Chinese Journal of Natural Medicines 2014, 12(12): 09010910

Chinese Journal of Natural Medicines

Chemical composition, antimicrobial, insecticidal, phytotoxic and antioxidant activities of Mediterranean Pinus brutia and Pinus pinea resin essential oils ULUKANLI Zeynep 1*, KARABÖRKLÜ Salih 1, BOZOK Fuat 1, ATES Burhan 2, ERDOGAN Selim 2, CENET Menderes 1, KARAASLAN Merve Göksin 2 1

Smaniye Korkut Ata University, Faculty of Science and Arts, Department of Biology, 80000 Osmaniye, Turkey;

2

İnönü University, Faculty of Science, Department of Chemistry, 44280 Malatya, Turkey Available online 20 Dec. 2014

[ABSTRACT] Essential oils of the resins of Pinus brutia and Pinus pinea were evaluated for their biological potential. Essential oils were characterized using GC-MS and GC/FID. In vitro antimicrobial, phytotoxic, antioxidant, and insecticidal activities were carried out using the direct contact and the fumigant assays, respectively. The chemical profile of the essential oils of the resins of P. pinea and P. brutia included mainly α-pinene (21.39% and 25.40%), β-pinene (9.68% and 9.69%), and caryophyllene (9.12% and 4.81%). The essential oils of P. pinea and P. brutia exerted notable antimicrobial activities on Micrococcus luteus and Bacillus subtilis, insecticidal activities on Ephestia kuehniella eggs, phytotoxic activities on Lactuca sativa, Lepidium sativum, and Portulaca oleracea, as well as antioxidant potential. Indications of the biological activities of the essential oils suggest their use in the formulation of ecofriendly and biocompatible pharmaceuticals. [KEY WORDS] Essential oil; Antimicrobial; Insecticidal; Phytotoxic; Antioxidant; Pinus brutia; Pinus pinea

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2014)12-0901-10

Introduction In the Conifers, responses to abiotic factors, as well as the biological factors such as potential herbivores, stem-boring insects, and pathogenic microorganisms, bring about the production of oleoresin secretions by preserving the wounded part of tree with sealing. The fundamental phytochemicals that have been reported in the resinous substance consist of mainly turpentine (volatile fraction) and the rosin (non-volatile fraction) [1-2]. Uses of the resins belonging to Pinus spp. have been well-known as a source of plant medicine in various cultures and traced back to earlier times. In Central and West Anatolia of Turkey, decoction of the resin of [Received on] 01-Dec.-2013 [Research funding] This project was supported by the Scientific Research Fund (OKU-BAP) of Osmaniye Korkut Ata University, Osmaniye, Turkey. [*Corresponding author] Zeynep ULUKANLI: Prof., Tel: +903288271000/2558, Fax: +903288251866, E-mail: [email protected], These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved

P. brutia is externally used on abscesses to promote suppuration [3-5]. The resin of P. brutia was used as a chewing gum to clean teeth and prevent bad breath. The resin plus hot water is externally applied on wounds and cuts in the Western part of Turkey [6]. The seeds of P. pinea were used as a tonic [7]. In traditional Chinese medicine, pine resin is used for the treatment of skin diseases, burn and scald wounds, trachitis, pulmonary tuberculosis, and as a good antiseptic [8]. Gum terpentine is the product from the steam distilled oleoresin of many Pinus species, and has been used in medicine, and in the food, cosmetic, and detergent industries [6, 9-10]. Essential oil constituents of the aerial parts belonging to P. brutia and P. pinea have been reported by several authors [11-16]. As of date, no report has been present on the chemical composition of P. pinea resin, and only a few reports are available on the chemical constituents of the gum turpentines from P. brutia in some Mediterranean countries [6, 17-18]. Furthermore, there are only a few reports available with regard to the antimicrobial, phytotoxic, and antioxidant activities of the essential oil of the aerial parts of P. brutia and P. pinea [12, 14, 19]. Today, a large amount of plants, their metabolites and various natural products remain to be explored in relation to

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their biological potential. Therefore, the aim of this study was i) to characterize the chemical constituents of the essential oils of the resins collected from P. brutia and P. pinea grown in the Eastern Mediterranean region of Turkey, and ii) to assess their antimicrobial, insecticidal, phytotoxic, and antioxidant activities. This is the first characterization of the essential oils of P. pinea and P. brutia, as well as testing their biological potential towards many organisms.

Materials and Methods Plant materials and extraction of the essential oils The species were selected and identified on the basis of their morphological properties by comparing the results with the Flora of Turkey and the East Aegean Islands before the collection of the resinous substances from the trees [20-21]. On July 2012, the resins were collected from Pinus brutia Ten. var. brutia (Pinaceae) and Pinus pinea L. (Pinaceae) grown in the Karacaoglan campus of Osmaniye Korkut Ata University, Osmaniye, which is 20 km away from the East Mediterranean Sea (37°02'303"N; 36°13'0.91"E) and 150 m above sea level. Essential oils were obtained through hydrodistillation of 100 g of cortex resin in distilled water (0.5 mL), using a Clevenger-type apparatus (3 h). Test oils were dried over anhydrous sodium sulfate and then filtered. The oil was bright yellow in color (7.6%, V/W and 6.4%, V/W, on a weight basis of the resins of P. pinea and for P. brutia, respectively). A 10 μL sample of the essential oil weighed 8.5 mg. Test oils were kept in dark vials at 4 °C for further use. Essential oils analyses The qualitative and quantitative analyses of the essential oils were carried out using gas chromatography (GC, Agilent Technologies 6890N Network system) with flame ionization detection (FID) detector GC/FID and gas chromatography coupled with a mass selective detector GC/MS [22]. The quantitative analyses of the constituents were based on the area percent data of FID. The calculations of RIs were also confirmed with those results obtained from alkanes (C8–C32). Retention indices (RI) of the constituents were compared with the authentic substances. In addition, a direct comparison between mass spectra data of the test oils and the earlier data in the GC/MS databases of the Wiley 2001 library data (NIST 02 version 2.62) and the published references [23] were also used. Microbial strains and antimicrobial assays Four Gram-negative bacteria (Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterobacter hormaechei ATCC 700323), and seven Gram-positive bacteria (Enterococcus casseliflavus ATCC 700327, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC BAA-977, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, Bacillus cereus EÜ, Micrococcus luteus NRRL B-4375) and the yeast species (Candida albicans ATCC 14053) were used as the test microorganisms. Antimicrobial activity was assessed using the disc diffu-

sion assay [24-25]. A sample (15 µL) of the essential oil in the antibiotic assay discs (6 mm in diameter, Oxoid, UK) was tested on bacterial and yeast species using Mueller Hinton agar and Sabaroud dextrose agar, respectively. Standard antibiotic assay discs (ciprofloxacin 5 μg (CIP5, Bioanalyse Ltd., UK), moxifloxacin 5 μg (MXF5, Bioanalyse Ltd. UK) and nystatin 100 U (NS 100 U, Oxoid, UK) were used as the controls for the microorganisms. Minimum inhibitory concentration (MIC) of the essential oil ranging from 0.5 to 64 mg/ml was determined in Mueller Hinton broth and Sabaroud dextrose broth plus Tween 80 (0.5%, V/V) using the broth dilution assay, respectively. Insect culture and fumigant toxicity The effect of the essential oils of P. brutia and P. pinea on Ephestia kuehniella eggs was determined with the fumigant assay [26-29]. The eggs (n = 40) younger than 24 h age were placed on a cardboard in a glass jar (300 mL). The blotting paper strips (3 cm × 3 cm) were fixed on the bottom of the lids and then were loaded with the oils ranging from 25 to 100 μLL–1 air doses. Immediately after application, jars were incubated at 27 ºC, on a 14 h light:10 h dark photoperiod and 60% ± 5% relative humidity and 16–48 h exposure time. Afterwards, the number of larvae emerging from the eggs, egg mortality (%), and lethal concentrations (LC50 and LC95) were calculated. Phytotoxicity assays Essential oils of P. brutia and P. pinea (4.25, 8.5, and 17 mgmL–1 in 0.5% Tween 80, V/V) and those of the herbicide (clodinafop-propargyl : Safener; 4 : 1) were tested on the seeds of Portulaca oleracea L. (Portulacaceae), Lepidium sativum L. (Brassicaceae), and Lactuca sativa L. (Asteraceae) using the direct contact assay [14, 30]. Surface-sterilized seeds from each species (n = 30) were aseptically transferred on two pre-sterilized layers of Whatman filter paper (No.1) in glass petri dishes (9 cm in diameter). After dispensing the essential oil solution to the lower side of the filter paper, the petri dishes were tightly sealed with Parafilm and then incubated at 24 °C, 12 h/12 h light/dark photoperiod of 1500 lux light and relative humidity of 80%. The seed germination in each plate was assessed for seven days. The lengths of radicle and plumule were measured after seven days of incubation. Fresh and dry weight of the emerging seedlings was determined at 70 °C for 24 h [31]. Antioxidant assays DPPH radical and reducing power assays The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical assay was applied according to Erdogan et al. (2011) [32]. Reducing power was assayed on the method of Oyaizu [33]. Total phenolic content Total phenolic content was determined using the Folin and Ciocalteu reagent method [34]. Statistical analysis The results obtained from different concentrations of the test oils were evaluated with the analyses of variance (one-way ANOVA) at the significance level of 5% by the least significant

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difference test (LSD) using SPSS statistical program for windows (Version 17, SPPS Inc., Chicago, IL, USA). The arcsine √x transformation of percentage data were used to meet the normality. The LC50 and LC95 values of the test oils on E. kuehniella eggs were estimated by using the probit analyses of SPSS. All biological assays were performed in triplicate.

Results and Discussion Chemical constituents of the essential oils of P. pinea and P. brutia resin The components, percentages, and retention index of the constituent of the essential oils from P. pinea and P. brutia resin are shown in Table 1. The GC chromatograms of the essential oils from P. pinea and P. brutia are also illustrated in Figs. 1 and 2. α-Pinene and β-pinene were found to be the

major compounds in the essential oil of P. brutia. The low and high percentages of these compounds in the resin of P. brutia were reported in previous studies [6, 17-18]. Likewise, differences in the essential oil composition could be observable in P. pinea. The essential oil of the resin of P. pinea had a higher percentage of α-pinene, β-pinene, and caryophyllene, but included a lower percentage of limonene [1,13-15] According to the obtained results, monoterpene hydrocarbons were found to be in lower percentages in the the essential oil of the resin of P. brutia and P. pinea, whereas oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes were higher than those of earlier findings [6, 11, 14, 17-19]. The differences between the present and previous reports could be attributed to the habitats of the species, the parts of the species, sampling time, and the method used [14].

Table 1 The chemical compositions (%) of the essential oils belonging to the P. pinea and P. brutia Compound

RT

Pinus pinea

Pinus brutia

Monoterpene hydrocarbons 3-Carene

10.56

0.05

0.16

α-Pinene

11.22

21.39

25.40

Camphene

12.07

1.30

2.16

β-Pinene

13.14

9.68

9.69

β-Phellandrene

13.39

0.36

0.10

β-Myrcene

14.36

0.98

0.11

α-Phellandrene

14.59

0.58

0.05

1, 5, 8-p-Menthatriene

15.42

0.39

0.12

D-Limonene

15.55

5.80

2.18

β-Phellandrene

15.83

1.57

0.21

1, 3, 8-o-Menthatriene

16.00

0.47

0.20

1, 3, 8-p-Menthatriene

16.14

0.18

0.18

α-Terpinolene

17.63

-

0.02

4-Carene

17.80

0.77

0.33

3, 8-o-Menthatriene

27.81

-

-

trans-Isolimonene

38.88

0.10

-

Verbenene

13.53

0.57

0.96

Filifolone

22.12

-

-

Oxygenated monoterpenes

Camphanone

22.42

0.75

1.21

trans-Chrysanthenone

22.98

0.02

-

Thujone

27.12

-

0.02

trans-Pinocarveol

27.65

0.08

0.09

trans-Verbenol

28.12

1.76

2.90

Borneol

28.83

0.34

1.04

α-Phellandren-8-ol

29.38

1.25

1.88

trans-Carveol

29.91

-

0.11

β-Isofenchol

31.29

0.07

0.16

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Continued RT

Pinus pinea

Pinus brutia

trans-3-Caren-2-ol

Compound

32.58

0.37

-

p-Cymen-8-ol

32.78

0.21

-

Nothosmyrnol

45.02

0.03

-

Thymol

45.63

-

0.09

α-Campholenic acid

48.00

-

0.08

Sesquiterpene hydrocarbons α-Cubebene

22.59

0.21

0.19

1, 4- Methenoazulene

24.97

8.63

0.91

γ-Patchoulene

25.81

-

-

Caryophyllene

26.41

9.12

4.81

γ-1-Cadinene

26.57

-

-

α-Caryophyllene

28.39

2.33

1.26

α-Farnesene

28.40

-

-

α-Muurolene

29.66

0.19

0.16

α-Bisabolene

39.46

0.10

0.06

Cedrane

42.96

-

-

Methanoazulene

43.46

0.25

0.1

α-Cedrane

44.47

0.08

-

Aromadendrene

45.04

-

-

α-Bisabolene

46.48

-

-

Caryophyllene oxide

38.36

3.26

1.54

Nerolidol

39.65

-

-

Aromadendrene oxide

49.56

0.63

0.49

1, 3, 6-Octatriene

16.29

0.21

-

2, 4, 6-Octatriene

21.66

-

0.31

Oxygenated sesquiterpenes

Aliphatic compounds

Other compounds exo-Methyl-camphenilol

26.06

-

-

α-Campholene

26.94

-

-

Seychelleme

29.81

0.43

0.10

Isopimaradiene

47.57

-

-

Cembrene

50.06

-

-

Anthracene

52.07

0.08

0.02

RT: Retention time

Fig. 1 The GC chromatogram of the essential oils from the resin of P. pinea. The peaks shown in the chromatogram belong to major components which were above 1% of the chemical composition of the essential oil. 1) α-Pinene, 2) Camphene, 3) β-Pinene, 4) D-Limonene, 5) β-Phellandrene, 6) 1, 4-Methenoazulene, 7) Caryophyllene, 8) trans-Verbenol, 9) α-Caryophyllene, 10) α-Phellandren-8-ol, and 11) Caryophyllene oxide

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Fig. 2 The GC chromatogram of the essential oil from the resin of P. brutia. The peaks shown in the chromatogram belong to major components which were above 1% of the chemical composition of the essential oil. 1) α-Pinene, 2) Camphene, 3) β-Pinene, 4) D-Limonene, 5) Camphanone, 6) Caryophyllene, 7) trans-Verbenol, 8) α-Caryophyllene, 9) Borneol 10) α-Phellandren-8-ol, and 11) Caryophyllene oxide

P. brutia. As shown in Table 2, the essential oils revealed same inhibitory activities towards E. faecalis, E. casseliflavus, E. coli, and C. albicans. Nevertheless, the essential oil of P. brutia was more prominent against S. aureus ATCC 29213 than those of P. pinea. None of the essential oils exerted antibacterial activity towards P. aeruginosa and K. pneumoniae. Activities of the essential oils towards the test microorganisms were found to be lower than those of the known antibiotics.

Antimicrobial activities The disc diffusion assay indicated that the essential oils of P. brutia and P. pinea were most active towards M. luteus and B. subtilis. Essential oils were also active against S. aureus ATCC 29213, S. aureus BAA, B. cereus, E. casseliflavus, E. faecalis, E. hormaechei, E. coli, and C. albicans (Table 2). The essential oil of P. pinea towards M. luteus, B. subtilis, B. cereus, S. aureus BAA, and E. hormaechei seemed to be more potent than

Table 2 Antimicrobial activities of the essential oils obtained from P. pinea and P. brutia’s resins on twelve microorganisms using the disc diffusion assay Inhibition zones (mm) Species

Essential oils (15 μL) P. brutia

Antibiotics

P. pinea a

CIP5

34.66 ± 0.57

a

M. luteus NRRL B-4375

31.00 ± 1.00

B. subtilis ATCC 6633

16.00 ± 1.00b

30.33 ± 1.52b

c

c

17.33 ± 0.57

MXF5

43.33 ± 1.52

a

35.33 ± 1.15bc

NT

32.33 ± 0.57c

NT

42.66 ± 2.51

NT NT

13.00 ± 1.00

S. aureus 29213

13.33 ± 0.57c

8.66 ± 0.57efg

29.00 ± 1.00e

32.00 ± 1.00c

S. aureus ATCC BAA-977

13.33 ± 0.57c

8.33 ± 0.57fg

29.00 ± 1.73e

34.66 ± 0.57b

NT

E. faecalis ATCC 29212

9.00 ± 1.00de

9.33 ± 1.15ef

19.66 ± 1.15h

23.00 ± 1.00e

NT

E. casseliflavus ATCC 700327

9.66 ± 0.57d

9.33 ± 0.57ef

19.00 ± 1.00h

24.00 ± 0.00e

NT

e

d

d

d

NT NT

E. hormaechei ATCC 70323

8.33 ± 0.57

E. coli ATCC 25922

8.33 ± 0.57e

8.00 ± 0.00g

34.00 ± 2.00c

31.66 ± 0.57c

P. aeruginosa ATCC 27853

0.00 ± 0.00f

0.00 ± 0.00h

37.00 ± 0.00b

27.33 ± 0.57d

NT

f

h

g

17.00 ± 0.00f

NT

NT

21.33 ± 0.57a

K. pneumoniae ATCC 700603

0.00 ± 0.00

C. albicans ATCC 14053

9.00 ± 0.00c

0.00 ± 0.00

9.66 ± 0.57e

31.00 ± 0.00

27.66 ± 1.15

d

B. cereus EÜ

12.33 ± 0.57

24.33 ± 0.57

f

NS 100 a

21.66 ± 0.57 NT

27.33 ± 0.57

Different letters above columns indicate significant differences among the microbial species at P < 0.05 (ANOVA followed by LSD test). SE: Standard error. CIP5: Ciprofloxacin 5 μg; MXF5: Moxifloxacin 5 μg; NS 100 U: Nystatin 100 U. NT: Not Tested

In the present assay, the MIC value was 34 mgmL–1 for M. luteus, B. subtilis and B. cereus, and a value higher than 68 mgmL–1 was found for S. aureus ATCC 29213, S. aureus ATCC BAA-977, B. cereus, E. casseliflavus, E. faecalis, E. hormaechei, E. coli. P. aeruginosa, K. pneumoniae, and C. albicans. Overall, essential oils revealed antimicrobial activities on both Gram-positive bacteria and Gram-negative bacteria and the yeast species; however, significant antibacterial activities were observed on Gram-positive bacteria rather

than Gram-negative ones, which might be as a consequence of bacterial cell wall differences. The results of the disc diffusion and MIC assays in the present assay revealed some differences from those of Loizzo et al. [12], who tested the essential oil of the cones and flowers of P. brutia on microorganisms. In their study, the cones and flowers of P. brutia consisted of α-pinene (40.7% and 24.2%), β-pinene (28.27% and 35.18%), Δ-3-carene (13.36% and 11.20%), β-myrcene (2.29% and 11.90%), limonene (3.69% and 5.50%),

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α-terpinolene (2.0% and 1.7%), α-terpineol (2.94% and 0.65%), and β-caryophyllene (2.74% and 1.85%), respectively [18]. Differences between the present study and those of Loizzo et al. [18] could be possibly attributed to the plant part and the percentage of the constituents residing in the essential oil. Insecticidal activities Essential oils of the resins of P. pinea and P. brutia on E. kuehniella eggs indicated that increasing the exposure time (16–48 h) caused a slight increase in the mortality (Figs. 3 and 4). Essential oil of P. pinea caused 12.5% mortality on the eggs at 100 µLL–1 air concentration at the end of 48 h; however, this was 14.13% in the case of P. brutia. LC50 and LC95 values were 343.57 and 719.84 µLL–1 air in the case of P. pinea; however, these values were 299.90 and 632.21 µLL–1 in the treatment of P. brutia essential oil air for 48 h, respectively (Table 3). A slight increase in the mortality of E. kuehniella eggs might be due to the changes in the permeability of the chorion and/or vitelline membrane, which occur during embryogenesis and facilitate the diffusion of vapors into older eggs so that vital physiological and biochemical processes are affected [35]. The present findings suggest that essential oils derived from the cortex resins revealed insecticidal activity; however, increasing concentration and exposure could be suggested as a source of bio-pesticides for further use.

Fig. 3 Percent mortality of the E. kuehniella eggs after exposure to the essential oil of P. pinea. Different letters above bars indicate significant differences between exposure periods for each concentration at P < 0.05 (ANOVA followed by LSD test). Error bars indicate SE of means

Fig. 4 Percent mortality of the E. kuehniella eggs after exposure to the essential oil of P. brutia. Different letters above bars indicate significant differences between exposure periods for each concentration at P < 0.05 (ANOVA followed by LSD test). Error bars indicate SE of means

Table 3 LC50 and LC95 values of P. pinea and P. brutia essential oils against E. kuehniella eggs LC50 (µLL–1)

LC95 (µLL–1)

2

16 h

752.90

1535.92

6.88a

32 h

543.95

1100.44

4.32

48 h

343.57

719.84

7.22

16 h

479.33

947.50

15.34

32 h

327.12

665.23

14.74

48 h

299.90

632.21

7.57

Essential oil P. pinea

P. brutia

a

Since goodness-of-fit test is significant (P < 0.150), a heterogenity factor is used in the calculation of fiducial limits (f.l)

Phytotoxic activities The use of the essential oils in understanding the dynamics of plant-plant interactions as well as exploring their potential to find biologically and environmentally safe drugs for controlling the crops and weeds in agricultural fields are of scientific and industrial importance [14, 30, 36-39]. The phytotoxic effects of the essential oils of P. brutia and P. pinea and those of the herbicide (Clodinafop-propargyl: Safener) were tested on the seeds of L. sativa, P. oleracea, and L. sativum using the direct contact assay. The differences between the control and the treatment groups were statistically evident (Fig. 5). The highest dose of P. brutia and P. pinea essential oils caused inhibitory effects in the germination by 53% and 22% in L. sativa, 60% and 33% in L. sativum, and 13% and 3% in P. oleracea, respectively. It appeared that the essential oil of P. brutia resin was more effective in inhibiting the germination of all test seeds than those of P. pinea resin. The notable inhibition in the germination of L. sativa and L. sativum was also evident with the treatment of P. pinea essential oil, whereas, the same effect was not observed on P. oleracea. Radicle length (RL) and plumule length (PL) of all test seeds were significantly inhibited by increasing the doses of the essential oils. The inhibition at the highest dose of the essential oils of P. brutia and P. pinea were 65% and 54% in the RL, and by 75% and 40% in the PL of L. sativa, respectively. Under the same experimental conditions, inhibition of the essential oils of P. brutia and P. pinea were 79% and 37% in the RL, and by 74% and 37% in the PL of P. oleracea. These percentages were 87% and 28% in the RL, and by 86% and 46% in PL of L. sativum, respectively. The present data indicated that the essential oil of P. brutia was clearly more effective in inhibiting the growth of plumule in L. sativa than those of their radicle; however, the inhibition of the RL was more prominent in those of P. oleracea and L. sativum. In contrast, the effect of the essential oil of P. pinea in the RL of L. sativa was more prominent. In addition, the inhibition of RL and PL was the same as in P. oleracea, whereas PL of L. sativum was more inhibited than those of their RL. Fresh and dry weight of all test species decreased by increasing the

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Fig. 5 Phytotoxic effects of different concentrations (mgmL–1) of P. brutia and P. pinea essential oils and herbicide on germination, radicle and plumule length of L. sativum, P. oleracea, and L. sativum seeds. Different points represent means of independent bioassays. Mean with different letter in curves is significant at P < 0.05 (ANOVA followed by LSD test). RL: Radicle Length, PL: Plumule Length, P: P. pinea, B: P. brutia, C: Control, H: Herbicide, FW: Fresh Weight, DW: Dry Weight

essential oil concentration. The highest oil concentration of P. brutia and P. pinea decreased the fresh weight by 74% and 55% in L. sativa, and 70% and 36% in P. oleracea, and by 70% and 44% in L. sativum; however, the dry weight was reduced by 52% and 30% in L. sativa, 50% and 42% in P. oleracea, and 60% and 53% in L. sativum. In the case of the herbicide, inhibition of the germination at the highest concentration were clearly evident and the percentages were 100%, 90%, and 69% in L. sativa, L. sativum and P. oleracea, respectively. Inhibition of the RL at the highest dose of the herbicide was the same as those in both P. oleracea and L. sativum (91%). Similarly, PL was inhibited by 90% in P. oleracea and 92% in L. sativum. The highest herbicide concentration decreased the fresh and dry weight by 86% and 89% in P. oleracea, and by 86% and 86% in L. sativum. Throughout the assays, RL, PL, fresh and dry weight parameters of L. sativa were not recorded for the seeds of L. sativa because of the total inhibition of germination. In a previous study, the needle part of P. pinea showed complete inhibition at 2 μL·mL−1 in the germination and the seedling growth of Sinapis arvensis, Lolium rigidum, and Raphanus raphanistrum [14]. The significant phytotoxic effects of twelve different essential oils were reported on the seeds of L. sativa, L. sativum, and Raphanus sativa by inhibiting the germination, as well as the early root formation[40]. Inhibitory activities of α-pinene, limonene, and camphor were

found on the mitochondrial respiration activities of corn radicles [41] and beans plumules [42-43]. Seeds of R. sativa were found to be more susceptible to twenty-seven monoterpene compounds than those of L. sativum [44]. The present findings indicate that the essential oils of P. brutia and P. pinea resin with α-pinene, β-pinene, and limonene inhibit the germination and the seedling growths of L. sativa and L. sativum, which is in line with previous reports [14, 40-43]. As in Fig. 5, the inhibitory effect of the essential oil of P. brutia was also observed in the germination activities of P. oleracea. The levels of the major compounds in the essential oil of P. pinea were close to those of P. brutia. Nevertheless, the essential oil of P. pinea resin did not statistically inhibit the germination of P. oleracea. The phytotoxic effects of the essential oil appeared to be species-dependent. The poor effect on the germination of P. oleracea could be possibly attributed to the interaction among the multiple compounds. Antioxidant activities The antioxidant properties of plants could be correlated with radical scavenging capacity and oxidative stress defense [45-46] . The DPPH scavenging assay is a feasible method for the evaluation of the antioxidant activity of a compound [47]. The hydrogen atom or electron donation capacity of the corresponding extracts was computed from the bleaching property of the purple-colored methanol solution of 2, 2-diphenyl1-picrylhydrazyl (DPPH). In this assay, -tocopherol was

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used as a standard for comparison with the DPPH scavenging activities of the essential oils. As shown in Fig. 6, the DPPH radical scavenging activity of P. pinea and P. brutia essential oil (20 mg) and α-tocopherol (10 µg) were 52.10%, 76.33% and 65.7%, respectively. Test results indicated that radical scavenging activities of the essential oils seemed to be on the moderate level, which was consistent with an earlier report [19].

Fig. 8 Total phenolic contents as gallic acid equivalents of the essential oils of P. pinea and P. brutia. Each value is the average of triplicate experiments with error bars indicating STDEVs (n-1)

Fig. 6 DPPH radical scavenging power of the essential oils of P. pinea and P. brutia. Twenty mg of extract solution and 10 µg of α-tocopherol were used in the reaction mixture. Each value is the average of triplicate experiments in with error bars indicating STDEVs (n-1). a) Significantly different from P. brutia vs P. pinea (P < 0.05)

Reducing power of a compound is another supporting feature for its antioxidant activity [48]. The reducing power of P. pinea and P. brutia essential oil (20 mg) was found to be higher than that of α-tocopherol (10 µg). Likewise, essential oil of P. brutia (20 mg) revealed higher reducing power than that of ascorbic acid (10 µg) (Fig. 7). The results of the reducing power of the oils appeared to be better than those of the DPPH scavenging activity. The higher activity could be attributed to the higher specificity of the assay for hydrophilic compounds. Phenolic compounds are known to be natural antioxidants, which display their effects by suppressing the

formation of free radicals [32]. Total phenolic contents of P. pinea and P. brutia were 21.43 and 17.02 mg gallic acid equivalent/g essential oil (EO), respectively (Fig. 8). The results of total phenolic content revealed that the phenolic compounds had a good level of contribution to the antioxidant capacity of P. brutia and P. pinea essential oil. On the other hand, the observed antioxidant properties were not entirely related to the total phenolic contents of the essential oil extracts.

Conclusion The essential oils of the resins of P. brutia and P. pinea grown in the Mediterranean region of Turkey had various levels of antimicrobial activities on Gram-positive, Gramnegative bacteria, and the yeast species, and hence, confirming the traditional uses of these resinous substances. In addition, insecticidal, phytotoxic, and antioxidant potential residing in the essential oils of the resinous substances could suggest their possible uses in the formulation of biological control agents. Bioassay guided research is needed, in conjuction with the recovering, identifying, and assaying of the individual compound and/or multiple compounds to determine their biological effects on the test organisms.

Acknowledgements Special thanks to Assoc. Prof. Abdurrahman Ayvaz for providing insect culture and laboratory facilities at Erciyes University, Kayseri, Turkey.

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Fig. 7 Reducing power of the essential oils of P. pinea and P. brutia. Twenty mg of extract solution and 10 µg of -tocopherol and ascorbic acid were used in the reaction mixture. Each value is the average of triplicate experiments with error bars indicating STDEVs (n-1). a) Significantly different from P. brutia vs P. pinea (P < 0.05)

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Cite this article as: ULUKANLI Zeynep, KARABÖRKLÜ Salih, BOZOK Fuat, ATES Burhan, ERDOGAN Selim[0], CENET Menderes, KARAASLAN Merve Göksin. Chemical composition, antimicrobial, insecticidal, phytotoxic and antioxidant activities of Mediterranean Pinus brutia and Pinus pinea resin essential oils [J]. Chinese Journal of Natural Medicines, 2014, 12(12): 901-910

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Chemical composition, antimicrobial, insecticidal, phytotoxic and antioxidant activities of Mediterranean Pinus brutia and Pinus pinea resin essential oils.

Essential oils of the resins of Pinus brutia and Pinus pinea were evaluated for their biological potential. Essential oils were characterized using GC...
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