2004). Traditionally, as per Indian Ayurveda it has been used in cancer treatment (Balachandran and Govinrajan, 2005; Williamson, 2002); local application of guduchi extracts was done to cure various tumors (Dash and Kashyap, 1987). In northern part of Gujarat in India, tribal people of Khedbrahma region consume the powdered root and stem/bark of this plant with milk for treating cancer (Bhatt, 1987). Juice and powder of the stem possesses anticancer effect in case of throat cancer ( Chauhan, 1995a ; Chauhan, 1995b) and breast cancer ( Chauhan, 1995a ; Chauhan, 1995b). Recently guduchi extracts have shown interesting results in experimental In vivo metastasis ( Leyon and Kuttan, 2004), inhibiting skin carcinogenesis (Chaudhary et al., 2008), antineoplastic effects in In vivo studies on Ehrlich ascites carcinoma ( Jagetia and Rao, 2006b) and cytotoxic effects in HeLa cells (Jagetia and Rao, 2006a). It's a well-known authentic immunomodulatory herb and is involved in helping the immune system in understanding the nature of cancer cells and ways to control them. It increases the immunity by promoting stem cell proliferation, elevating the levels of immunoglobulins and those of white blood cells. Toxicity study results have proven it to be non-toxic and with negligible side effects (Prakashananda, 1992). Guduchi powder/ Guduchi satva or Giloy Satva has exhibited promising effects on cancer which are either similar or even better than doxorubicin, a renowned chemotherapy drug which is used in the treatment of almost all cancers but is also associated with a number of side effects including cardiac myopathy in the later years after the treatment. 58.8% of reduction in the tumour volume by guduchi powder is quite comparable to cyclophosphamide which is another widely used anticancer drug (Sohini and Bhatt, 1996; Kapil and Sharma, 1997; Mathew and Kuttan, 1999). These remarkable properties of this herb can be utilized in cases where immunosuppression is mediated by cancer and hence could be a promising future drug of choice against various cancers, one such being colon cancer which is the third most common cause of cancer-related death and it is expected that in 2017, more than 90,000 new cases are to occur in the United States (U.S.) Medicinal value of any herb is directly related to the nature of the chemical constituents present in it. Extensive evidence based research done on the chemical constituents responsible for the various medicinal properties of this plant has revealed that myriad of biologically active phytoconstituents like alkaloids, tannins, phenolics, cardiac glycosides, flavanoids, sesquiterpenoids, saponins and steroids isolated from different parts of the plant have been attributed to the various pharmacological activities of Tinosporacordifolia ( Sudha et al., 2011; Rout, 2006; Ahmed et al., 2006; Kiem et al., 2010; Fukuda et al., 1993; Singh et al., 2005 ; Meghna et al., 2008; Jeychandran et al., 2003; Kumar et al., 2016a ; Kumar et al., 2016b; Grover et al., 2000; Premnath et al., 2010; Singh et al., 2005; Singh et al., 2003a ; Singh et al., 2003b; Kumar et al., 1981; Khuda et al., 1964; Sarma et al., 2009; Chi et al., 1994;Bisset et al., 1983;Khan et al., 1989). On the basis of the recent researches done, many articles have been cited to state the fact that the said plant has been used for treating various cancers. But as far as its anticancer chemical constituents are concerned, not much work has been done on this plant. Since it's a proven fact that the major breakthroughs in cancer drug discovery have been owed either to the natural products or the natural product scaffolds; vincristine, vinblastine, podophyllotoxin, taxol are few to be quoted. These discoveries- inspired by traditional and folk medicine clearly gives an indication that natural products are the future source for lead structures, and these will be used as templates for the development of more promising novel compounds with improved biological properties. Therefore, it is essential to use the available traditional knowledge and investigate the active plant extracts for the isolation of new, less toxic and highly efficacious molecules. Thus, in the course of our ongoing research and in a bid to arrive at more potential anticancer molecules, we investigated the bioactive aqueous fraction of Tinospora cordifolia via bioassay guided isolation and landed up with a new clerodane furano diterpenoid (TC-2) with potent activity against HCT-116 cells along with six known molecules. Apoptosis – an organized cell death causes specific morphological changes and cell death. However, exaggerated apoptosis and the impairment in the cell cycle can result in aberrant ability of multiplication in cancer cells. Thus, induction of apoptosis can be a treatment for cancer by reducing accumulation of cancer cells. Autophagy, the programmed cell death type-II also plays a part in tumour suppression ultimately self- destruction of cells in the body. Thus, the initiation of autophagic cell death via potent molecule can be an important approach for cancer prevention (Cotter, 2009). To ascertain whether the isolated new molecule, TC-2 induced cytotoxicity in HCT-116 cells could be the result of cell apoptosis, we examined the induction effect of this molecule on cell apoptosis of colon cancer cells, HCT-116 by DAPI staining, annexin-V/PI dual staining and on mitochondrial membrane potential studies. To investigate whether TC-2 also caused autophagy in HCT-116 cells, it was examined by monodansylcadaverine (MDC) staining by fluorescence microscopic studies and also detection of LC3b was carried out by the immunofluorescence confocal microscopic studies. 2. Materials and methods 2.1. General experimental procedures, chemical and biochemicals Buchi, B-542 was used for recording the melting points. UV spectras were measured with Shimadzu UV-2600 UV–Vis Spectrophotometer and the IR spectras were obtained with Perkin Elmer FT-IR, Spectrum Two. 1H and 13C NMR data was recorded on Bruker-Advance DPX FT NMR 500 and 400 MHz instruments. LCESIMS data was acquired on Agilent UHD-6540 LCMS/MS (HRMS) system. Silica gel of #60–120 and #100–200 mesh size from Merck Germany, SupelcoDiaion HP-20SS, U.SA, Sephadex and LH-20 from Sigma Aldrich were used for chromatographic separation, isolation and purification of the compounds. 2.2. Plant material Fresh stems of Tinospora cordifolia were procured from Indian Institute of Integrative Medicine (IIIM), Jammu, India. A voucher specimen (RJM/0010) was identified by the botany division and deposited in the herbarium of the Institute. 2.3. Cell culture, growth conditions and treatment conditions Human lung carcinoma cell line (A549), Prostate (PC-3), SF-269 (CNS), MDA-MB-435 (Melanoma), HCT-116 (Colon) and Breast (MCF-7) were procured from NCI: National Cancer Institute, USA. Culturing of the cancer cells was done in RPMI-1640 medium consisting of 10% FCS, penicillin (100 units/ml) and streptomycin (100 μg/ml). Standard culture conditions were employed. The cell cultures were grown in CO2 incubator (New Brunswick, Galaxy 170 R, Eppendorf)) at37 °C with 98% humidity and 5% CO2 gas environment. 2.4. Extraction and isolation Fresh stems (1.5 kg) of Tinospora cordifolia were crushed, soaked in 4.5 L of ethyl acetate: water in the ratio of 1:1 and mechanically stirred for 2 h. The mixture was double filtered through muslin cloth and distilled on rota vapour at 55 °C. 59.2 g of the aqueous portion (coded as TCE) obtained was freeze dried. Freeze dried extract was divided into two parts, 20 g of which was used for column chromatography on Diaion HP-20 (400 g, column size - 90 cm × 6 cm). The column was eluted successively with 100% water (9 × 500 ml), 25% MeOH/ H2O (6 × 500 ml), 50% MeOH/ H2O (4 × 500 ml), 100% MeOH (15 × 500 ml) and 50% EtOAc/MeOH (3 × 500 ml). A total of 47 fractions, each of 500 ml volume were collected. Fractions 11–14 yielded 125 mg of residue with one major spot on the T.L.C. On repeated column chromatography of the pooled 11–14 fractions on Sephadex LH-20, 14 mg of white amorphous compound was obtained which was identified as cordifolioside A (coded as TC-1) on the basis of spectral data obtained with that reported in the literature. 560 mg of fraction 20–22 (coded as TCFR) was taken up for further purification for flash chromatography on RP-18 silica gel (33.5 g). Column was eluted with water (3 × 100 ml), 10% ACN in water (2 × 100 ml), 20% ACN in water (2 × 100 ml), 30% ACN in water (2 × 100 ml), 40% ACN in water (2 × 100 ml), 50% ACN in water (2 ×100 ml), 100% ACN (2 × 100 ml), 100% MeOH (3 × 100 ml) and finally with isopropyl alcohol. On the basis of similar TLC pattern obtained, fraction no. 8-12 (TCFR-3) from a total of 30 fractions were pooled and dried. 121 mg of the residue so obtained was re-chromatographed on RP-18 silica gel. Elution with 100% ACN and repeated crystallization in methanol led to the isolation of 76 mg of creamish colored pure compound, coded as TC-2. Remaining 39.2 g form the freeze-dried extract dissolved in water was extracted with butanol and further used for isolation. Butanol extract was chromatographed over silica gel column (100–200 mesh, column size – 90 cm × 15 cm) and eluted with a combination of 1–100% MeOH/CHCl3 with 112 fractions. Initial fractions eluted in chloroform yielded octacosanol, TC-3 (10 mg) followed by the isolation of β-sitosterol, TC-4 (25 mg). Fractions 33–39 (eluted with 10% MeOH/CHCl3) and 48–56 (eluted with 15% MeOH/CHCl3), crystallized in methanol and acetone afforded clerodadiene-diolide, TC-5 (15 mg) and Ecdysterone, TC-6 (10 mg). 82–91 of the total 112 fractions on repeated column chromatography yielded Tinosporaside, TC-7 (32 mg) and crystallized in acetone respectively. The identification of the compound structures isolated from the column were determined on the basis of the spectroscopic data with that reported in the literature. All the collected fractions were subjected to preliminary screening for their anticancer activity in order to identify the bioactive fractions for the isolation of potent anticancer molecules. 2.5. Structure elucidation 2.5.1. Spectral analysis TC-2 was obtained as creamish white amorphous solid. Analysis of the 13 C NMR and DEPT-135 spectra revealed 28 resonances along with a pseudo molecular ion peak [M-H3O]+ at m/z 605. Its IR spectrum showed absorptions corresponding to hydroxyl and furan moieties at 3384 cm−1 and 771.62 cm-1. Signals at 1729.05 cm-1 indicated the presence of lactone which was confirmed by the presence of resonance at 172.062 ppm in the 13 C NMR spectrum. The UV absorption at 209 nm supported the presence of an α, β-unsaturated ketone group. 1H-NMR of TC-2 displayed signals at δ 7.42 (t, 1H, J = 3.3 Hz), δ 7.43 (d, 1H, J = 0.6 Hz) and δ 6.40 (dd, 1 H, J = 0.9 Hz) suggested the presence of protons of the β-substituted furan moiety commonly reported in clerodanes isolated from different Tinospora species. One angular methyl group at C-9 was observed as three proton singlet at 1.09 respectively. The signals at δ 3.40 (m) were assigned to the C-12 proton bearing the β-substituted furan moiety. The signals at the aliphatic region δ 1.94 (m) and δ 1.74 (m) were attributed to the C-3 methylene protons (Fig. 1, 2 and 3; Table 1). Fig. 1 Fig. 1. 1H NMR of compound TC-2. Figure options Fig. 2 Fig. 2. 13C NMR of compound TC-2. Figure options Fig. 3 Fig. 3. DEPT-135 spectral images of compound TC-2. Figure options Table 1. NMR Data for TC-2 in CDCl3 (δ in ppm, J in Hz in parentheses), bs- broad signal. Position δC δH HMBC Correlation observed in 1 COSY NOESY 1 76.31 3.41 m – 3.4(C-12) 2 70.47 3.53 m – 3 30.49 1.94b, 1.74a m – I)2.4(C-11),2.10(3′,4′)-II)2.2(C-7) 4 75.51 3.31 m – 3.57(C-2) 5 34.58 Q – – 6 25.17 1.74a,1.447b m – 7 21.39 2.36a,1.73b m – 8 51.10 2.31 m – CH3-9 9 35.14 Q – – 10 39.06 1.67 m CH3-9, 5′, 4′, 2′ 11 43.53 2.42a,1.73b d at 2.42(3.25) & m at 1.73 – 12 73.28 3.40 m – 3.54(C-1), 4.27(C-1′) 13 125.38 Q – – 14 108.16 6.40 d,1H, (0.9) C-13,C-15,C-16 15 139.22 7.44 d,1H, (0.6) C-13,C-14,C-16 6.4(C-14) 16 143.901 7.43 in HSQC t,1H,(3.3) C-13,C-15,C-14 17 172.062 Q – – 1′ 99.91 4.26 d,H, (7.7 Hz) – 3.38(C-4) 2′ 68.61 5.46 dd,1H,(3.15) and (2.95) – 3′ 71.02 5.61 dd, 1H, (3.10) and (3.20) – 1.76(C-3 or C-6) 4′ 73.37 4.96 dd, 1H, (3.25) and (3.25) – 5′ 73.79 3.84 m, bs – 6′ 62.30 3.83b,3.78a m, bs – 9-CH3 21.53 1.09 s,3H C-11,C-8,C-2 1.65 (C-10) H8, H10 2′-OCOCH3 21.06 1.94 s, 3H – 3′-OCOCH3 20.94 2.13 s, 3H – 2.42 (C-11), 4′-OCOCH3 21.147 2.36 s, 3H – 2′-OCOCH3 172.06 Q – – 3′-OCOCH3 170.60 Q – – 4′-OCOCH3 169.57 Q – – Table options The HMBC correlations observed from H-14 (δ 6.4) to C-13 (δ 125.38), C-15 (δ 139.22) & C-16(δ 143.90); H-15 (δ 7.44) to C-13 (δ 125.38), C-14 (δ 108.16) & C-16(δ 143.90); H-16 (δ 7.43) to C-13 (δ 125.38), C-14 (δ 108.16) & C-15 (δ 139.22) and CH3-9 (δ 1.09) methyl protons to C-2 (δ 70.47), C-8 (δ 51.10) and C-11 (δ 43.53) augmented the above argument (Fig. 4a and Fig. 4a ; Fig. 4bb). Fig. 4a Fig. 4a. Major HMBC correlations between protons of C-14, 15 & 16 with Carbons 13, 14,15 & 16 in compound TC-2. Figure options Fig. 4b Fig. 4b. Major HMBC correlations (blue arrows) for compound TC-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Figure options Of the three hydroxyl resonances, the one at δ 3.10 was allocated to the carbon at position 1. The other deshielded hydroxyl peak at δ 3.53 was assigned to the C-2 based on HMBC correlations. The relative configuration of TC-2 was deduced based on NOESY experiment. A cross peak was found from H-8 (δ 2.31) to CH3-9 (δ 1.09); H-10 (δ 1.67) to CH3-9 (δ 1.09), H-5′ or 6′ (δ 3.83), H-2′ (δ 5.46), H-4′(δ 4.96); CH3-9 (δ 1.09) to H-8 (δ 2.31) and H-10 (δ 1.67) indicated similar configurations between different groups which was confirmed by the X-ray crystallography. Thus TC-2 was assigned the following structure (Fig. 7). HPLC chromatogram and Mass spectrum of TC-2 has been represented in Fig. 5 and 6. The other compounds from TC-1, TC-3 to TC-7 were identified on the basis of comparing their spectral data with the ones already reported in the literature (Supplementary data: S1-S20). Fig. 5 Fig. 5. HPLC chromatogram of TC-2. Figure options Fig. 6 Fig. 6. Mass spectra of TC-2. Figure options Fig. 7 Fig. 7. Chemical structure of TC-2. Figure options 2.6. Acetylation of TC-2 TC-2 was reacted with acetic anhydride in catalytic amount of pyridine to obtain acetate crystals of the isolated molecule for X-ray structure determination and confirmation. 10 mg of TC-2 was dissolved in 1 ml of pyridine and 2 ml of acetic anhydride was added to the solution. The reaction mixture was heated on a steam bath for 2 h under dry conditions. Usual work up followed by crystallization yielded triacetate, TC-2acetate (Fig. 8). In 1H NMR (500 Hz, CDCL3), signal shifts from δ 3.41, 3.53, 3.84 and 3.778 to δ and increase in number of 9 protons at δ 2.1 confirmed the formation of three acetate groups (Fig. 9). The triacetate so formed was confirmed by X-ray crystallography. Fig. 8 Fig. 8. Molecular structure of TC-2 acetate with atomic labelling. Figure options Fig. 9 Fig. 9. 1H NMR of compound TC-2acetate. Figure options 2.7. X-ray crystal studies of TC-2 2.7.1. Crystal structure determination and refinement X-ray intensity data of 7288 reflections (of which 4004 unique) were collected on X′calibur CCD area-detector diffractometer equipped with graphite monochromated MoKa radiation (λ = 0.71073 Å). The dimensions of the TC-2 acetate crystal used were 0.30 × 0.20 × 0.20 mm. The intensities were measured by w scan mode for q ranges 3.55–26.00. 1149 reflections were treated as observed (I > 2σ(I)). Data were corrected for Lorentz, polarization and absorption factors. The structure was solved by direct methods using SHELXS97 (Sheldrick, 2008). All non-hydrogen atoms of the molecule presented the best E-map locations. Full-matrix least-squares refinement experiment was performed using SHELXL97 (Sheldrick, 2008). The final refinement cycles converged to an R = 0.0614 and wR(F2) = 0.1204 for the observed data. Residual electron densities ranged from −0.175 < Δρ < 0.409 eÅ-3. Atomic scattering factors were referred from International X-ray Crystallography tables (1992, Vol. C, Tables 4.2.6.8 and 6.1.1.4). The crystallographic data has been summarized in Table 2. The molecular structure of the TC-2 acetate with atomic labelling is shown in Fig. 8. Table 2. Crystallographic data of TC-2 acetate. CCDC no. 1545869 contains the crystallographic data. The data can be obtained free of cost via www.ccdc.cam.ac.uk/data_request/cif by e-mailing data request @ ccdc.cam.ac.uk, or by contacting The Cambridge Crystallography Data Centre, 12 Union Road, Cambridge, CB2 IEZ, UK. Fax: +44(0) 1223-336033. Computer programs: SHELXL97 (Sheldrick, 1997). CCDC deposition No. 1545869 Chemical formula C36H46O17 Mr 750.73 Temperature (K) 293 a, b, c (Å) 12.4621 (15), 7.4751 (6), 21.069 (3) α, β, γ (deg.) 90, 105.277 (13), 90 V (Å3) 1893.3 (4) Z 2 Radiation type Mo Kα µ (mm−1) 0.11 No. of measured, independent and observed [I > 2σ(I)] reflections 7288, 4004, 2173 Rint 0.049 (sin θ/λ)max (Å−1) 0.617 Refinement R[F2 > 2σ(F2)], wR(F2), S 0.061, 0.147, 0.99 No. of reflections 4004 No. of parameters 486 No. of restraints 1 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρmax, Δρmin (e Å−3) 0.41, −0.18 Absolute structure Flack H D (1983), Acta Crystal. A39, 876–881 Absolute structure parameter 10 (10) Table options 2.7.2. Crystal packing The packing of the molecules in the unit cell is shown in Fig. 10. From the figure, it is evident that the molecules related by twofold screw are packed in layers. The stability of the crystal structure was due to the the presence of O-H···O and C-H···O intra- and intermolecular hydrogen bonds. Details of O–H···O and C–H···O hydrogen bonds are given in Table 3 and those details on the bond lengths and bond angles of the non-hydrogen bonds has been provided in the Supplementary section; S21-S23. Fig. 10 Fig. 10. The packing arrangement of molecules in TC-2 acetate, viewed down the b-axis. Figure options Table 3. Geometry of intermolecular hydrogen bonds in TC-2 acetate. D-H…A D-H (Å) D…A(Å) H…A(Å) D-H…A,(deg.) C8-H18-O3 0.98 3.495(7) 2.59 153 C33-H39B-O12 0.96 3.436(8) 2.50 165 C31-H42A…O7 0.96 3.136(11) 2.57 118 Symmetry codes: (i) -x+1, y+1/2,-z (ii) –x+z, y-1/2, -z+1 (iii) x+1, y-1, z. Table options An ORTEP view of TC-2 with atomic labelling and the unit cell packing view of the TC-2 molecules down the b-axis is shown in Fig. 10 (Farrugia, 2012) and Fig. 11. The molecular geometry was analyzed and calculated using the WinGX (Farrugia, 1997), PLATON (Spek, 2009) and PARST (Nardelli, 1995) softwares. Fig. 11 Fig. 11. ORTEP view of molecule TC-2 acetate with displacement ellipsoids drawn at 40%. H atoms are shown as small spheres of arbitrary radii. Figure options 2.8. In vitro cytotoxicity assay Sulforhodamine B (SRB) testing was performed, in which cell suspension of cell density 7500−15,000 cells/100 μL was seeded. Cultures were incubated with 1 µM, 10 µM, 30 µM and 50 µM concentrations of test material in complete growth medium (100 μL) after 24 h of incubation. Paclitaxel, mitomycin, 5-fu and doxorubicin were used as positive controls. After further 48hr incubation, cells were fixed with ice-cold TCA for 1hr at 4 °C. Plates were washed atleast five times with distilled water (D.W) and kept for air drying. Further 100 μL of 0.4% sulphorhodamine-B (SRB) solution added into each well of the dried plates was allowed for 30 min staining at room temperature. SRB solution was detached by quickly washing of the plates with 1% v/v acetic acid in order to remove the excess unbound dye. The bound SRB dye was solubilised by adding 100 μL of 10 mM unbuffered Tris Base (pH 10.5) to each well and with continuous shaking for 5 min on a shaker platform to solublize the dye completely, and finally the reading was taken at 540 nm on microplate reader (Thermo Scientific). IC50 was determined by plotting OD against concentration from Graph PAD Prism version 5. 2.9. Nuclear morphology studies by DAPI staining The presence of apoptotic cells was ascertained by staining human colon cancer HCT-116 cells with DAPI. Seeded HCT-116 cells (2 × 105/ml/well) in 60 mm culture dishes. After 24 h, cells were incubated with various concentrations of TC-2 and paclitaxel (positive control) for 24 h. Media was collected and cells were rinsed with PBS. Trypsinization was done in order to detach the cells followed by their back addition to the conditioned media to ascertain the incorporation of the floating and poorly attached cells in the analysis. Air dried smears of HCT-116 cells were fixed in methanol at −20 °C for 20 min, air dried and stained with DAPI at 1 µg/ml in PBS at room temperature for 20 min in the dark and the slides were placed in glycerol-PBS (1:1) and examined in an inverted fluorescence microscope (Olympus, 1X81) (Rello et al., 2005) 2.10. Detection of apoptosis by Annexin V-FITC and PI Annexin V-FITC and propidium iodide (PI) dual staining technique is generally employed to detect the early and late stages of apoptosis (PCD type 1). For evaluating apoptosis, HCT-116 cells were splitted and made confluent in six-well plates (2 × 105 cells) and treated with TC-2 for 24 h and paclitaxel (1 μM) was used as a positive control. 24 h after treatment, cells were collected, washed with PBS and suspension was made in binding buffer. Following that, staining of the cells was done with Annexin V/FITC and PI for 15 min in dark and studied by laser scanning confocal microscope using appropriate lasers (Olympus Fluoview FV 1000) (Zhang et al., 2013; Bai et al., 2015; Dai et al., 2008; Munafo et al., 2001; Acharya et al., 2009;Kumar et al., 2016a ; Kumar et al., 2016b) 2.11. Detection of intracellular reactive oxygen species (ROS) accumulation Intracellular ROS levels were examined by fluorescence microscopy after staining with DCFH-DA (dichloro dihydro-fluorescein diacetate). HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h and examined under fluorescence microscope using 40X lens (Olympus, 1X81). H2O2 (0.05%) was used as positive control (Bai et al., 2015; Kumar et al., 2016a ; Kumar et al., 2016b). 2.12. Loss of mitochondrial membrane potential (MMP) Loss in mitochondrial membrane potential (∆ψm) as a result of mitochondrial disturbance was examined using confocal microscopy after staining with Rhodamine 123 (Rh123). Human colon cancer (HCT-116) cells (2 × 105/ml/well) were seeded in six well plate and treated for 24 h with different concentrations of TC-2 and paclitaxel (positive control). Cells untreated and treated with test materials were trypsinized and washed twice with PBS. Thereafter cell pellets were then suspended in fresh medium (2 ml) containing Rh123 (1.0 μM) and incubated at 37 °C for 20 min with gentle shaking. Following that, the cells were centrifuged, collected and washed twice with PBS, then examined by laser scanning confocal microscope (Olympus Fluoview FV1000) (Dai et al., 2008; Kumar et al., 2016a ; Kumar et al., 2016b) 2.13. Analysis of autophagy by monodansylcadaverine (MDC) staining MDC, Monodansylcadaverine is a prescribed marker for labelling of autophagic vacuoles. HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h. MDC labelling of the autophagic vacuoles was done with by incubating the cells for 1 h with 0.05 mM MDC in PBS at 37 °C. After incubation, cells were washed thrice with PBS and forthwith examined by fluorescence microscope using 40X lens (Munafo et al., 2001;Kumar et al., 2016a ; Kumar et al., 2016b). 2.14. Immunofluorescence microscopic studies for detection of cytochrome C and LC3B HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h. MDC labelling of the autophagic vacuoles was done with by incubating the cells for 1 h with 0.05 mM MDC in PBS at 37 °C. After incubation, cells were washed thrice with PBS and forthwith examined by fluorescence microscope using 40X lens (Munafo et al., 2001;Kumar et al., 2016a ; Kumar et al., 2016b). HCT-116 cells were seeded over cover slips in 35 mm culture dishes, incubated with different concentrations of TC-2 for 24 h. Cells were washed two times in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Forthwith 10 min permeabilization of the cells in 0.1%TritonX-100/PBS at room temperature was done. Cells were incubated in 10% BSA to block the nonspecific binding sites followed by their incubation with primary antibodies cytochrome c and LC3B diluted 1:100 in 0.1% Triton X-100 in PBS for 1hr at room temperature, followed by washing with PBS and incubation with respective Alexa Fluor 555and 488 conjugated secondary antibodies diluted 1:500 in PBS for 1 h at room temperature in dark. Cells were then washed thrice in PBS and stained with DAPI (1 µg/ml) in PBS. The cover slips were mounted over glass slides and images of cells were taken using a laser scanning confocal microscope (Olympus Fluoview FV1000) employing 60X oil immersion objective lens (Acharya et al., 2009; Kumar et al., 2016a ; Kumar et al., 2016b) 3. Results Out of all the fractions from the aqueous extract of Tinospora cordifolia evaluated for their cytotoxicity against different cancer cell lines, TCFR fraction was found to be more active than the other fractions, Table 4a. The bioactive fractions were subjected to repeated column chromatography for the expected isolation of more potent molecules. All the isolated molecules were screened for their anticancer potential where TC-2 exhibited more promising results than the other molecules. Using SRB assay, we evaluated the cytotoxicity of TC-2 on a panel of human cancer cell lines of various origin for a period of 48 h incubation. The following cell lines were used: Lung (A549), Prostate (PC-3), SF-269 (CNS), MDA-MB-435 (Melanoma), HCT-116 (Colon) and Breast (MCF-7). From the screening experiments, compound TC-2 was found to be most active on Prostate (PC-3), MDA-MB-435 (Melanoma) and HCT-116 (Colon) cancer cell lines. To determine the IC50 values, cells were treated with different concentrations of the compound. The results showed that the incubation of different cancer cells with 1 µM, 10 µM, 30 µM and 50 µM concentrations of TC-2 for 48 h imparted varied effects on cellular viability. IC50 values were of the order of 8 µM (HCT-116) in colon carcinoma, 10.4 µM (PC-3) Prostate, 14.8 µM (MDA-MB-435) for melanoma, 23 µM (SF-295) in CNS carcinoma, 33 µM (A549) in lung cancer and 40 µM (MCF-7) in breast carcinoma, Table 4b. Table 4a. In vitro cytotoxicity of the extract, bioactive fraction and the isolated compounds against human cancer cell lines. Cell line type HCT-116 A549 PC-3 Tissue Colon Lung Prostate S.NO. Code Conc. (µg/ml) % Growth inhibition 1. T.C.E 50 23 0 27 100 52 5 48 2. TCFR-3 50 62 2 72 100 94 9 97 3. TC-1 50 0 12 5 100 20 19 14 4. TC-2 50 94 77 71 100 97 78 78 5. TC-3 50 3 11 26 100 9 25 43 6. TC-4 50 7 13 11 100 24 16 20 7. TC-5 50 12 0 0 100 27 2 10 8. TC-6 50 0 0 3 100 0 0 17 5-Fluorouracil 20 µM 52 – – Paclitaxel 1 µM – 76 – Mitomycin 1 µM – – 66 Table options Table 4b. In vitro cytotoxicity of TC-2 against 6 human cancer cell lines. TISSUE LUNG CNS PROSTATE MELANOMA COLON BREAST CELL LINE A549 SF-295 PC-3 MDA-MB-435 HCT-116 MCF-7 CODE CONC.(µM) %CYTOTOXICITY TC-2 1 0 0 0 23 0 0 10 16 41 51 42 57 0 30 45 49 62 69 88 20 50 73 92 78 86 99 74 IC50 33 23 10.4 14.8 8 40 Paclitaxel 1 77 < 0.01 – – – – Mitomycin-C 1 – – 63 – – – 5-FU 20 – – – – 52 – Doxorubicin 1 – – – – – 65 Table options To examine, whether TC-2 treatment killed cancer cells by inducing apoptosis, we analyzed the human colon cancer (HCT-116) cells for nuclear morphological changes, by staining nuclei with DAPI. TC-2 induced chromatin condensation and fragmentation of nuclei of few cells in concentration dependent manner, typical of apoptosis (Fig. 12). Annexin V/PI dual staining suggested the significant externalization of PS (phosphatidylserine) in the events of early cell death after 24 h in the present studies. The concentration dependent increase in percentage of early and late stage apoptosis with treatment of TC-2 was observed (Fig. 13). We evaluated mitochondrial membrane potential (MMP) changes with laser scanning confocal microscope which exhibited considerable loss of MMP in human colon cancer (HCT-116) cells with different concentrations of TC-2 (Fig. 14). Further, Cytochrome c localization was determined by immunofluorescence with a cytochrome c specific antibody. Cytochrome c was colocalized in mitochondria of untreated cells. In contrast, TC-2 and paclitaxel treatments displayed decrease in functional mitochondria and release of cytochrome c to the cytosol respectively (Fig. 15). Fig. 12 Fig. 12. Nuclear morphology analysis of HCT-116 cells (2 × 105/ml/well) using DAPI. After treatment with indicated concentrations of TC-2 for 24 h and examined using fluorescence microscopy (40X). Paclitaxel (1 μM) was used as positive control. With increase in concentration of TC-2 there is significant increase in nuclear condensation and formation of apoptotic bodies. Figure options Fig. 13 Fig. 13. The representative images of TC-2 treatment on the exposure of phosphatidylserine (PS) in HCT-116 cells after 24 h treatment. Phosphatidylserine exposure was assessed by the Annexin V/propidium iodide assay and analyzed by confocal microscopy using 40× oil immersion lens. Histogram showing the percentage of cells in early and late stages of apoptosis obtained by analysis of the cell images. Data are mean ± S.D. of three similar experiments; statistical analysis was done with *p < 0.05. Figure options Fig. 14 Fig. 14. Loss of mitochondrial membrane potential (∆ψm) was measured in human colon cancer (HCT-116) cells (2 × 105/ml/well) treated with indicated concentration of TC-2 in 6 well plates for 24 h and incubated with Rodamine-123 (1.0 µM) in serum free media for 20 min at 37 °C and washed with PBS. The loss of mitochondrial membrane potential (MTP) in HCT-116 cells was observed under laser scanning confocal microscope using 40X lens (Olympus Fluoview FV1000). Figure options Fig. 15 Fig. 15. Colocalization of cytochrome c and mitochondria was determined by confocal microscopy using 60× oil immersion lens. Human colon cancer (HCT-116) cells were immunostained for cytochrome c release (green) and the mitochondria of the cells were stained with MitoTracker (red). HCT-116 cells were treated with different concentrations of TC-2 and paclitaxel (1 µM) for 24 h and stained with anticytochrome c antibody and Alexa Fluor 488-labeled secondary antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). Figure options TC-2 increased ROS/oxidative stress in HCT-116 cells in concentration dependent manner (Fig. 16). We also ascertained changes in autophagic activity by examining the fluorescence of MDC, which has been known as a specific marker for autophagic vacuoles. The number of autophagic vacuoles stained by MDC in the TC-2 treated HCT-116 cells was much higher than in the untreated cells (Fig. 17). Next, to further confirm the induction of autophagy by TC-2, a set of autophagy- related factors including LC3-I and LC3-II in the HCT-116 cells after treatment with different concentrations of TC-2 for 24 h were studied by immunofluorescence microscopy. The microtubule associated protein light chain 3 (LC3) is an additional signature marker of autophagosomes. Cleavage of the 18 kDa full length LC3, known as LC3-I, to a 16 kDa form, known as LC3-II, results in recruitment of LC3-II to double layered membrane of autophagosomes and this is a key step in autophagy. The immunofluorescence confocal microscopic studies of TC-2 treated HCT-116 cells too displayed concentration dependent induction of autophagy (Fig. 18). Fig. 16 Fig. 16. Intracellular ROS level was detected by fluorescence microscopy using 2′,7′- dichlorofluorescein diacetate (DCFH-DA) after 24 h treatment. HCT-116 cells were treated with indicated concentrations of TC-2 and 0.05% H2O2 and incubated with 5 μM 2′,7′-dichlorofluorescein diacetate and examined by fluorescence microscope (40X). Figure options Fig. 17 Fig. 17. TC-2 induces autophagy in HCT-116 cells. The autophagic vacuoles were observed under fluorescence microscope (40×) with MDC staining. The treatment of compound and BEZ235 (positive control group) induced concentration-dependent formation of autophagic vacuoles in HCT-116 cells after 24 h. Figure options Fig. 18 Fig. 18. Detection of autophagy with LC3b antibody by confocal microscopy using 60X oil immersion lens. Immunocytochemical staining was conducted using anti-LC3b antibody and Alexa Flour-555-labeled secondary antibody. Nuclei were stained with DAPI. Figure options 4. Discussion TC-2 revealed highest activity against colon cancer (HCT-116) cells and least activity against breast cancer (MCF-7) cells with IC50 values of 8 and 40 μM respectively. To understand the interesting potency revealed by this new clerodane diterpenoid, detailed mechanistic studies were carried out. The differential cytotoxicity exhibited by the compound may be due to the varying molecular characteristics of these cells. Additionally the differential cytotoxicity of TC-2 against different human cancer cell lines exhibits that its use against different types of cancers might present promising results. These findings substantiate the findings in Polyalthia longifolia ( Verma et al., 2008) and Ocimum basilicum ( Manosroi et al., 2006). The property of cancer cells is their resistance to apoptosis induction (Hanahan et al., 2000). Therefore, inducing apoptosis is the aim of many anticancer therapeutic approaches as it makes it possible to kill cancer cells without causing inflammation (Reed, 2002). Mitochondria are intermediate to the intrinsic apoptotic pathway and thus are important targets for curing cancer (Fulda et al., 2010). Mitochondrial membrane permeabilization (MMP) and release of pro-apoptotic proteins (e.g. cytochrome c) from the intermembrane space to the cytosol are the characteristic features of the mitochondrial pathway of apoptosis. These events lead to the activation of the initiator caspase 9, thereby triggering the caspase cascade causing DNA condensation/fragmentation and ultimately cell death (Kroemer et al., 2009). MMP involves pore formation such as Bax/Bak oligomers and the permeability transition pore complex (PTPC) (Kroemer et al., 2007). Mitochondria plays a major role in energy generation and are also significant sensors for apoptosis. ROS is generated during cellular metabolism through leakage of electrons by mitochondrial electron transport and is a mediator in apoptosis. To gain insight into the mechanism by which TC-2 results in cell death we next examined ROS production, since excessive generation of ROS results in cell injury and death. Mitochondria play a significant role in apoptosis. Mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cells. The apoptosis induction in the present studies causes the activation of the mitochondrial pathway and is subsequently associated with ROS production, Cyt c release, and nuclear fragmentation. The other modes of non-apoptotic cell death by plant-derived anti-cancer drugs are also emerging, and mainly comprise autophagy etc. In the current study, TC-2 also induces autophagy in HCT-116 cells. The present studies are in conformity with the earlier findings wherein the synthesized natural alkaloid berberine derivatives have also been reported to induce autophagy in human colon carcinoma HCT-116 and SW613-B3 cells (Guaman et al., 2015). Similarly natural compounds have also been reported to induce autophagy, such as rottlerin (Torricelli et al., 2012), chrysin-organotin based on chrysin (Xuan et al., 2016), betanin/isobetanin (Nowacki et al., 2015), cucurbitacin B (Ren et al., 2015). In conclusion, we demonstrated that the new clerodane diterpenoid, TC-2 induces apoptosis of colon cancer (HCT-116) cells mainly by triggering ROS production. This new natural compound thus shows potential for the treatment of colon cancer. Autophagy was also observed after the treatment. Taken together, our study identified a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. The results from the present studies will be very advantageous in the further development of new chemotherapeutic agents. Acknowledgements Two authors, Neha Sharma and Ashok Kumar are highly thankful to the Department of Science and Technology, New Delhi for the award of INSPIRE fellowship. Acknowledgements Author's contribution NKS and MKV conceived and designed the study. NS carried out the bioassay guided isolation of the new molecule along with six known compounds from T.cordifolia. Compounds were characterized by NKS, MKV and NS. PD conducted the purity profile of the isolated molecules. via HPLC.VG carried out the X-ray crystallographic experiments and interpreted the crystal data. AQ and SKS conducted the preliminary screening of the extracts and the molecules on different cancer cell lines. AK and PRS carried out the detailed mechanistic study of the new clerodane diterpenoid on colon cancer cell lines. SP and RAV helped in drafting the manuscript. 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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. 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