In Vitro Antimicrobial, Antioxidant and Anti-Inflammatory Evaluation Of

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

RESEARCH ARTICLE In vitro antimicrobial, antioxidant and anti-inflammatory evaluation of Eucalyptus globulus essential oil Gamze Göger1,*, Nursenem Karaca2, Betül Büyükkılıç Altınbaşak3, Betül Demirci4 and Fatih Demirci4,5 1Department of Pharmacognosy, Faculty of Pharmacy, Trakya University, Edirne, TURKEY 2Graduate School of Health Sciences, Anadolu University, Eskişehir, TURKEY 3Department of Pharmaceutical Botany, Faculty of Pharmacy, Bezmialem Vakıf University, İstanbul, TURKEY 4Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, TURKEY 5Faculty of Pharmacy, Eastern Mediterranean University, 99628, Famagusta, N. CYPRUS

*Corresponding author. Email: [email protected] Submitted: 30.06.2020; Accepted: 18.08.2020

Abstract Eradication of Propionibacterium acnes and associated skin pathogenic species such as Staphylococcus aureus and S. epidermidis involve anti-oxidant as well as anti-inflammatory effects besides antimicrobial action. For this purpose, Pharmacopoeia Grade (PhEur) Eucalyptus globulus essential oil was evaluated against the human pathogenic species such as P. acnes ATCC 6919, P. acnes ATCC 11827, S. aureus ATCC 6538 and S. epidermidis ATCC 12228 using an in vitro microdilution method. The composition and quality of the essential oil was confirmed both by GC/FID and GC/MS techniques, respectively. The in vitro radical-scavenging activity was evaluated using the photometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay; the anti-inflammatory activity assay performed by using the in vitro lipoxygenase (5-LOX) enzyme inhibition assay. Essential oil analysis confirmed the presence of 1,8-cineole (80.2 %), p-cymene (6.6 %), and limonene (5 %) as main components. The antibacterial performance of the tested oil was more susceptible against Staphylococcus species (MIC=625 µg/mL) compared to P. acnes (MIC=1250 µg/mL). 5-LOX inhibitory activity was determined as IC50 = 58 ±1,4 µg/mL for the essential oil, compared to the inhibition of the standard nordihydroguaiaretic acid = NDGA. The preliminary experimental results suggest that the Eucalyptus essential oil and its major constituent 1,8-cineole acts against skin pathogenic bacteria as a mild natural antimicrobial with anti-inflammatory effects, for further potential topical applications.

Keywords: Antibacterial, anti-inflammatory, Eucalyptus globulus, Propionibacterium, Staphylococcus

Introduction Inflammation is a physiological response, which occurs in the human system against tissue damage caused by various physical, biochemical effects, and microbial infections. Lipoxygenases (LOX) are well known as an important group of enzymes associated with inflammatory processes. The ethiology of acne is an inflamed and painful swollen unpleasant topical situation, which is associated with Propionibacterium sp. and Staphylococcus sp. etc. pathogenic microorganisms located on the skin pores (Athikomkulchai et al., 2008; Lertsatitthanakorn et al., 2006; Perry & Lambert, 2006; Serpi et al. 2012). Today, resistance of P. acnes to antibiotics (Abascal& Yarnell, 2002; Chomnawang et al., 2005; Ergin et al., 2007; Akolade et al., 2012), and the extensive use of external antimicrobials may cause dryness, irritation and peeling among others which urged the need for new treatment approaches (Lertsatitthanakorn et al., 2006). For many years, essential oils (EOs) are used to treat and protect from diseases due to their pharmacological effects. EOs and their preparations can be used orally after dilution, by inhalation and by topical routes (Başer & Buchbauer, 2020; Babar et al., 2015). Herbal preparations or cosmetics containing Eucalyptus oils are also used in several countries for the treatment of infectious diseases and several conditions (Maccioni et al., 2002). Additionally, it is also documented that in some countries, Eucalyptus preparations are used for the

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

treatment of acne (Dey et al., 2014). On the basis of traditional use, the present study aimed to evaluate the combined in vitro antibacterial, antioxidant, and anti-inflammatory activity of the Pharmacopoeia (PhEur) quality Eucalyptus globulus Labill. (Myrtaceae) essential oil (EGEO). To the best of our knowledge, there is only limited data on EGEO against P. acnes and skin pathogenic microorganisms, as well as on 5-LOX inhibitions. Antibacterial activity was studied against the skin pathogens Propionibacterium acnes, Staphylococcus aureus, and S. epidermidis by in vitro microdilution method. In addition, antioxidant activity of EGEO was examined using the in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and the anti- inflammatory effect was evaluated using the lipoxygenase (LOX) inhibition. Materials and Methods Materials Pharmacopoeia Grade (PhEur) EGEO was kindly provided by Enafarma Ltd., Istanbul, Turkey. All bacteria were obtained from Microbiologics, USA. Antibacterial activities of the EGEO against P. acnes ATCC 6919 and ATCC 11827 (American Type Culture Collection), S. aureus ATCC 6538 and S. epidermidis ATCC 12228 strains were screened. The antibacterial compounds [tetracycline, ampicillin and cefuroxime] were provided by Deva Pharmaceutical Company, Istanbul, Turkey. Lipoxygenase (1.13.11.12, type I-B, soybean), linoleic acid Nordihydroguaiaretic acid (NDGA), resazurin sodium salt and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were acquired from Sigma-Aldrich. All chemical substances were used analytical grade if not otherwise stated. GC-FID analysis The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 °C. To obtain the same elution order with GC/MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. GC/MS analysis The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column [60 m x 0.25 mm, 0.25 mm film thickness] was used with helium as carrier gas [0.8 ml/min]. GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min, and kept constant at 220°C for 10 min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. Identification of components Identification of the EGEO components was carried out by comparison of their relative retention times (RRT) with those of authentic samples or by comparison of their relative retention index [RRI] to a series of n- alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (McLafferty & Stauffer, 1989; Koenig et al., 2004) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils. Additionally, MS literature data (Joulain & Koenig, 1998; ESO, 2000) was also used for the identification.

Antibacterial activity (MIC, µg/mL) Antibacterial activity of EGEO against P. acnes ATCC 6919, P. acnes ATCC 11827, S. aureus ATCC 6538 and S. epidermidis ATCC 12228 were screened in vitro by the modified CLSI microdilution method (CLSI, 2006). The

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

tests were carried out in 96-well micro plates. The sample (100 µL per well) was diluted two fold, with a final concentration range of 5000 to 9.76 µg/mL, respectively. Standard antibacterial agents ampicillin, cefuroxime (64 to 0.125 µg/mL) and tetracycline (16 to 0.03 µg/mL) were used under the same conditions as positive controls. P. acnes ATCC 6919 and P. acnes ATCC 11827 were inoculated in Reinforced Clostridium Medium

(RCM), and cultured % 5 CO2 at 37°C for 72 h under anaerobic conditions. S. aureus ATCC 6538 and S. epidermidis ATCC 12228 were incubated in Mueller Hinton Broth (MHB) overnight at 37°C for 24h or 48h. Cultures, with a final inoculum size of 1 x 106 colonies forming units (CFU/mL) were used. Microbial growth was observed by adding 20 µL of resazurin of 0.01% with minor modifications of CLSI standards (Pfaller et al., 2008). A change from blue to pink indicated the reduction of resazurin and, therefore, microbial growth. The minimum inhibitory concentration (MIC) was determined as the lowest drug concentration that prevented the color change. All experiments were repeated in triplicate, and average results were reported. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity Antioxidant activity was measured by using the microplate spectrophotometric method for EGEO and its main compound 1,8-cineole (Sigma Aldrich). Stock solutions of test samples (10 mg/mL) were dissolved initially in methanol. Serial dilutions (100 µL) were prepared using the stock solutions of the EGEO and 1,8- cineole. A stock solution of the DPPH (2 mg /25 mL) in methanol was prepared freshly. 100 µL DPPH solution was then added each well and mixed, and after 30 min storage in a dark environment at 25°C the UV absorbance was recorded at 517 nm. Blank samples containing the same amounts of methanol and DPPH solution were also prepared. The experiment was performed in duplicate for the EGEO, and 1,8-cineole with positive standard control, vitamin C. The average of the absorptions was noted for each concentration. The percentage inhibition (I%) was calculated using the Equation. The IC50 value, which is the concentration of the test material that inhibits 50% of the free radical concentration, was calculated as mg/mL using Sigma Plot statistical program (Kumarasamy et al., 2007).

Percentage Inhibition (I%) = [(Abscontrol - Abssample)/ Abscontrol] x100 Equation

Abscontrol: Control absorbance Abssample: Sample absorbance

In vitro lipoxygenase [LOX] enzyme inhibition assay The anti-inflammatory activity was evaluated by modifying the spectrophotometric method developed by Baylac and Racine (Baylac & Racine, 2003). Potassium phosphate buffer (1,94 mL; 100 mM; pH 9.0), 40 μL of test compound solution and 20 μL of lipoxygenase solution were mixed and incubated for 10 min at 25°C. The reaction was then initiated by the addition of 50 μL linoleic acid solution, the change of absorbance at 234 nm was followed for 20 min. Test compounds and the positive control Nordihydroguaiaretic acid (NDGA) were dissolved in methanol. All the kinetic experiments were performed in quartz cuvette. The concentration that gave 50% inhibition (IC50) for test samples was calculated. All experiments were repeated in triplicate and average results were reported.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

Results and Discussion Chemical analysis of the essential oil The compounds of Eucalyptus globulus essential oil were determined by GC/FID and GC/MS, the results are listed in Table 1. Twelve main compounds were identified in the oil representing 100%. The major compounds were characterized as 1,8-cineole (80.2%), p-cymene (6.6%), and limonene (5%) as shown in Figure 1. The analytical results met the required Pharmacopoeia limits (Anonymous, 2008). Numerous studies exist on the Eucalyptus globulus oil composition, where varying amouts of 1,8-cineole were reported (Damjanovic-Vratnica et al., 2001; Harkat-Madouri et al., 2015; Luis et al., 2016). 1,8-Cineole (85.8%), α-pinene (7.2%), and β-myrcene (1.5%) were the main components (Damjanovic-Vratnica et al., 2001). In another report, 1,8-cineole (55.3 %), spathulenol (7.4 %) and α-terpineol (5.5 %) were found as main components (Harkat-Madouri et al., 2015). In another study, major oil components were reported as 1,8- cineole (63.8 %), α-pinene (16.1 %), aromadendrene (3.7 %), and o-cymene (2.4 %) (Luis et al., 2016).

Table 1. Chemical composition of E. globulus Labill. essential oil

RRI Compounds % IM 1032 α-Pinene 1.0 RRI, MS 1118 β-Pinene 0.4 RRI, MS 1174 Myrcene 0.2 RRI, MS 1203 Limonene 5.0 RRI, MS 1213 1,8-Cineole 80.2 RRI, MS 1280 p-Cymene 6.6 RRI, MS 1611 Terpinen-4-ol 0.1 RRI, MS 1706 α-Terpineol 0.4 RRI, MS 2100 Heneicosane 0.8 RRI, MS 2131 Hexahydrofarnesyl acetone 2.6 MS 2300 Tricosane 1.9 RRI, MS 2492 Ethyl oleate 0.8 MS Total 100 RRI: Relative retention indices calculated against n-alkanes, %: calculated from FID data, IM: Identification method based on the relative retention indices (RRI) of authentic compounds on the HP Innowax column; MS, identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

Figure 1. GC chromatogram of E. globulus essential oil

Antibacterial activity (MIC, µg/mL) In the present study; P. acnes ATCC 6919, P. acnes ATCC 11827, S. aureus ATCC 6538 and S. epidermidis ATCC 12228, were selected as test microorganisms based on their pathological potential. Minimum inhibitory concentrations (MIC) of test samples are listed in Table 2. The EGEO showed more antibacterial effect against Staphylococcus (MIC=625 µg/mL) species compared to Propionibacterium species (MIC=1250 µg/mL). In addition, the major component 1,8-cineole was also tested and was relatively more effective against S. epidermidis compared to other strains.

Table 2. Eucalyptus globulus essential oil Minimum Inhibitory Concentration (MIC, µg /mL) results Samples S. aureus S. epidermidis P. acnes P. acnes ATCC 6538 ATCC 12228 ATCC 6919 ATCC 11827 EGEO 625 625 1250 1250 1,8-Cineole 1250 312.5 1250 2500 Ampicillin >64 2 0.125 > 0.125 > Cefuroxime >64 4 - - Tetracycline 4 4 2 4 EGEO: Eucalyptus globulus essential oil Acne vulgaris is a skin disorder, and pathogenesis of acne include many factors such as follicular hyperkeratosis, increased sebum secretion, inflammation and P. acnes, which is one of the main causative microorganisms. In addition, S. epidermidis and S. aureus may be present in acne lesions. P. acnes secretes several proinflammatory products, which play an important role in the development of inflammation. These include lipases, proteases, hyaluronidases, and chemotactic factors (Kanlayavattanakul & Lourith, 2011; Das & Raynolds, 2014). Azelaic acid, salicylic acid, retinoic acid and derivatives, benzoyl peroxide, macrolides, tetracylines are extensively used in acne treatment (Kanlayavattanakul & Lourith, 2011). However, widespread use of antibiotics in the treatment of acne has led to the development of resistance against

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

strains. To overcome the problem of antibiotic resistance, EOs have been extensively studied for the treatment of acne and other skin diseases. (Orafidiya et al., 2002; Orchard et al., 2018; Hammer, 2015; Taleb et al., 2018). Currently, a systematic review demonstrated clinical use of EOs for the treatment of topical infections (Deyno et al., 2019). Clinical studies showed the efficacy of tea tree oil, against many diseases including acne, oral candidiasis, tinea, Onychomycosis, and Molluscum contagiosum (Hammer, 2015). Additionaly, it was reported that a topical preparation containing 2% Ocimum oil in cetomacrogol blend base is more effective in the treatment of Acne vulgaris than a 10 % benzoyl peroxide lotion (Orafidiya et al., 2002). The in vitro antibacterial activity of different EOs against acne associated bacteria like P. acnes, S. epidermidis and Multi drug resistant of S. aureus (MRSA) was reported (Orchard et al., 2017). Antibacterial activity of E. globulus leaf EO against Staphylococcus, Streptococcus, Escherichia, Haemophilus, Bacillus, Klebsiella species, as well as its anti-candidal and antifungal activities were the subject of several reports (Bachir&Benali, 2012; Ghalem&Mohamed, 2008; Salari et al., 2007; Tohidpour et al., 2010; Noumi et al., 2011; Bansod&Rai, 2008; Vilela et al., 2009). MIC =15.75 mg/mL was found against S. aureus and S. epidermidis for EGEO, where the main component was 1,8-cineole (63 %) by microdilution (Mekonnen et al., 2016). In this present study, MIC=625 µg/mL was obtained against S. aureus and S. epidermidis. According to the literature survey, a few studies were reported against P. acnes, S. aureus, and S. epidermidis. Antibacterial activity of EGEO by disc diffusion and agar dilution method were performed against P. acnes strains. Inhibition zones were not observed, and 4% concentration was reported as active concentration (Luangnarumitchai et al., 2007). Furthermore, EGEO containing γ-terpinene and p-cymene as the major components with a MIC=9375 µg/mL against P. acnes was reported (Athikomkulchai et al., 2008). In another study, antimicrobial activity of EGEO was evaluated against acne pathogenic microorganisms such as, S. aureus 2079 and S. aureus 5021 (Bhatt, 2011). The possible reason for differences may be due to the characteristic compositions of EO or the microorganism strains used. Most of the antimicrobial activity of EOs were attributed to the oxygenated monoterpenes, while some hydrocarbons may also exhibit antimicrobial effects (Bassole & Juliani, 2012; Chouhan, 2017). The antimicrobial activity of Eucalyptus EO could be associated with the presence of 1,8-cineole, and linalool which are well-known constituents with pronounced antimicrobial properties (Damjanovic-Vratnica et al., 2001). 1,8-cineole, α-pinene, β-pinene, limonene, and linalool which have been known to exhibit antimicrobial activity against Bacillus subtilis, Enterococcus faecalis, S. aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, Saccharomyces cerevisiae and Aspergillus niger (Sonboli et al., 2006). In recent years, there is an increased interest in the use of EOs and antimicrobial combination studies to control resistant microorganisms. Combinations of EOs and antimicrobial drugs may lead to synergistic, additive or antagonistic effects. Therefore, EGEO were combined with different EOs such as; Citrus limon, Cedrus atlantica, Lavandula angustifolia, Melaleuca alternifolia, Pinus sylvestris, Rosmarinus officinalis, Thymus vulgaris, Mentha piperita, Mentha spicata against P. acnes ATCC 11827 and S. epidermidis ATCC 2223 (Orchard et al., 2018). The combinations E. globulus and Thymus mastichina EOs were found to be synergistic against S. aureus (Vieira et al., 2017).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity DPPH assay was used for antioxidant activity of EGEO, 1,8-cineole and vitamin C. EGEO and 1,8-cineole did not inhibit the DPPH free radicals at the tested concentrations (10 mg /mL), while significant DPPH radical inhibition was obtained for vitamin C. IC50 values are shown in Table 3. Table 3. Antioxidant evaluation of E. globulus essential oil and 1,8-cineole

Samples IC50 (mg/mL) EGEO >10 1,8-Cineole >10 Vitamin C 0.017 EGEO: Eucalyptus globulus essential oil

Antioxidant activities of EGEOs were reported previously extensively (Akolade et al., 2012; Noumi et al., 2011; Sacchetti et al., 2005). Previous work on the radical scavenging activity of EGEO was not significantly active (Sacchetti, et al., 2005; Wei & Shibamoto, 2012). These results are in agreement with our results. Noumi and • colleagues tested in vitro DPPH antioxidant activity of EGEO and determined IC50=57 µg/mL (Noumi et al., 2011). In another study, EGEO exhibited a weak antioxidant capacity. The activity of the tested oils is lower

(IC50 = 33.33 ± 055 mg/mL) than that of the standard BHA (IC50 = 0.033 ± 0.002 mg/mL) (Harkat-Madouri et al., 2015). Fruit essential oil of E. globulus exhibited a weak antioxidant capacity (Said et al., 2016). Essential oils with relatively higher monoterpenic compound amounts showed that they are relatively ineffective in many assays (Wannes et al., 2010; Dzamic et al., 2013). EGEO were rich in monohydroxylated compounds such as 1,8-cineole, which is not capable to chelate ferrous ions. α-Pinene, β-pinene, limonene, β-myrcene, terpinolene and sabinene are known to have good antioxidant properties, however, individually they may exhibit low antioxidant activity depending on the mechanism involved (do Rosario Martins et al., 2014). So, the low activity of the tested EO can be explained by the abundance of the ineffective compounds (Wannes et al., 2010). In vitro lipoxygenase (LOX) enzyme inhibition The oil was evaluated for its potential in vitro anti-inflammatory activity compared to the standard NDGA.

The essential oil inhibited the enzyme with IC50= 58 ± 1,41 µg/mL, while IC50= 1,65 ± 0,05 μg/mL was observed for NDGA under the same conditions. The results showed that the oil is a moderate anti-inflammatory agent according to the in vitro evaluation. As it is well known, inflammation plays an important role in different pathophysiological conditions. 5-LOX is a lipid peroxidase enzyme, which is found in plants and animals, it plays a role in the synthesis of strong proinflammatory mediators at the second major arachidonic acid pathway (Kuhn 2000; Leone et al., 2007). EGEO was previously evaluated for its 15-LOX inhibitory activity where 53% inhibition was reported at 0.5 µg/mL (Wei & Shibamoto, 2012). Also, three Eucalyptus oils were evaluated for their in vivo anti- inflammatory effects, on the rat paw edema induced assay, where neutrophil migration into rat peritoneal cavities was induced by carrageenan (Silva et al., 2003). Conclusion In the present study, pharmaceutical grade E. globulus essential oil was evaluated for its in vitro antibacterial, antioxidant and 5-LOX inhibitory activities. The evaluated essential oil showed in vitro antibacterial activity,

Nat. Volatiles & Essent. Oils, 2020; 7(3): 1-11 Goger et al. DOI: 10.37929/nveo.759607

especially against the human pathogen S. epidermidis. Further detailed in vitro studies are required to confirm the effect of essential oil with different formulations against other pathogens as well.

ACKNOWLEDGEMENT

The authors would like to thank Anadolu University Scientific Research Project 1301S005 for the financial support. Part of this work was presented during “XXI. Bitkisel İlaç Hammaddeleri Toplantısı”, 28 May-01 June 2014, in Nevşehir Turkey. REFERENCES

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Nat. Volatiles & Essent. Oils, 2020; 7(3): 12-17 Salleh & Khamis DOI: 10.37929/nveo.746965

RESEARCH ARTICLE Chemical composition of the essential oil of Diospyros wallichii King & Gamble (Ebenaceae) Wan Mohd Nuzul Hakimi Wan Salleh1, * and Shamsul Khamis2

1Department of Chemistry, Faculty of Science and Mathematics, University Pendidikan Sultan Idris (UPSI), 35900 Tanjung Malim, Perak, MALAYSIA 2School of Environmental and Natural Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, MALAYSIA

*Corresponding author. Email: [email protected] Submitted: 02.06.2020; Accepted: 18.08.2020

Abstract The chemical composition of the essential oil from the leaves of Diospyros wallichii (Ebenaceae) growing in Malaysia was investigated for the first time. The essential oil was obtained by hydrodistillation and fully characterized by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 34 components (95.8%) were successfully identified in the essential oil which were characterized by high proportions of β-eudesmol (28.5%), caryophyllene oxide (9.5%), β-caryophyllene (7.2%), α-eudesmol (6.5%) and germacrene D (6.2%).

Keywords: Ebenaceae, Diospyros wallichii, essential oil, hydrodistillation, β-eudesmol, GC-MS

Introduction Essential oils are complex mixtures of volatile compounds, mainly terpenes and oxygenated aromatic and aliphatic compounds, such as phenols, alcohols, aldehydes, ketones, esters, ethers, and oxides, biosynthesized and accumulated in many plants (Dhifi et al., 2016). These naturally occurring mixtures of volatile compounds have been gaining increasing interest because of their wide range of applications in pharmaceutical, sanitary, cosmetics, perfume, food, and agricultural industries (Jugreet et al., 2020). The Ebenaceae family contains approximately 5 genera and 500 species. The genus Diospyros contains about 200 species of trees, which are mainly distributed in tropical and subtropical regions (Yang & Liu, 1999). The genus has versatile uses including edible fruits, valuable timber, and ornamental uses. The leaves of D. lotus are approved as Chinese Medicine for the treatment of stroke and apoplexy syndrome in China or used as a hypotensive drug in Japan (Yang et al., 2020). The dry leaves of D. kaki are used in the treatment of hypertension, angina and internal haemorrhage in China, and have been used traditionally in Korea to promote maternal health (Han et al., 2002). In Thailand, the roots of D. filipendula are used as treatment for croup in children and fever (Wisetsai et al., 2019). The biological activities from the leaves of these plants such as antioxidant, anti-inflammatory, analgesic, antidiabetic, antibacterial, and antihypertensive have been validated by means of an in vitro, in vivo, and clinical tests (Tameye et al., 2020; Byun et al., 2020; Sulub-Tun et al., 2020). The chemical analyses of Diospyros species revealed the presence of bioactive molecules such as benzoic acid derivatives, coumarins (Wisetsai et al., 2019), ent-kaurane diterpene (Feusso et al., 2017), and triterpenes (Rauf et al., 2016). Diospyros wallichii commonly known as tuba buah in Malaysia, is grows in lowland mixed dipterocarp forests and limestone hills from sea-level to 2,300 ft altitude. It is distributed mainly in India, Thailand, Peninsular Malaysia, Sumatra, and Borneo (Utsunomiya et al., 1998). This plant grows to about 10-20 m in height, whose round fruits are poisonous and used for fishing. The twigs are rusty- hairy when young. The inflorescences bear up to nine flowers. The fruits are round, up to 2.5 cm in diameter. The leaves are alternate, simple, penni-veined, and usually hairy below. The flowers ca. 2.7 mm diameter,

Nat. Volatiles & Essent. Oils, 2020; 7(3): 12-17 Salleh & Khamis DOI: 10.37929/nveo.746965

white-cream, with corolla tube, and placed in bundles in leaf axils (Smitinand, 2001). The Plant List includes two synonymous plant names of D. wallichii which are Diospyros bakhuisii Boerl. & Koord.-Schum. and Diospyros pulchrinervia Kosterm (The Plant List, 2012). There has been no information of this plant in traditional or folk medicine practice. Previous phytochemical investigations of D. wallichii have resulted in the isolation of naphthalene derivatives, naphthoquinones, coumarins, and triterpenes (Salae et al., 2010). In addition, the fruits extract of D. wallichii shown cytotoxicity effect against HTC116 colon carcinoma cell line (Nematollahi et al., 2012). Meanwhile, D. wallichii has not been previously studied for its essential oils. As a continuation part of our systematic evaluation of the aromatic flora of Malaysia (Salleh et al., 2014a, 2014b, 2014c, 2015, 2016a, 2016b, 2016c), we here report the volatile components of D. wallichii leaves. Materials and Methods Plant material Sample of Diospyros wallichii was collected from Gambang, Pahang in September 2019, and identified by Dr. Shamsul Khamis from Universiti Kebangsaan Malaysia (UKM). The voucher specimen (SK358/19) was deposited at UKMB Herbarium, Faculty of Science and Technology UKM. Isolation and analysis of essential oil The fresh leaf (300 g) was subjected to hydrodistillation in Clevenger-type apparatus for 4 hours. The essential oil obtained was dried over anhydrous magnesium sulfate and stored at 4-6°C. Gas chromatography (GC-FID) analysis was performed on an Agilent Technologies 7890B equipped with HP-5MS capillary column (30 m long, 0.25 μm thickness and 0.25 mm inner diameter). Helium was used as a carrier gas at a flow rate of 0.7 mL/min. Injector and detector temperatures were set at 250 and 280°C, respectively. The oven temperature was kept at 50°C, then gradually raised to 280°C at 5°C/min and finally held isothermally for 15 min. Diluted samples (1/100 in diethyl ether, v/v) of 1.0 μL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percent were reported as means ± SD of triplicates. Gas chromatography-mass spectrometry (GC-MS) analysis was recorded using a Hewlett Packard Model 5890A gas chromatography and a Hewlett Packard Model 5989A mass spectrometer. The GC was equipped with an HP-5 column. Helium was used as carrier gas at a flow rate of 1 mL/min. The injector temperature was 250°C. The oven temperature was programmed from 50°C (5 min hold) to 280°C at 10°C/min and finally held isothermally for 15 min. For GC-MS detection, an electron ionization system, with ionization energy of 70 eV was used. A scan rate of 0.5 s (cycle time: 0.2 s) was applied, covering a mass range from 50-400 amu. Identification of components For identification of essential oil components, co-injection with the standards (major components) were used, together with correspondence of retention indices and mass spectra with respect to those reported in Adams (2007). Semi-quantification of essential oil components was made by peak area normalization considering the same response factor for all volatile components. Percentage values were the mean of three chromatographic analyses. Results and Discussion The essential oil had a spicy odour and yielded 0.15% calculated from the fresh weight of the leaves. The GC- FID (Figure 1) and GC-MS analysis of the essential oil revealed the presence of 34 chemical components with the constitution of 95.8%.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 12-17 Salleh & Khamis DOI: 10.37929/nveo.746965

Table 1. Chemical composition of the essential oil of Diospyros wallichii RRIa RRIb Components Percentagec Identificationsd 935 932 α-Pinene 2.5 ± 0.2 RI, MS 978 975 β-Pinene 2.2 ± 0.1 RI, MS 1014 1015 α-Terpinene 1.4 ± 0.2 RI, MS 1025 1029 Limonene 1.2 ± 0.2 RI, MS 1095 1097 Linalool 1.8 ± 0.2 RI, MS 1185 1189 α-Terpineol 2.5 ± 0.2 RI, MS 1351 1350 α-Cubebene 0.2 ± 0.1 RI, MS 1386 1385 δ-Elemene 2.0 ± 0.2 RI, MS 1425 1420 β-Caryophyllene 7.2 ± 0.2 RI, MS, Std 1434 1435 α-Bergamotene 0.2 ± 0.1 RI, MS 1455 1453 α-Humulene 0.2 ± 0.1 RI, MS 1458 1458 Aromandendrene 1.2 ± 0.2 RI, MS 1472 1470 β-Selinene 1.0 ± 0.2 RI, MS 1478 1480 Germacrene D 6.2 ± 0.1 RI, MS, Std 1500 1501 Bicyclogermacrene 2.4 ± 0.2 RI, MS 1502 1500 α-Muurolene 1.8 ± 0.1 RI, MS 1528 1528 α-Calacorene 0.4 ± 0.1 RI, MS 1529 1530 δ-Cadinene 4.2 ± 0.2 RI, MS 1563 1565 (E)-Nerolidol 3.2 ± 0.2 RI, MS 1580 1582 Caryophyllene oxide 9.5 ± 0.2 RI, MS, Std 1580 1581 Spathulenol 0.5 ± 0.1 RI, MS 1592 1595 Viridiflorol 0.8 ± 0.2 RI, MS 1600 1602 Guaiol 0.2 ± 0.2 RI, MS 1635 1639 α-Eudesmol 6.5 ± 0.1 RI, MS, Std 1640 1640 τ-Cadinol 1.2 ± 0.1 RI, MS 1650 1650 β-Eudesmol 28.5 ± 0.1 RI, MS, Std 1688 1685 α-Bisabolol 1.6 ± 0.2 RI, MS 1700 1700 Heptadecane 0.4 ± 0.1 RI, MS 1720 1722 Dodecanal 0.2 ± 0.1 RI, MS 1762 1765 (E)-2-Undecenal 0.8 ± 0.2 RI, MS 1945 1940 Phytol 1.5 ± 0.2 RI, MS 2400 2400 n-Tetracosane 0.2 ± 0.1 RI, MS 2500 2500 n-Pentacosane 0.2 ± 0.1 RI, MS 2931 2930 Hexadecanoic acid 1.9 ± 0.2 RI, MS Monoterpene hydrocarbons 7.3 ± 0.1 Oxygenated monoterpenes 4.3 ± 0.1 Sesquiterpene hydrocarbons 27.0 ± 0.2 Oxygenated sesquiterpenes 52.0 ± 0.2 Others 5.2 ± 0.1 Total identified 95.8 ± 0.2 a b Linear retention index, experimentally determined using homologous series of C6-C30 alkanes. Linear retention index taken from Adams (2007). cRelative percentage values are means of three determinations ±SD. dIdentification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, Adams, FFNSC2, and NIST08 MS databases; RI, based on comparison of calculated RI with those reported in Adams, FFNSC2 and NIST08.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 12-17 Salleh & Khamis DOI: 10.37929/nveo.746965

Figure 1. GC chromatogram of essential oil of Diospyros wallichii King & Gamble

β-Caryophyllene Caryophyllene oxide

Germacrene D β-Eudesmol α-Eudesmol

Oxygenated sesquiterpene hydrocarbons were the most dominant components in the essential oil accounting for 52.0%, followed by sesquiterpene hydrocarbons indicated as 27.0%. Besides, monoterpene hydrocarbons were present in appreciable amounts which accounted for 1.3-7.3% of the total composition. The most abundant components of the essential oil were β-eudesmol (28.5%), caryophyllene oxide (9.5%), β-caryophyllene (7.2%), α-eudesmol (6.5%), and germacrene D (6.2%). The other minor components detected in the essential oil in more than 2% were δ-cadinene (4.2%), (E)-nerolidol (3.2%), α-pinene (2.5%), α-terpineol (2.5%), bicyclogermacrene (2.4%), β-pinene (2.2%), and δ-elemene (2.0%). The genus Diospyros is still poorly explored as far as its essential oil composition is concerned. A review of the existing literature on essential oils of the genus Diospyros revealed the presence of a few studies. The essential oils of D. blancoi (Pino et al., 2008) and D. malabarica (Viswanathan et al., 2002) shown the foremost components were benzyl butyrate (33.9%) and trans-α-methyl isoeugenol (31.5%), respectively. In addition, two studies have been reported from D. discolor essential oils, which discovered benzyl salicylate (26.9%) and (2Z,6E)-farnesol (35.0%) as the major components (Su et al., 2015; Smith & Belardo, 1992). The most abundant component D. wallichii essential oil are endowed with important biological activities. For instance, β-eudesmol, which is one of the main volatile components of the Chinese traditional herb Atractylodis lancea (Asteraceae). It has shown to induce neurite outgrowth in rat pheochromocytoma cells, thus being a promising lead compound for potential neuronal function (Obara et al., 2002). Besides, it shows antiepileptic action in electroshock seizure mice (Chiou et al., 1997) and ameliorated conditions in mice after intoxication from organophosphorus anticholinesterase agents (Chiou et al., 1995). The oral administration of β-eudesmol ameliorated gastrointestinal motility in mice (Kimura & Sumiyoshi, 2012). In another study, β- eudesmol showed anticancer (Plengsuriyakarn et al., 2015) and antiangiogenic (Tsuneki et al., 2005) activities, whereas the intravenous administration of β-eudesmol caused a dose-dependent decreasing of blood pressure in rats (Lim & Kee, 2005). In conclusion, this is the first report of the chemical composition of the essential oil from Diospyros wallichii and the results of this study could contribute to the valorisation of this Malaysian aromatic and medicinal plant.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 12-17 Salleh & Khamis DOI: 10.37929/nveo.746965

ACKNOWLEDGMENT

The authors would like to thank the Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris for research facilities. REFERENCES

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Nat. Volatiles & Essent. Oils, 2020; 7(3): 18-28 Sen et al. DOI: 10.37929/nveo.747855

RESEARCH ARTICLE Essential oil composition of different parts of Tanacetum cilicicum (Boiss.) Grierson Ali Şen1*, Mine Kürkçüoğlu2, Leyla Bitiş1, Ahmet Doğan3 and Kemal Hüsnü Can Başer2,4

1 Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, İstanbul, TURKEY 2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, TURKEY 3 Department of Pharmaceutical Botany, Faculty of Pharmacy, Marmara University, Istanbul, TURKEY 4 Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, N. CYPRUS

*Corresponding author. Email: [email protected]; [email protected] Submitted: 04.06.2020; Accepted: 18.08.2020

Abstract The aim of this study was to investigate and compare the chemical composition of essential oils obtained by Clevenger-type apparatus from capitula (TCC) and aerial parts including leaves and stems (TCA) of Tanacetum cilicicum (Boiss.) Grierson from Turkey. TCC and TCA essential oils were separately analysed by GC-FID and GC/MS. Forty and thirty-two compounds were identified, representing 99% and 94.4% of the oils of TCC and TCA essential oils, respectively. The main constituents of the oil of the TCC were camphor (28.8%), (E)-nerolidol (16.9%), trans-chrysanthenyl acetate (12.8%) and 1,8-cineole (8.9%), whereas the oil from TCA comprised mainly trans-chrysanthenyl acetate (22.2%), borneol (19.4%), camphor (11.2%), 1,8-cineole (10.2%) and (E)- nerolidol (9.2%). The results showed that TCC and TCA essential oils were rich in oxygenated monoterpenes and oxygenated sesquiterpenes. This study is the first to study the essential oil composition of different parts of Tanacetum cilicicum separately. Also, to the our best of our knowledge, (E)-nerolidol was found in the Tanacetum species as the major compound for the first time.

Keywords: Tanacetum cilicicum, essential oil, 1,8-cineole, borneol, camphor, (E)-nerolidol, trans-chrysanthenyl acetate

Introduction The genus Tanacetum comprises forty-six species in Turkey and nineteen of which are endemic (NGBB, 2019). Tanacetum species are used in the treatment of abdominal pain, ulcer, lung diseases, colds, shortness of breath, bronchitis, flu, throat diseases, hoarseness, diabetes, scabies, boils, inflammatory skin diseases, itching, kidney stone, rheumatism, migraine, infertility, menstrual disorders and also as carminative, facilitating digestion appetizing, expectorant, wound healing, antipyretic, tonic, anthelmintic. Some Tanacetum species have been reported to possess anticancer, antimicrobial, anti-inflammatory and antioxidant activities (Mantle et al., 2000; Rosselli et al., 2012). Tanacetum species contain essential oils (Table 1), flavonoids and sesquiterpene lactones as secondary metabolites (Tabanca et al., 2007; Gören et al., 2002; Polatoğlu et al., 2010a). Previous phytochemical studies reported that Tanacetum cilicicum, known as “Kaba pireotu” in Turkey, contained phenolic compounds [p-Coumaric acid, ferulic acid, gallic acid, gentisic acid, chlorogenic acid, chrysin, galangin, quercetin, naringenin, catechin] and essential oils [Bicyclo (3,1,1) hept-2-en-4-ol, camphor, 1,8-cineole, 1,6,10-dodecatrien-3-ol, linalool, sesquisabinene hydrate] (Gecibesler et al., 2016; Ulukanli et al., 2017). Also, this species was found to have antioxidant (Gecibesler et al., 2016; Arıtuluk et al., 2016; Yıldırım et al., 2019), anti-inflammatory (Yıldırım et al., 2019), cholinesterase inhibitory (Orhan et al., 2015), antimicrobial (Ulukanli et al., 2017) and phytotoxic (Ulukanli et al., 2017) activities. Although there are reports on the chemical composition of essential oil obtained from the aerial parts of Tanacetum cilicicum, the current study was aimed to investigate comparatively for the first time the

Nat. Volatiles & Essent. Oils, 2020; 7(3): 18-28 Sen et al. DOI: 10.37929/nveo.747855

chemical composition of essential oils obtained separately from capitulum and aerial parts excluding capitulum.

Table 1. Previous studies on essential oil of Tanacetum species from Turkey

Tanacetum species Part distilled Main components (%) Reference T. cilicicum (Boiss.) Grierson Aerial parts Bicyclo(3,1,1)hept-2-en-4-ol (Gecibesler et al., (21.9%) 2016) Camphor (15.6%) 1,8-cineole (13.5%) 1,6,10-dodecatrien-3-ol (7.9%) T. cilicicum (Boiss.) Grierson Aerial parts Eucalyptol (5.1%) (Ulukanli et al., 2017) Linalool (7.0%) Sesquisabinene hydrate (6.9%) T. mucroniferum Hub.-Mor. & Grierson Capitula 1,8-cineole (21.9%) (Polatoglu et al., Camphor (6.4%) 2012a) T. nitens (Boiss. & Noë) Grierson Aerial parts 1,8-cineole (27.6%) (Bagci et al., 2010) T. argenteum (Lam.) Willd. subsp. argenteum (L.) Aerial parts α-pinene (27.9%) (Bagci et al., 2010) All Santolinatriene (8.8%) 1,8-cineole (6.8%) T. argenteum (Lam.) Willd. subsp. flabellifolium Aerial parts β-thujone (47.1%) (Kose et al., 2017) (Boiss. & Heldr.) Grierson α-pinene (19.1%) α-thujone (10.5%) T. kotschyi (Boiss.) Grierson Capitula Artemisia ketone (54.6%) (Polatoglu et al., Longiverbenone (vulgarone B) 2011a) (9.2%) Stem Artemisia ketone (26.5%) Longiverbenone (vulgarone B) (8.9%) Artemisia alcohol (5.2%) Intermedeol (9.0%) T. tomentellum (Boiss.) Grierson Aerial parts Camphor (9.4%) (Tabanca et al., 2016) Linalool (7.6%) α-terpineol (7.1%) Trans-pinocarveol (5.3%) T. zahlbruckneri (Náb.) Grierson Capitula Germacrene D (29.7%) (Polatoğlu et al., Spathulenol (12.0%) 2011b) T. tabrisianum (Boiss.) Sosn. & Takht. Capitula 1,8-cineole (17.6%) (Polatoğlu et al., Hexadecanoic acid (10.3%) 2011b) Decanoic acid (5.8%) Trans-linalooloxide acetate (5.3%) Stem 1,8-cineole (22.5%) Hexadecanoic acid (8%) T. alyssifolium (Bornm.) Grierson Aerial parts Borneol (35.2%) (Kandemir et al., α-thujone (24.6%) 2008) Camphor (12.4%) β-eudesmol (6.1%) T. argenteum (Lam.) Willd. subsp. flabellifolium Aerial parts α-Pinene (29.0%) (Tabanca et al., 2007) (Boiss. & Heldr.) Grierson (E)-sesquilavandulol (16.0%) Camphor (14.0%) T. chiliophyllum (Fisch. & Mey.) Schultz Bip. var. Capitula Camphor (32.5%) (Polatoğlu et al., chiliophyllum (Fisch. & Mey.) Schultz Bip. from Chamazulene (9.2%) 2012b)

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Muradiye sample 1 Stem 1,8-Cineole (16.1%) Camphor (36.2%) T. chiliophyllum (Fisch. & Mey.) Schultz Bip. var. Capitula 1,8-Cineole (12%) (Polatoğlu et al., chiliophyllum (Fisch. & Mey.) Schultz Bip. from p-Cymene (5.4%) 2012b) Muradiye sample 1 Terpinen-4-ol (10.3%) (E)-Sesquilavandulol (5.8%) Stem 1,8-Cineole (18.4%) p-Cymene (5.4%) Terpinen-4-ol (9.0%) T. chiliophyllum (Fisch. & Mey.) Schultz Bip. var. Capitula α-Pinene (5.3%) (Polatoğlu et al., chiliophyllum (Fisch. & Mey.) Schultz Bip. from 1,8-Cineole (22.1%) 2012b) Güzeldere Terpinen-4-ol (6.5%) Stem 1,8-Cineole (28.9%) Terpinen-4-ol (5.6%) T. densum (Labill.) Sch.Bip. subsp. eginense Capitula Camphor (30.9%) (Polatoğlu et al., Heywood 1,8-Cineole (12.4%) 2012c) Camphene (10.6%) α-pinene (7.0%) Neodihydrocarveol (5.1%) An unidentified compound (11.5%) Stem Camphor (25.7%) Bornyl acetate (9.4%) Borneol (5.1%) An unidentified compound (27.2%) Leaf Camphor (27.7%) Camphene (7.0%) Bornyl acetate (11.8%) α-pinene (5.3%) Borneol (5.2%) An unidentified compound (20.5%) T. parthenium (L.) Sch.Bip. (Sample 1) Capitula and Camphor (49.0%) (Polatoğlu et al., stem Trans-chrysanthenyl acetate 2010b) (22.1%) Camphene (9.4%) T. parthenium (L.) Sch.Bip. (Sample 2) Capitula and Camphor (60.8%) stem Camphene (6.8%) T. argyrophyllum (C. Koch) Tvzel. var. Leaf 1,8-Cineole (11.1%) (Gören et al., 2001) argyrophyllum (C. Koch) Tvzel. α-Thujone (51.8%) T. argyrophyllum (C. Koch) Tvzel. var. Capitula α-Thujone (62.8%) argyrophyllum (C. Koch) Tvzel. T. argenteum (Lam.) Willd. subsp. canum (C. Leaf α-Thujone (11.9%) Koch) Grierson var. canum (C. Koch) Grierson β-Caryophyllene (5.1%) Caryophyllene oxide (12.6%) T. praeteritum (Horwood) Heywood subsp. Aerial parts β-Pinene (5.7%) praeteritum (Horwood) Heywood 1,8-Cineole (12.3%) Bornyl acetate (10.0%) Terpinen-4-ol (7.1%) Borneol (28.1%) T. praeteritum (Horwood) Heywood subsp. Aerial parts α-Thujone (51.1%) massicyticum Heywood β-Thujone (10.0%)

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T. argenteum (Lam.) Willd. subsp. argenteum (L.) Leaf α-Pinene (36.7%) (Polatoğlu et al., All β -Pinene (27.5%) 2010a) 1,8-Cineole (9.8%) T. densum (Labill.) Sch.Bip. subsp. amani Leaf α-Pinene (9.7%) Heywood β -Pinene (27.2%) 1,8-Cineole (13.1%) p-Cymene (8.9%) Lavandulyl acetate (8.1%) T. macrophyllum (Waldst. & Kit.) Sch. Bip. Aerial parts β-eudesmol (89.5%) (Javidnia et al., 2010) T. armenum (DC.) Sch.Bip. Leaves 1,8-Cineole (31.3%) (Başer et al., 2001) Camphor (8.6%) α-Terpineol (5.5%) T. armenum (DC.) Sch.Bip. Aerial parts 1,8-Cineole (11.3%) Camphor (26.7%) Borneol (10.6%) γ-Eudesmol (5.5%) T. balsamita L. Aerial parts α-Thujone (11.7%) Carvone (52.4%) T. chiliophyllum Fisch. & Mey.) Schultz Bip. var. Capitula α-Thujone (12.5%) chiliophyllum Fisch. & Mey.) Schultz Bip. Camphor (16.8%) cis-Chrysanthenyl acetate (16.3%) T. haradjani (Rech.fil.) Grierson Leaves 1,8-Cineole (10.0%) Camphor (15.9%) Terpinen-4-ol (6.7%) T. cadmeum (Boiss.) Heywood (using Direct Leaves p-Cymene (11.9%) (Gogus et al., 2009) Thermal Desorption Technique- DTD) Eucalyptol (27.0%) Ascaridol (22.4%) Unknown (5.0%) T. cadmeum (Boiss.) Heywood (using Direct Capitula 2-Carene (6.2%) Thermal Desorption Technique- DTD) p-Cymene (9.3%) γ-Terpinene (15.3%) Ascaridol (39.8%) T. cadmeum (Boiss.) Heywood (using Leaves and p-Cymene (7.2%) hydrodistillation-HD) capitula Eucalyptol (12.0%) Verbenone (5.7%) Ascaridol (46.0%) Unknown (5.2%) T. cadmeum (Boiss.) Heywood subsp. orientale Aerial part 1,8-Cineole (18.9%) (Özek et al., 2007) Grierson p-cymene (15.7%) Terpinen-4-ol (14.8%) Borneol (9.8%) T. macrophyllum (Waldst. & Kit.) Sch.Bip. Aerial part Camphor (5.8%) (Demirci et al., 2007) cis-chrysanthenol (12.0%) Copaborneol (5.6%) β−eudesmol (21.4%) T. argyrophyllum (C. Koch) Tvzel. var. Capitula 1,8-Cineole (8.4%) (Polatoğlu et al., argyrophyllum (C. Koch) Tvzel. Camphor (29.7%) 2010c) Bornyl acetate (6.1%) Borneol (12.0%) T. argyrophyllum (C. Koch) Tvzel. var. Stem 1,8-Cineole (17.5%) argyrophyllum (C. Koch) Tvzel. Camphor (26.6%)

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Borneol (15.0%) T. macrophyllum (Waldst. & Kit.) Sch.Bip. Capitula (E)-Sesquilavandulol (20.3%) (Polatoğlu et al., γ-Eudesmol (21.5%) 2015) Copaborneol (8.5%) Leaf 1,8-Cineole (11.0%) β-Thujone (9.0%) Bornyl acetate (9.6%) Borneol (6.3%) Copaborneol (14.1%)

Materials and Methods Plant material Aerial parts of plant were collected in the flowering period from Pülümür district of Tunceli province of Turkey in 2017 and identified by one of us (AD). Voucher specimens were deposited in the Herbarium of the Faculty of Pharmacy, Marmara University (MARE No: 17768). Isolation of essential oil Capitula (100 g) and aerial parts including leaves and stem (200 g) were separately hydrodistilled for 4 hours using a Clevenger apparatus. The essential oils obtained were stored at 4°C in the dark until analysed. GC and GC-MS conditions The oils were analysed by Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC/MS) using an Agilent GC-MSD system (Mass Selective Detector- MSD). GC-MS analysis The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system (Agilent, USA; SEM Ltd., Istanbul, Turkey). Innowax FSC column (60m x 0.25mm, 0.25m film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60C for 10 min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10 min and then programmed to 240C at a rate of 1C/min. Split ratio was adjusted 40:1. The injector temperature was at 250C. The interphase temperature was at 280C. MS were taken at 70 eV. Mass range was from m/z 35 to 450. GC-FID analysis

GC-FID analyses were performed using an Agilent 6890N GC system. FID temperature was set to 300C and the same operational conditions were applied to a triplicate of the same column used in GC–MS analyses. Simultaneous auto injection was done to obtain equivalent retention times. Relative percentages of the separated compounds were calculated from integration of the peak areas in the GC-FID chromatograms. Identification of compounds The components of essential oils were identified by comparison of their mass spectra with those in the Baser Library of Essential Oil Constituents, Adams Library (Adams, 2007), MassFinder Library (Hochmuth, 2008), Wiley GC/MS Library (McLafferty & Stauffer, 1989) and confirmed by comparison of their retention indices. These identifications were accomplished by comparison of retention times with authentic samples or by comparison of their relative retention index (RRI) to a series of n-alkanes. Alkanes (C8-C29) were used

Nat. Volatiles & Essent. Oils, 2020; 7(3): 18-28 Sen et al. DOI: 10.37929/nveo.747855

as reference points in the calculation of relative retention indices (RRI) (Curvers et al., 1985). Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The analysis results are expressed as mean percentage ± standard deviation (SD) (n = 3) as listed in Table 2 and 3. Results and Discussion The aim of our study was to comparatively investigate the chemical composition of water-distilled essential oils obtained from capitula (TCC) and aerial parts including leaves and stems (TCA) of Tanacetum cilicicum (Boiss.) Grierson from Turkey. Forty and thirty-two compounds were identified, representing 99% and 94.4% of the oils of TCC and TCA, respectively. The main compounds of the oil of the TCC were identified as camphor (28.8%), (E)-nerolidol (16.9%), trans-chrysanthenyl acetate (12.8%) and 1,8-cineole (8.9%), whereas trans-chrysanthenyl acetate (22.2%), borneol (19.4%), camphor (11.2%), 1,8-cineole (10.2%) and (E)-nerolidol (9.2%) were found as major compounds in the essential oil of TCA (Table 2 and 3).

Table 2. Essential oil composition of capitula of Tanacetum cilicicum IM No RRI Compounds % Identification method 1 1014 Tricyclene 0.1 ± 0.0 MS

2 1032 α-Pinene 0.9 ± 0.1 tR, MS 3 1035 α-Thujene 0.2 ± 0.0 MS

4 1076 Camphene 2.0 ± 0.1 tR, MS

5 1118 β-Pinene 0.4 ± 0.0 tR, MS

6 1132 Sabinene 0.8 ± 0.0 tR, MS

7 1188 α-Terpinene 0.1 ± 0.0 tR, MS

8 1203 Limonene 0.6 ± 0.0 tR, MS

9 1213 1,8-Cineole 8.9 ± 0.1 tR, MS

10 1255 γ-Terpinene 0.3 ± 0.0 tR, MS

11 1280 p-Cymene 0.7 ± 0.0 tR, MS 12 1445 Filifolone 0.2 ± 0.0 MS 13 1484 trans-Chrysanthenol 2.7 ± 0.0 MS

14 1532 Camphor 28.8 ± 0.1 tR, MS 15 1538 trans-Chrysanthenyl acetate 12.8 ± 0.1 MS

16 1553 Linalool 2.7 ± 0.0 tR, MS

17 1590 Bornyl acetate 0.3 ± 0.1 tR, MS

18 1611 Terpinen-4-ol 1.6 ± 0.0 tR, MS 19 1667 cis-Verbenol 0.4 ± 0.1 MS 20 1683 trans-Verbenol 1.5 ± 0.0 MS

21 1706 α-Terpineol 2.9 ± 0.1 tR, MS

22 1719 Borneol 3.9 ± 0.1 tR, MS 23 1747 p-Mentha-1,5-dien-8-ol 0.6 ± 0.1 MS

24 1772 δ-Cadinene 0.4 ± 0.0 tR, MS 25 1783 Campholene alcohol 0.4 ± 0.0 MS 26 1875 Benzyl 2-methylbutyrate 0.4 ± 0.0 MS

27 1916 Benzyl isovalerate 0.6 ± 0.0 tR, MS 28 1988 Nerolidol oxide I 0.5 ± 0.0 MS

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29 2008 Caryophyllene oxide 0.6 ± 0.1 tR, MS 30 2016 Nerolidol oxide II 0.5 ± 0.1 MS

31 2041 (E)-nerolidol 16.9 ± 0.2 tR, MS 32 2096 Elemol 1.0 ± 0.1 MS 33 2174 Fokienol 0.3 ± 0.0 MS 34 2187 γ-Eudesmol 1.0 ± 0.1 MS 35 2191 T-Cadinol 1.1 ± 0.1 MS 36 2246 α-Eudesmol 0.5 ± 0.0 MS

37 2255 α-Cadinol 0.6 ± 0.1 tR, MS 38 2256 β-Eudesmol 0.5 ± 0.1 MS 39 2273 Selina-11-en-4α-ol 0.6 ± 0.1 MS 40 2296 Decanoic acid 0.2 ± 0.0 MS Total identified compounds (%) 99.5 Monoterpene hydrocarbons 6.1 Oxygenated monoterpenes 67.7 Sesquiterpene hydrocarbons 0.4 Oxygenated sesquiterpenes 24.1 Aromatic ester 1.0 Saturated fatty acid 0.2 RRI; Relative retention indices calculated against n-alkanes C8-C29. %; calculated from the FID chromatograms and expressed as mean ± SD (n = 3). tr; Trace (<0.1 %); tR, identification based on the retention times (tR) of genuine compounds on the HP Innowax column; MS, identified on the basis of computer matching of the mass spectra.

Table 3. Essential oil composition of aerial parts including leaves and stem of Tanacetum cilicicum IM No RRI Compounds % Identification Method 1 1014 Tricyclene 0.1 ± 0.0 MS

2 1032 α-Pinene 1.1 ± 0.0 tR, MS 3 1035 α-Thujene 0.2 ± 0.0 MS

4 1076 Camphene 1.8 ± 0.0 tR, MS

5 1118 β-Pinene 1.9 ± 0.0 tR, MS

6 1132 Sabinene 0.7 ± 0.0 tR, MS

7 1203 Limonene 0.5 ± 0.0 tR, MS

8 1213 1,8-Cineole 10.2 ± 0.1 tR, MS

9 1255 γ-Terpinene 0.4 ± 0.1 tR, MS

10 1280 p-Cymene 0.6 ± 0.0 tR, MS 11 1484 trans-Chrysanthenol 1.7 ± 0.0 MS

12 1532 Camphor 11.2 ± 0.1 tR, MS 13 1538 trans-Chrysanthenyl acetate 22.2 ± 0.1 MS

14 1553 Linalool 0.5 ± 0.0 tR, MS 15 1586 Pinocarvone tr MS

16 1590 Bornyl acetate 2.2 ± 0.1 tR, MS

17 1611 Terpinen-4-ol 1.4 ± 0.0 tR, MS 18 1667 cis-Verbenol tr MS 19 1683 trans-Verbenol 1.5 ± 0.0 MS

20 1706 α-Terpineol 2.9 ± 0.0 tR, MS

21 1719 Borneol 19.4 ± 0.1 tR, MS

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22 1772 δ-Cadinene 0.4 ± 0.1 tR, MS 23 1786 ar-Curcumene 0.4 ± 0.1 MS 24 1988 Nerolidol oxide I tr MS

25 2008 Caryophyllene oxide 1.8 ± 0.0 tR, MS 26 2016 Nerolidol oxide II tr MS

27 2041 (E)-Nerolidol 9.2 ± 0.2 tR, MS 28 2191 T-Cadinol 1.6 ± 0.0 MS 29 2246 α-Eudesmol tr MS

30 2255 α-Cadinol 0.3 ± 0.1 tR, MS 31 2256 β-Eudesmol 0.7 ± 0.1 MS 32 2273 Selina-11-en-4α-ol 1.1 ± 0.1 MS Total identified compounds (%) 96.0 Monoterpene hydrocarbons 7.3 Oxygenated monoterpenes 73.2 Sesquiterpene hydrocarbons 0.8 Oxygenated sesquiterpenes 14.7 RRI; Relative retention indices calculated against n-alkanes C8-C29. %; calculated from the FID chromatograms and expressed as mean ± SD (n = 3). tr; Trace (<0.1 %); tR, identification based on the retention times (tR) of genuine compounds on the HP Innowax column; MS, identified on the basis of computer matching of the mass spectra.

The terpenoids consist of the main portion of essential oils and oxygenated monoterpenes (67.7%) were the main group of constituents of TCC oil, followed by oxygenated sesquiterpenes (24.1%), monoterpene hydrocarbons (6.1%), aromatic ester (1%), sesquiterpene hydrocarbons (0.4%), saturated fatty acid (0.2%). The major groups of compounds of TCA oil were oxygenated monoterpenes (73.2%), followed by oxygenated sesquiterpenes (14.7%), monoterpene hydrocarbons (7.3%), sesquiterpene hydrocarbons (0.8%) (Table 2 and 3). There are only two reports on the composition of essential oil of Tanacetum cilicicum. In these studies, the chemical content of the essential oils obtained from the aerial parts containing the capitulum, stem and leaves of the plants was studied. Gecibesler et al. (2016) found bicyclo (3,1,1) hept-2-en-4-ol (21.9%), camphor (15.6%), 1,8-cineole (13.5%), 1,6,10-dodecatrien-3-ol (7.9%) as major compounds in oil, while Ulukanli et al. (2017) reported 1,8-cineole (eucalyptol) (5.1%), linalool (7.0%), sesquisabinene hydrate (6.9%) as major compounds. Contrary to these studies, in our current study, the chemical analysis of the essential oils T. cilicicum was performed separately for capitula and aerial parts (stem and leaves). However, similar to previous studies, camphor and 1,8-cineole were found to be major compounds in the essential oils (Gecibesler et al.,2016). The plant materials used in both studies were collected from the same areas. This may explain the similarity of chemical compositions of both oils. In addition, the major components of T. cilicicum essential oils have also been previously reported from other Tanacetum species. Briefly, camphor in the essential oils of T. mucroniferum (Polatoglu et al., 2012a), T. tomentellum (Tabanca et al., 2016), T. alyssifolium (Kandemir et al., 2008), T. argenteum subsp. flabellifolium (Tabanca et al., 2007), T. chiliophyllum var. chiliophyllum (Polatoğlu et al., 2012b; Başer et al., 2001), T. densum subsp. eginense (Polatoğlu et al., 2012c), T. parthenium (Polatoğlu et al., 2010b), T. armenum (Başer et al., 2001), T. haradjani (Başer et al., 2001), T. macrophyllum (Demirci et al., 2007), T. argyrophyllum var. argyrophyllum (Polatoğlu et al., 2010c); trans-chrysanthenyl acetate in the essential oil of T. parthenium (Polatoğlu et al., 2010b); 1,8-cineole in the essential oil of T. mucroniferum (Polatoğlu et al., 2012a), T. nitens (Bagci et al., 2010), T. argenteum subsp. argenteum (Bagci et al., 2010), T. tabrisianum

Nat. Volatiles & Essent. Oils, 2020; 7(3): 18-28 Sen et al. DOI: 10.37929/nveo.747855

(Polatoğlu et al., 2011b), T. chiliophyllum var. chiliophyllum (Polatoğlu et al., 2012b), T. densum subsp. eginense (Polatoglu et al., 2012c), T. argyrophyllum var. argyrophyllum (Gören et al., 2001), T. praeteritum subsp. praeteritum (Gören et al., 2001), T. argenteum subsp. argenteum (Polatoğlu et al., 2010a), T. densum subsp. amani (Polatoğlu et al., 2010a), T. armenum (Başer et al., 2001), T. haradjani (Başer et al., 2001), T. cadmeum (Gogus et al., 2009), T. cadmeum subsp. orientale (Özek et al., 2007), T. argyrophyllum var. argyrophyllum (Polatoğlu et al., 2010c), T. macrophyllum (Polatoğlu et al., 2015); borneol in the essential oils of T. alyssifolium (Kandemir et al., 2008), T. densum subsp. eginense (Polatoglu et al., 2012c), T. praeteritum subsp. praeteritum (Gören et al., 2001), T. armenum (Başer et al., 2001), T. cadmeum subsp. orientale (Özek et al., 2007), T. argyrophyllum var. argyrophyllum (Polatoğlu et al., 2010c), T. macrophyllum (Polatoğlu et al., 2015) have been found in previous studies. On the other hand, although (E)-nerolidol was found as a minor compound in the essential oil of different Tanacetum species (Ulukanli et al., 2017; Polatoğlu et al., 2012a; Polatoğlu et al., 2012b; Başer et al., 2001; Özek et al., 2007; Demirci et al., 2007; Polatoğlu et al., 2010c; Başer et al., 2001), it was found to be a major compound in the current study. Generally, essential oils of Tanacetum species are characterized by high amounts of monoterpene compounds (Table 1). The essential oil of T. cilicicum in our current study was found to be rich in monoterpene compounds. Conclusion The present study showed that essential oils of TCC and TCA were rich in oxygenated monoterpenes and oxygenated sesquiterpenes. Also, the results showed that (E)-nerolidol was found, for the first time, as a major compound in the Tanacetum oils. REFERENCES

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RESEARCH ARTICLE Chemodiversity of wild populations of aromatic plants as source of valuable essential oil profiles. A study on Thymus vulgaris L. from Valencia (Spain) Juan Antonio Llorens-Molina1,*, Sandra Vacas2, Enrique Burgals Royo3, María Pilar Santamarina Siurana4, Mercedes Verdeguer Sancho1

1Mediterranean Agroforestry Institute, Universitat Politècnica de València, CPI Bldg. 8E (acc.F), Camí de Vera, s/n, 46022, València, SPAIN 2Centre for Agricultural Chemical Ecology (Mediterranean Agroforestry Institute), Universitat Politècnica de València, Bldg. 6C, Camí de Vera, s/n, 46022, València, SPAIN 3School of Agricultural Engineering and Environment, Universitat Politècnica de València, Bldg. 3P, Camí de Vera, s/n, 46022, València, SPAIN 4Department of Agroforest Ecosystems, Universitat Politècnica de València, Bldg. 3J, Camí de Vera, s/n, 46022, València, SPAIN

*Corresponding author. Email: [email protected] Submitted: 17.04.2020; Accepted: 23.08.2020

Abstract Chemodiversity of wild populations of aromatic plants is a valuable source of essential oils, whose composition may be suitable for specific purposes according their biological activity. Furthermore, knowing the intrapopulational variability based on individual analysis has allowed characterizing atypical profiles, which can reach high levels of active compounds. Obviously, it requires the treatment of a high number of individual samples. In this work, a methodology to characterize T. vulgaris profiles in an area of recognized biodiversity was proposed and applied. After Thin Layer Chromatography (TLC) screening data of 85 individual samples, 7 groups, and 13 individuals were classified. Then, 20 samples were subjected to GC/MS and GC/FID analysis, respectively. These data were subjected to Hierarchical Agglomerative, Discriminant Analysis and ANOVA, which finally highlighted five profiles: (1) based on the camphane skeleton (camphene, camphor and borneol), (2) rich in the oxygenated sesquiterpenic fraction, (3) rich in 1,8- cineole, with appreciable amounts of camphor and borneol (typical chemotype from Eastern Iberian Peninsula), (4) camphor and terpinen-4-ol as major compounds, and (5) linalool chemotype. It should be noted that the percentages of the main compounds in these groups were higher than some of those described in the literature for similar chemotypes. In summary, the preliminary screening by TLC, grouping individuals with similar profiles, allowed establishing a quick first approximation to the chemodiversity of T. vulgaris in the studied area. Furthermore, the analysis of unclassified and potentially atypical individuals has also provided valuable information to establish the final profiles.

Keywords: T. vulgaris, thin layer chromatography, chemodiversity, atypical individuals, wild populations

Introduction Over millions of years of evolution, the nature has provided us with an enormous amount of secondary plant metabolites whose biological activity makes them potentially useful natural resources (Verpoorte, 1998; Cordell, 2000; Bakkali et al., 2008). Among them, essential oils (EOs) are particularly promising from the perspective of sustainability and environmental protection. Indeed, during the last two decades an extensive review literature is available on specific fields such as food processing and conservation (Sanchez-González et al., 2011; Calo et al., 2015, Anupama et al., 2019), pest and weed control in agriculture (Tworkoski, 2002; Tripathi et al., 2009), or aromatherapy and health care (Edris, 2007; Koo, 2017). Comparing the cultivation of aromatic plants for EOs production with their collection from wild populations, the first allows to fulfil quality requirements due to the standardization of their chemical profiles (Kindlovits & Németh, 2012). On the other hand, it also contributes to improve the biodiversity conservation, especially

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

as a consequence of the protection of certain species, sometimes endemic. In this way, the selection and domestication of plants from wild populations to create chemically uniform cultivars has been widely applied in numerous aromatic plants (Bernáth, 2002; Dudai, 2011). With respect to Thymus species, some experiences have been reported in a great diversity of environmental conditions (Salas, 2014). Concerning Thymus vulgaris L. (thyme), the work reported by Delpit (2000) on the clonal selection of sabinene hydrate rich individuals, and the improvement of its micropropagation in vitro (El-Banna, 2017), can be cited as representative examples. The high intrapopulational chemodiversity of EOs coming from wild populations of many species of aromatic and medicinal plants can be considered as an opportunity for finding particularly useful genotypes (Németh, 2016). Furthermore, to study in which way each genotype is affected by environmental and ontogenetic factors is also required to ensure the chemical identity of new developed cultivars (Figueiredo et al., 2008). This methodological approach demands, beyond the study of EO chemical profiles from bulk population samples, a research focused on the analysis of individual plants. It has revealed the existence of atypical profiles, apart from the known chemotypes, which may be of particular interest for their propagation and cultivation (Judzentiene, 2016). Thus, the key point is how to identify these valuable profiles in such a way that a significant number of samples could be processed. It requires simple and fast methods to achieve a first path to explore the chemodiversity of wild populations. In that respect, as reported by Pothier et al., (2001); Taylor, (2001); Njenga, (2005); Chapman, (2009) and Franz, (2010); thin layer chromatography (TLC) can be considered as a useful methodology because of its simplicity, speed, reliability, economy and possibility of using in field conditions. Many recent reviews enhanced the increasing interest of thyme cultivation due to its wide range of applications derived from its biological activity and sensory characteristics: it is used in medical and pharmacological applications (Sáez, F. & Stahl-Biskup, 2002; Reddy et al., 2014; Hosseinzadeh, 2015; Miraj & Kiani, 2016; Kuete, 2017), and in food preservation (Embuscado, 2015), for example. On the other hand, T. vulgaris shows a noticeable chemical polymorphism which has been extensively studied and several main chemotypes based on the oxygenated monoterpenic compounds have been reported. They can be grouped in two main types specifically related to their potential applications: (a) phenolic, with thymol and carvacrol as more representative compounds, typical of milder and drier Mediterranean environment; and (b) non- phenolic chemotypes, which are characterized by the predominance of cyclic and acyclic aliphatic oxygenated monoterpenes, adapted to a wider range of habitats, even to extreme climates (Kulevanova et al., 1996) As reported by Thomson et al. (2003), four of these non-phenolic chemotypes were found in Southern France and European countries (Satyal et al., 2016), defined by the predominance of geraniol, α-terpineol, thujan- 4-ol and linalool. In addition, another nonphenolic chemotype rich in 1, 8-cineole was reported as endemic of Iberian Peninsula (Guillem & Manzanos, 1998; Jordan et al., 2006, Torras et al., 2008), although its occurrence in Southern France has also been cited (Keefover-Ring, 2009). Other chemotypes based on the bornane skeleton (camphene, borneol and camphor) have also been reported from several countries (Giordani et al (2004), such as Morocco (Imelouane et al., 2009); Iran (Kazemi, 2015); Brazil (Kohiyama et al., 2015) or Italy (Mancini et al. 2015). The aim of this work was to study the chemodiversity of T. vulgaris in a relatively small area located in La Safor, a coastal region in the Valencian Community (Spain), which is characterized by its high biodiversity due to the wide range of edaphoclimatic conditions. A methodological approach is proposed in order to carry out this type of studies. It can be summarized according the following process:

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

(1) A first stage of field work, in which a high number of individuals is selected trying to obtain a representative set of the different environmental conditions that can influence the composition of the EO, mainly altitude and orientation, due to its relationship with the possible microclimatic differences. (2) Screening by TLC of individual extracts. Afterwards, TLC profiles are visually grouped according the presence of discriminant spots, whose chemical composition is identified both by comparing with pure standards and preparative TLC followed by the GC/MS. Unclassified or doubtful individuals are considered as potentially atypical, to be subsequently independently analysed. (3) Once the classification has been performed, the plant material coming from the grouped and potential atypical individuals is subjected to hydrodistillation or simultaneous distillation extraction (SDE) according the amount of available material. Then, these samples are analysed by GC/MS and GC/FID. Material and Methods Area of study The study area was located in the Mondúver mountain, a calcareous mountainous massif. From the geomorphological point of view, Mondúver is a cretacic massif with Jurassic stratums, in which karstic landforms are widely represented. All these geographical features, together with a complex orography, give rise to a great diversity of microclimates and soil types, which lead to a high plant biodiversity. It covers an area of approximately 25 km2, where a protected natural area of 650 ha (Parpalló-Borrell) and a micro- reserve of flora (0.95 ha) are placed, as well as a meteorological station (Barx-La Drova, 39° 0' 18.00" N, 00° 17' 24.36" W, 379 m asl), to which the following data correspond: yearly average temperature: 15,9 ºC; yearly average precipitation: 928,6 mm; compensated thermicity index = 360 (Rivas, 2004). According to these data, this area can be classified belonging to upper thermomediterranean bioclimatic floor. As for characterizing its climatic conditions, it is important to indicate the irregularity of rainfall, largely influenced by the NE-SW orientation, with a marked summer drought and relatively frequent episodes of torrential rains (which can exceed 200 mm daily). In addition, frosts are not frequent (eight days a year, on average) due to maritime influence. Plant material The sampling of plant material was performed in such a way that different orientations and altitudes were considered, avoiding areas with special protection (Figure 1). Samples formed by small cuts of 85 individuals were collected during full flowering stage (August 2018). Each individual plant was marked in order to carry out possible subsequent analysis or to obtain material for its propagation. A voucher specimen of each individual sample was submitted to identification in the Mediterranean Agroforestry Institute (IAM) of Universitat Politècnica de València (UPV). After TLC screening, vouchers of material from grouped individuals according the similarity of their TLC profiles and unclassified ones were deposited at the herbarium of IAM, UPV (VALA 9581-9600). The rest of material was kept in sealed bags at -40 ºC for further analysis. It should be noted that in the studied area had been reported two species of the genus Thymus: Thymus piperella (phenolic, and clearly distinguishable morphologically) and T. vulgaris, of which three subspecies have been identified: T. vulgaris ssp. vulgaris, T. vulgaris ssp. aestivus and T. vulgaris ssp. mansanetianus, with certain specific morphological characteristics (Gallego et al., 2013; Mateo & Crespo, 2014).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 1. Geographical distribution of the samples

TLC screening Extraction The extraction and TLC analysis were performed according to Wagner & Bladt (1996). Small amounts of individual samples (0,2 g) with 2 mL of dichloromethane (Sigma-AldrichTM, for GC analysis) were subjected to stirring in 5 mL glass vials for 30 min. Afterwards, they were dried with anhydrous sodium sulphate and filtered with a syringe filter (TeknokromaTM). Dried extracts were transferred to other vials and placed opened in fume hoods up to total solvent evaporation. Finally, 100 L of toluene were added to each vial. They were sealed and kept in refrigerator at 4º C until TLC analysis. TLC analysis From each extract, 10 L were spotted with capillary tubes (Blaubrand intraMark, 10 μL) on the silica gel plates (DC-Fertigfolien Alugram Sil G/UV 254). They were developed in duplicate using toluene:ethyl acetate (93:7) as a mobile phase, and further stained with sulphuric vanillin and p-anisaldehyde (UV 265 nm), respectively, as stain reagents, as described by Wagner & Bladt (1996). From the visual analysis of plates, spots showing different colour and/or retention factor (Rf value) were marked for further validation to ensure their chemical identity. TLC method validation

TLC plates were spotted with 25 L of sample extracts, and developed in the same conditions, but they were not sprayed with any visualization reagent. Silica gel layer, matching to measured range for discriminant spots, was scrapped and extracted with 0.5 mL of dichloromethane. The filtered extract was allowed to evaporate, until its volume was reduced to approximately 300 L, which was kept and sealed in 350 L insert vials until GC/MS analysis. These extracts were analysed by GC/MS in order to identify their composition. The presence of 1,8-cineole, borneol, camphor and linalool were also confirmed by comparing with pure standards.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Characterization of chemical profiles SDE extraction Plant material coming from grouped individuals was put together and homogenized to obtain representative samples (5-10 g). These samples and those originated from each of the unclassified individuals, were subjected to simultaneous extraction distillation using a Godefroot type apparatus (De Frutos et al., 1988) using dichloromethane (≥99.9%, capillary GC grade, Sigma-AldrichTM) as solvent, for 3 h. The extracts were dried with anhydrous sodium sulphate and concentrated under reduced pressure at room temperature up to 1.5 mL. They were kept in sealed chromatographic vials and stored at -18ºC until GC analysis. GC and GC/MS analysis The analysis of samples was carried out by gas chromatography with flame ionization detector (GC-FID) and mass spectrometry (GC-MS). A Clarus 500 GC (Perkin-Elmer Inc. Wellesley. PA. USA) chromatograph equipped with a FID detector and capillary column ZB-5 (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Phenomenex Inc. Torrance. CA. USA) was used for the quantitative analysis. The injection volume was 1 μL. The GC oven temperature was programmed from 50°C to 250°C at a rate of 3°C min−1. Helium was the carrier gas (1.2 mL min−1). Injector and detector temperatures were set at 250°C. The percentage composition of the EO was calculated from GC peak areas without correction factors by means of the software Total Chrom 6.2 (Perkin-Elmer Inc., Wellesley. PA. USA). Analysis by GC-MS was performed using a Clarus 500 GC-MS (Perkin-Elmer Inc.) apparatus equipped with the same capillary column, carrier and operating conditions described above for GC-FID analysis. Ionization source temperature was set at 200°C and 70 eV electron impact mode was employed. MS spectra were obtained by means of total ion scan (TIC) mode (mass range m/z 45-500 uma). The total ion chromatograms and mass spectra were processed with the Turbomass 5.4 software (Perkin-Elmer Inc.). Retention indices were determined by injection of C8–C25 n-alkanes standard (Supelco, Bellefonte, PE, USA) under the same conditions. The EO components were identified by comparison of calculated retention indices and high probability matches according to mass spectra computer library search (NIST MS 2.0) and available data from literature (Adams, 2007). A shorter run was applied to identify discriminant spots. Identification of the following compounds was also confirmed by comparison of their experimental lineal retention index (LRI) with those of authentic reference standards (Sigma-AldrichTM): α-pinene, β-pinene, camphene, myrcene, limonene, (Z)- β-ocimene, camphor, terpinolene and terpinen-4-ol. Statistical analysis All the data obtained from GC-FID analysis were processed using Statgraphic Centurion XVI. Agglomerative Hierarchical Clustering (Square Euclidean Distance and Wards method as aggregation criterions) was performed on percentages of compounds (over 5 % at least on three samples) in order to group the most characteristic EO profiles. Then, these data were subjected to Discriminant Analysis to validate the classification. The significance of differences among the final defined profiles was determined by means of analysis of variance (ANOVA) using Statgraphics 5.1. Software. Tukey’s HSD multiple-range test at P < 0.05 was used to find out significant differences among average values. As the original data were expressed like percentage (%) peak areas, they were subjected to arcsin [square root (%/100)] transformation and previously their homocedasticity was tested.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Results and Discussion Preliminary screening From the 85 individual profiles, 47 were found clearly similar, showing a pattern of spots like the one displayed in Fig. 2A (majority profile). Among the other ones, some groups could be visually established based on the presence with remarkable intensity or the absence of certain discriminant spots which are described in Fig. 2. Their GC/MS analysis allowed to determine their composition, so that the following groups were defined according the occurrence of the following representative compounds: B. Linalool (5 individuals: 9,49,51,52,53), C. Borneol (4 individuals: 10, 56, 57, 67), D. α-Cadinol (8 individuals: 58, 60, 61, 71, 72, 73,74,75), E. 1,8-Cineole (2 individuals: 17, 18), F. Camphor and borneol, (2 individuals: 12,65) G. 1,8-Cineole and borneol (4 individuals: 15, 50, 62, 66).

Figure 2. TLC screening: (A) representative profiles, (B-G) discriminant spots (Rf range) and mass spectra.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 2. (Cont.)

As an example, individuals grouped according their high level of linalool are shown in Fig.3. Finally, the profiles of 13 individuals could not be clearly classified, so they were directly analysed by GC/MS and/or GC/FID.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 3. The five individuals grouped according their high amount of linalool

1: Representative TLC plate of predominant profile. 2a: stained with vanillin sulphuric. 2b: stained with anisaldehyde sulphuric.

This screening methodology was applied to select chemotypes of Mentha longifolia (L.) Huds. from wild populations (Llorens-Molina et al., 2017) in order to obtain chemical homogeneous plots of this species. Seasonal changes of these selected accessions have been recently studied (Llorens-Molina et al., 2020). To the best of our knowledge, this type of methodology has not been applied so far to study the chemodiversity of wild populations or to identify atypical EO profiles. GC results Samples from grouped individuals In summary, 20 samples (one from each group A-G and the 13 non-classified individuals) were subjected to GC/MS and GC/FID analysis. As example, Total Ion Chromatograms (TIC) of the samples A, B, D, F are displayed in Figures 4-7.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 4. Chromatographic profile (TIC) of group A (predominant composition).

Figure 5. Chromatographic profile (TIC) of group B (rich in linalool).

Main compounds (>0.5 % TIC peaks area): 1. Camphene (0,5 %); 2. 1,8-Cineole (2,3 %); 3. -Terpinene (0,7 %); 4. (Z)-linalool oxide furanoid (2,0 %); 5. (E)-linalool oxide furanoid (3,5 %); 6. Linalool (78,6 %); 7. Camphor (1,4 %); 8.  Borneol (1,0 %); 9. Terpinen-4-ol (4,5 %); 10. -terpineol (0,5 %); 10. Isobornyl acetate (0,5 %); 11. Spathulenol (0,9 %)

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 6. Chromatographic profile (TIC) of group D (rich in oxygenated sesquiterpenes).

Figure 7. Chromatographic profile (TIC) of group F (rich in camphene, 1,8-cineole, camphor and borneol).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

As aforementioned, the individuals not matching any of the established groups were independently subjected to GC-MS and GC-FID analysis. Figure 8 shows an atypical profile highly rich in 1,8-cineole.

Figure 8. Chromatographic profile (TIC) of an atypical individual highly rich in 1,8-cineole

GC analysis The composition of the 20 processed samples (one from each of the seven groups and the 13 non-classified individuals) expressed as percentage (%) peak areas in FID chromatograms, is detailed in Tables 1 and 2. A total of 73 compounds were identified accounting for 88.7-99.6 % of the entire area. The major profile, grouping 47 individuals, contained as main constituents: camphene (17 %), camphor (46.4 %) and borneol (11.5 %). This profile was found very similar to that reported by Imelouane et al. (2009) from Morocco. The group B, as well as the non-classified individuals: 2, 3, 4, 5, 8, exhibited a high amount of linalool (48.3-80.6 %). This composition could be related to chemotype linalool (Thomson et al. ,2003; Giordani et al., 2004). Other well-defined profile was E, which represents the 1,8-cineole chemotype, typical of Eastern Iberian Peninsula. The non-classified individual 1 could be characterized by its high amount of oxygenated sesquiterpenic fraction (-cadinol, 43.5 % and epi-α-cadinol, 9.8 %). The profile D showed some similarity with this composition (34.4 % oxygenated sesquiterpenes). The rest of groups and the non-classified individuals represented different relative rates of pinene isomers, camphene, camphor, linalool, borneol, terpinen-4-ol and oxygenated sesquiterpenic fraction (considered as a whole, which main constituents were found spathulenol and cadinol isomers). For a more accurate classification, the aforementioned components accounting for more than 5% in at least three samples, were taken as variables for the Hierarchical Clustering and the Discriminant Analysis. Camphene was not considered because it was highly correlated with camphor (r = 0.96).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

The Cluster analysis (Fig. 9) allowed to propose a first classification to be validated by Discriminant Analysis. The samples were grouped as follows: 1. A, C, F, NC11; 2. D, NC1, NC10; 3. E, G, NC12, NC13; 4. NC6, NC7, NC9; 5. B, NC2, NC3, NC4, NC5, NC8.

Table 1. Chemical composition of grouped samples of Thymus vulgaris L coming from Mondúver area (La Safor, València, Spain)

Compounds Grouped individual samples (% peak areas) LRI A B C D E F G Id. b LRIc (lit.)d Tricyclene 1,2 920 921 0.7 0.2 0.7 0.3 0.3 0.9 0.7 -Thujene 1,2 924 924 0.3 0.5 0.3 0.1 0.2 0.3 0.3 -Pinene 1,2,3 931 932 4.9 0.6 5.9 6.6 2.7 6.6 6.1 Camphene 1,2,3 946 946 17.0 1.8 15.1 6.1 0.9 16.9 11.6 Sabinene 1,2 969 969 0.2 0.2 0.1 2.7 2.6 0.2 0.8 -Pinene 1,2,3 975 974 1.2 0.4 1.2 0.4 5.4 1.3 2.5 1-Octen-3-ol 1,2 979 974 0.1 0.3 0.1 0.2 t 0.1 0.1 3-Octanone 1,2 984 979 -e tf t - 0.3 t t Myrcene 1,2,3 989 988 1.2 0.8 1.0 0.6 3.7 1.2 2.4 3-Octanol 1,2 998 994 - t t - - - - -Phellandrene 1,2 1003 1002 t 0.1 - - t - - -Terpinene 1,2,3 1016 1014 0.2 0.5 0.2 0.3 t 0.3 0.2 p-Cymene 1,2,3 1022 1020 1.1 1.3 0.8 - 0.2 1.2 0.6 Limonene 1,2,3 1026 1024 1.9 1.0 2.5 1.4 - 3.2 - 1,8-Cineole 1,2,3 1028 1026 3.4 3.3 0.2 2.1 56.0 0.4 25.8 cis--Ocimene 1,2 1035 1032 0.1 0.2 0.1 1.3 - 0.2 0.1 trans--Ocimene 1,2 1046 1044 0.5 0.4 1.0 0.6 0.5 2.1 1.0 -Terpinene 1,2,3 1055 1054 0.5 1.3 0.5 0.6 0.7 0.8 0.5 cis-Sabinene hydrate 1,2 1069 1065 0.5 3.9 0.5 0.2 0.8 0.7 0.5 Camphenilone 1,2 1079 1078 0.2 - 0.2 0.2 - 0.2 0.1 Terpinolene 1,2,3 1083 1086 0.1 2.9 0.1 0.5 0.2 0.2 0.1 trans-Linalool oxide (furanoid) 1,2 1085 1084 t ------Linalool 1,2 1100 1095 0.3 61.3 1.4 0.5 0.8 0.3 0.6 -Campholenal 1,2 1121 1122 0.1 0.3 0.3 0.3 0.2 0.5 0.3 Camphor 1,2,3 1144 1141 46.4 2.1 43.5 19.9 t 41.3 27.5 Sabina ketone 1,2 1152 1154 0.1 - 0.1 0.7 0.1 0.1 t Pinocarvone 1,2,3 1159 1160 0.3 - 0.2 0.2 0.4 0.2 0.2 Borneol 1,2,3 1170 1165 11.5 4.9 12.1 3.7 4.0 10.1 6.7 Terpinen-4-ol 1,2,3 1177 1174 2.3 4.1 2.4 2.0 1.8 2.9 2.0 -Terpineol 1,2 1192 1186 0.5 0.7 0.4 0.5 6.9 0.6 2.1 Dodecane 1,2 1203 1204 0.1 t t 0.7 0.2 0.1 0.1 Isobornyl methanoate 1,2 1223 1223g 0.1 0.1 0.8 - 0.1 0.2 0.1 Carvone 1,2 1237 1239 0.1 - - 0.4 0.1 0.1 t Linalool acetate 1,2 1254 1254 - 0.1 - - - - - Isobornyl acetate 1,2 1280 1283 1.0 0.4 1.6 2.4 0.1 1.4 0.6 -Terpinyl acetate 1,2 1309 1316 - - - - 0.4 - 0.1

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

-Elemene 1,2 1334 1335 - - 0.2 0.6 0.8 - 0.1 Isobornyl propanoate 1,2 1381 1383 - - 0.1 - - - t -Bourbonene 1,2 1381 1387 0.1 0.3 0.3 - 0.2 0.2 0.2 -Caryophyllene 1,2,3 1413 1417 - 0.2 0.3 - 0.4 0.4 0.5 -Copaene 1,2 1422 1430 - - t - - - t Aromadendrene 1,2 1430 1439 - t - - - - t -Humulene 1,2 1449 1452 ------Alloaromadendrene 1,2 1458 1458 ------Isobornyl n-butanoate 1,2 1471 1473 0.1 - 0.2 - - 0.1 0.1 Germacrene D 1,2 1476 1484 0.3 0.1 0.5 - - 0.3 0.3 Bicyclogermacrene 1,2 1490 1500 - 0.3 0.8 0.2 0.1 0.4 0.8 -Muurolene 1,2 1494 1500 0.1 - - - - - 0.1 -Cadinene 1,2 1508 1513 - - - 4.0 - 0.1 0.1 -Cadinene 1,2 1512 1522 - 0.1 - 0.1 - 0.1 - Isobornyl 3-methylbutanoate 1,2 1520 1521 - - 0.1 - - 0.2 t Isobornyl 2-methylbutanoate 1,2 1524 1524 -------Calacorene 1,2 1542 1544 - 0.7 - 0.2 1.3 0.1 - Elemol 1,2 1545 1548 ------Amorph-4-ene <1α,10α-epoxy-> 1,2 1559 1572 - - - 0.2 t - t Spathulenol 1,2 1570 1577 1.6 2.8 2.9 2.2 0.7 1.9 3.0 Caryophyllene oxide 1,2 1576 1582 0.2 - - 0.4 - t - -Oplopenone 1,2 1600 1607 - t t 0.3 - - t Cubenol <1,10-di-epi> 1,2 1610 1618 - t - 4.2 - t t -Muurolol 1,2 1623 1644 t 0.2 0.1 - 0.5 0.1 t epi--Cadinol 1,2 1638 1638 t - - 12.9 0.1 0.5 0.1 -Eudesmol 1,2 1640 1652 -------cadinol 1,2 1650 1652 - 0.8 - 1.7 1.9 0.3 0.2 cis-Calamenen-10-ol 1,2 1659 1660 - 0.2 - 0.4 - - - Elemol acetate 1,2 1670 1680 ------Eudesma-4(15),7-dien-1--ol 1,2 1676 1687 - - 0.1 4.2 0.1 t 0.1 Eudesm-7(11)-en-4-ol 1,2 1683 1700 - 0.1 0.1 2.5 - 0.2 t Muurol-5-en-4-one 1,2 1692 1688 - - - 0.1 - - t Isobicyclogermacrenal 1,2 1725 1733 - - - 0.5 - - - Cyclocolorenone 1,2 1732 1759 - - - 3.0 - 0.1 t 1-Octenyl acetate 1,2 1108 1110 - - - 0.1 - - - (2Z)-Hexenyl isovalerate 1,2 1234 1241 - 0.2 0.1 0.2 0.2 0.1 0.1 Manool oxide 1,2 1959 1987 - 0.1 0.1 0.1 0.1 t t Grouped compounds Hydrocarbon monoterpenes 29.8 12.1 29.4 21.5 17.5 35.3 26.8 Oxygenated monoterpenes 66.9 81.2 64 33.1 71.7 59.1 66.7 Hydrocarbon sesquiterpenes 0.4 1.7 2.0 5.0 2.8 1.6 2.0 Oxygenated sesquiterpenes 1.9 1.6 3.3 34.2 4.6 3.2 3.5 Other 0.2 0.6 0.3 1.4 0.7 0.3 0.4 Total identified 99.0 99.0 99.1 93.4 95.8 99.4 99.2

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Table 2. Chemical composition of individual samples (no classified) of Thymus vulgaris L coming from Mondúver area (La Safor, València, Spain). The listed compounds and therefore the identification methods and the LRI values are the same as in Table 1 Unclassified individual samples (% peak areas)

NC NC NC NC Compound NC1 NC2 NC3 NC4 NC5 NC6 NC7 NC8 NC9 10 11 12 13 Tricyclene - - - - 0.0 0.2 0.1 0.0 0.0 0.4 0.3 0.0 0.2 -Thujene - - - - t 1.0 0.5 - 1.3 0.3 0.2 0.2 0.8 -Pinene 1.3 0.3 0.1 0.3 0.1 5.7 2.1 0.1 5.0 4.7 3.1 2.3 2.0 Camphene 0.3 - 0.1 0.2 0.0 17.7 11.6 0.3 15.8 8.7 9.0 2.8 0.7 Sabinene - - - - - t 0.1 - 0.2 5.6 0.1 0.7 1.3 -Pinene 6.0 0.7 0.1 0.6 0.2 0.2 0.1 - 1.1 0.3 0.7 2.0 2.6 1-Octen-3-ol - - - 0.9 - 1.3 0.9 t 0.1 - 0.1 - t 3-Octanone ------0.1 - - - - Myrcene 0.8 1.1 1.1 0.1 0.4 0.1 - t 0.9 0.6 0.5 0.5 1.6 3-Octanol ------t - - - - -Phellandrene - - - 1.6 - 1.1 0.6 0.1 t - - - - -Terpinene 0.1 - - - t 0.5 0.2 0.1 0.5 0.4 - - 0.2 p-Cymene 0.6 t t - t - - - 1.6 3.2 1.0 1.3 2.0 Limonene 2.0 1.1 0.9 t 0.7 2.3 1.0 t - 2.3 2.7 3.1 1.6 1,8-Cineole 0.7 17.3 3.6 26.5 4.3 5.5 6.5 1.3 4.5 1.3 1.5 36.9 38.6 cis--Ocimene - - 1.4 - - 0.1 0.1 - 0.2 - - - - trans--Ocimene 3.9 1.2 t - t 0.5 0.5 t 1.3 0.7 0.7 - - -Terpinene 0.3 - 0.1 0.1 0.2 0.9 0.4 - 1.0 0.9 0.7 0.8 3.0 cis-Sabinen hydrate 0.2 0.3 0.8 1.0 1.4 0.7 0.3 1.3 0.7 0.8 1.4 1.8 8.5 Camphenilone ------0.5 - 0.2 - - Terpinolene - 0.2 0.5 - 0.4 0.2 0.2 1.0 0.1 - 0.4 - 0.3 trans-Linalool oxide - - - 0.3 - t ------(furanoid) Linalool 0.1 63.3 76.7 48.3 79.2 0.9 1.6 80.6 t 0.5 13.8 4.3 13.1 -Campholenal - t t - - 0.1 0.6 - t - 0.2 - 0.6 Camphor 1.3 t 1.0 0.4 t 36.3 42.0 1.3 41.1 27.5 35.4 11.5 2.6 Sabina ketone ------1.5 - - - Pinocarvone - - - - - 0.1 0.1 - - 0.2 - - - Borneol 0.2 t 0.4 2.9 0.6 0.1 0.1 0.4 0.2 5.4 11.4 4.9 2.2 Terpinen-4-ol 0.6 - 0.6 - 0.6 13.2 17.5 - 17.0 2.7 3.3 2.6 8.6 -Terpineol - - - 0.4 - 3.0 2.1 0.2 0.6 0.2 0.5 2.2 3.3 Dodecane ------0.2 0.5 0.2 - - Isobornyl methanoate ------0.2 - 0.9 0.1 1.0 Carvone ------0.2 - - - - Linalool acetate t - - - 0.1 0.1 ------Isobornyl acetate 0.1 - - 0.3 - 1.3 1.4 - 1.5 1.3 1.5 0.5 - -Terpinyl acetate ------0.8 - -Elemene - - - - 0.4 - - - - 0.3 - 2.0 0.1 Isobornyl propanoate - 1.1 0.4 - 0.6 0.1 - - t - - - - -Bourbonene - - - 0.6 - 0.2 0.6 0.7 0.1 - - - -

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

-Caryophyllene - 1.1 2.1 - 1.3 0.2 0.2 0.0 0.2 - - 0.4 - -Copaene - - - 0.7 - 0.3 0.4 1.3 - - - - - Aromadendrene ------0.1 - - - - - -Humulene ------0.2 - - - - - Alloaromadendrene 1.0 - - 0.3 - 0.1 0.3 0.2 - - - - - Isobornyl n-butanoate - - - - - 0.3 t - 0.2 - 0.2 - - Germacrene D 1.0 4.3 1.3 2.2 1.9 0.1 0.2 0.4 0.1 - - - - Bicyclogermacrene 0.2 0.7 1.3 - 2.2 - - - 0.2 - 0.4 - - -Muurolene - - - 1.0 - 0.5 0.4 0.8 0.1 - - - - -Cadinene 10.4 0.8 - - - - - 0.2 - 0.7 - - - -Cadinene ------Isobornyl 3------0.2 1.9 0.2 0.4 - methylbutanoate Isobornyl 2- 0.8 - - 0.2 - - 0.1 0.2 - - - - - methylbutanoate -Calacorene ------Elemol - - 4.4 - 1.3 ------Amorph-4-ene - 1.3 - 1.4 - - - 0.6 - 0.6 - - - <1α,10α-epoxy-> Spathulenol 0.3 0.2 0.4 0.4 0.9 2.8 4.1 5.1 2.2 3.6 3.8 3.2 1.5 Caryophyllene oxide - 0.4 0.8 0.2 0.4 0.6 1.2 1.2 - 0.8 - - - -Oplopenone ------3.3 1.3 6.3 0.4 Cubenol <1,10-di-epi> ------0.1 0.1 - - - - - -Muurolol ------0.1 - - - - epi--Cadinol 9.8 0.1 - 0.8 - - - 0.2 - 8.7 - - - -Eudesmol - - 0.2 - - 0.1 0.1 t - - - - - -cadinol 43.5 3.2 - 3.6 - - 0.1 0.1 - 0.7 - 0.1 - cis-Calamenen-10-ol 2.9 0.1 0.3 0.7 0.1 - 0.3 1.1 - - - - - Elemol acetate 0.3 - 0.8 - 0.2 - - - - 2.0 - - - Eudesma-4(15),7-dien- - - - 0.3 - - - - - 2.1 - - - 1--ol Eudesm-7(11)-en-4-ol - - 0.2 0.5 ------0.2 - - Muurol-5-en-4-one ------ Isobicyclogermacrenal ------2.3 - - - Cyclocolorenone ------1-Octenyl acetate - - - - - t - - t - - t - (2Z)-Hexenyl - - - - 1.3 0.4 0.4 - t 0.9 0.2 - 1.0 isovalerate Manool oxide ------t - 0.8 2.1 0.9 Grouped compounds Hydrocarbon 15.3 4.6 4.4 3.0 2.1 30.3 17.4 1.6 28.9 28.1 19.3 13.6 16.2 monoterpenes Oxygenated 3.2 82 83.5 80.1 86.8 61.7 72.2 85.1 66.7 41.4 70.3 65.6 78.5 monoterpenes Hydrocarbon 12.6 6.9 4.7 4.6 5.8 1.5 2.0 3.7 0.6 1.0 0.4 2.4 0.1 sesquiterpenes

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Oxygenated 57.5 5.4 7.1 8.0 2.9 3.5 5.9 8.4 2.3 24.3 5.3 10.3 1.8 sesquiterpenes Other - - - 0.9 1.3 1.7 1.3 t 0.5 1.4 1.4 2.2 1.9 Total identified 88.7 98.9 99.6 96.6 98.9 98.3 98.7 98.9 99.1 97.6 96.8 93,6 98.6 aCompounds listed in order of elution on DB5 column; bMethod of identification: 1. MS spectra, 2. LRI, 3. Co-injection with pure standards, cExperimental linear retention index; dLinear retention index according Adams (2007); eNo detected; fTraces (peak area < 0.05 %); gCao et al. (2011)

Figure 9. Dendogram of Hierarchical Clustering of selected variables

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

Figure 10. Grouped samples according the two first discriminant functions. Relative eigenvalue percentage: F1: 70,40 %; F2: 17,36 % (P = 0.0000).

Both F1 and F2 allows to consider well established differences among the defined groups according (Wilks) values: F1 ( = 0,00005, p=0,0000), F2 ( = 0,003, p = 0,0000), (100 % samples well classified). Standardized coefficients for both factors are the following:

F1= 0,487*1,8-cineole - 0,642*Camphor + 1,101*Borneol - 0,581*Terpinen-4-ol - 0,062*Oxygenated sesquiterpenes - 0,078*+ pinene - 1,278*Linalool F2= 1,055*1,8-cineole + 0,831*Camphor – 0,624*Borneol + 0,630*Terpinen-4-ol -0,032*Oxygenated sesquiterpenes +0,335*+ pinene +0,950*Linalool Based on the selected variables for cluster classification, a discriminant analysis (AD) was performed in order to evaluate it. The first two factors explained 87,8 % of the variability. According F1 and F2 coefficients, borneol, 1,8-cineole and linalool were the most powerful discriminant compounds in order to define the EO profiles. The relationships camphor-borneol and camphor-terpinen-4- ol in each one of the factors expressed clearly the presence of two well defined profiles in which camphor and each one of these two compounds were the dominant ones. The best defined profile can be related with the chemotype linalool as expressed when the coefficients values in both factors are compared. The same, but in a less pronounced way, can be said of 1,8-cineole. Oxygenated sesquiterpenes showed negative and low coefficients in both factors, constituting also a well classified group. On the other hand, + pinene did not seem to be relevant as discriminatory compounds. The composition

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

of the final defined profiles and the significance of the contributions of the main compounds is displayed in Table 3.

Table 3. Average amounts of considered variables of groups defined from Cluster Analysis. Different letters on the same row mean significant differences according the Tukey’s HSD test, (P <0.05)

GROUP CLUSTER Compounds 1 2 3 4 5 + Pinene 6,2±1,8 a 6,4±1,2 a 6,4±2,2 a 4,7± 2,2 a 0,6±0,4 b 1,8-Cineole 1,4 ± 1,5 b 1,4 ± 0,7 b 39,3 +12,5 a 5,5±1,0 b 9,4±10,2 b Linalool 3,9±6,6 b 0,4±0,3 b 4,7±5,9 b 0,8±0,8 b 68,2±12,8 a Camphor 41,7± 4,6 a 16,2±13,5 b 10,4±12,4 bc 39,8±3,1 a 0,8±0,8 c Borneol 11,3 ± 0,8 a 3,1±2,6 bc 4,4±1,8 b 0,1±0,1 d 1,6±1,9 cd Terpinen-4-ol 2,7 ± 0,5 b 1,8±1,0 b 3,7±3,3 a 15,9±2,4 b 0,9±1,6 a Oxygenated 3,4±1,4 b 38,8±17,0 a 5,0±3,7 b 3,9 ± 1,9 b 5,6±2,8 b sesquiterpenes

The profiles A, C, F and NC 11 (group 1) could be considered as major profiles. In this case, the preliminary screening did not allow for significant distinctions, probably due to differences in the concentration of the plant extracts subjected to the TLC analysis. As displayed in Table 1, these samples also exhibited important amounts of camphene (9,0 %-16,9 %). This way, group 1 could be considered as the representative profile based on the camphane skeleton (camphene, camphor and borneol as the most important components). If compared profile A (predominant in terms of number of individuals) with that described by Imelouane et al. (2009), a higher content of the major compounds was in general observed (17.0%; 46.4% and 11.5% versus 17.19%, 38.54% and 4.92% of camphene, camphor and borneol, respectively). The group 2 exhibited a relative high amount of sesquiterpenic fraction including NC1, which could be considered as an atypical individual because of its great amount of cadinol isomers. To the best of our knowledge, a similar profile has not been referred to so far in thyme. Only samples accounting up to 14,1 % of -cadinol were reported by Mancini et al. (2015) in phenolic (thymol) T. vulgaris. It is also worth mentioning its reported pharmacological applications (Zygmunt et al., 1993). The group 3 was characterized by its high rate of 1,8-cineole, although profile C showed a noticeable amount of camphene and camphor. This profile was mostly observed in a previous study with samples from the East of the Iberian Peninsula (Teruel and Valencia) (Llorens et al., 2017). On the other hand, the content of 1,8- cineole raised up to 56,0 % in sample E, whereas it accounted for a 36,42 % as reported by Jordán et al. (2006) on Spanish T. vulgaris, chemotype 1,8-cineole. The composition of the group 4 exhibited a noticeable presence of terpinen-4-ol, which was not considered in the preliminary screening. It may explain why these individuals were not classified. Its composition could be considered as a specific chemotype: camphor, terpinen-4-ol. It is important referring to the composition described by Quesada et al. (2016) in an oil marketed by a company located close to the area studied. Its composition (camphene (4,78 %); 1,8-cineole (12,3 %), camphor (11,23 %), terpinen-4-ol (5,50 %), borneol (8,87 %)) seems to be balanced between those described for groups 3 and 4. The group 5 was the best-defined and it can be clearly related to the chemotype linalool, already cited above (Thomson et al., 2002), the same way that the reported samples from Catalonia (Spain) (Torras et al., 2007),

Nat. Volatiles & Essent. Oils, 2020; 7(3): 29-50 Llorens-Molina et al. DOI: 10.37929/nveo.722313

in which the variation range of linalool was 32.92 - 74.55% versus 48.3 - 80.6% found in this group. This increase could be attributed to the prior selection of individuals. As mentioned when describing the plant material, it should be noted that in the studied area three T. vulgaris subspecies have been reported: T. vulgaris ssp vulgaris, T. vulgaris ssp. aestivus and T. vulgaris ssp. mansanetianus. Nevertheless, the composition reported of T. vulgaris ssp. aestivus collected in a close geographical area (1,8-cineol (22.18%), geraniol (17.43%) and geranyl acetate (20.01%) as major compounds) is quite different to profiles identified in this work (Blazquez et al., 1990). No information has been described when regards to the chemical composition of T. vulgaris ssp. mansanetianus EO. Conclusion From the final classification obtained through the statistical treatment of the results, the validity of the preliminary TLC screening has been proved as a useful tool to carry out a first classification of the individuals studied. In spite of the difficulties derived from the similarity between the colours and the Rf values of the spots corresponding to the main compounds, the most notable differences among the EO profiles could be identified. On the other hand, it should be noted that the aforementioned difficulties led to validate the identification of the spots by preparative TLC and GC/MS, instead of the simple comparison with the development of the plates with reference pure standards. In this way, it was possible to group the 85 individuals so that those with a similar profile were part of a single sample. Thus, taking into account that individuals which could not be clearly classified were analysed independently, the number of samples to be processed could be reduced from 85 to 20. Moreover, this prior classification gives a first orientation about the relative abundance of each profile in the population. Finally, after the statistical treatment of the data, five profiles were defined. As it has been demonstrated by comparing the range of variation of the main compounds in the defined groups and those reported from studies on non-phenolic samples of T. vulgaris, a more defined composition can be observed in terms of the proportion of the major components. Insofar as these compounds are related to potentially applicable biological activities, this fact leads to consider the interest of individual screening of populations in order to identify valuable genotypes to create more profitable cultivars. Another interesting aspect of this methodology is that certain compounds of interest can be obtained with similar percentages in more advantageous species from the agronomic point of view, as can happen, for example, with linalool-rich oils. The results of this work may be the starting point for further investigations, for example, to establish the possible taxonomic implications of the identified profiles in relation to the different subspecies of T. vulgaris present in the area. Moreover, given that the established groups of individuals as well as those considered atypical are localized, those with the greatest potential interest could be propagated. REFERENCES

Adams, R. P. (2007). Identification of essential oil components by gas chromatography/mass spectrometry (Vol. 456). Carol Stream, IL: Allured publishing corporation U.S.

Anupama, G., Netravathi, D. K., & Avinash, M. (2019). Essential oils: A novel source for food preservation. Journal of Pharmacognosy and Phytochemistry, 8(1), 2098-2101.

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Nat. Volatiles & Essent. Oils, 2020; 7(3): 51-54 Hanoğlu et al. DOI: 10.37929/nveo.767913

RESEARCH ARTICLE Microdistilled essential oil of Thymus integer Griseb., endemic in Cyprus Duygu Yiğit Hanoğlu1, *, Azmi Hanoğlu2, Betül Demirci 3, Sami Tamson4, Banu Kesanli5 and Kemal Hüsnü Can Başer2

1Department of Pharmaceutical Botany, Faculty of Pharmacy, Near East University, 99010, Lefkoşa (Nicosia), TURKISH REPUBLIC OF NORTHERN CYPRUS 2Department of Pharmacognosy, Faculty of Pharmacy, Near East University, 99010, Lefkoşa (Nicosia), TURKISH REPUBLIC OF NORTHERN CYPRUS 3Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, Türkiye 4Kurtuluş High School, 99010, Güzelyurt (Morphou), TURKISH REPUBLIC OF NORTHERN CYPRUS 5Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Near East University, 99010, Lefkoşa (Nicosia), TURKISH REPUBLIC OF NORTHERN CYPRUS

*Corresponding author. Email: [email protected] Submitted: 14.07.2020; Accepted: 21.08.2020

Abstract The aim of the present study was to characterize the essential oil composition of Thymus integer Griseb. (Lamiaceae), an endemic species in Cyprus. Microdistilled essential oil was analysed simultaneously both by Gas Chromatography-Flame Ionization Detector (GC-FID), and Gas Chromatography/Mass Spectrometry (GC/MS). Overall, 49 components were determined and the major components were characterized as borneol (22%), p-cymene (10.1%),terpinene (9.6%), -pinene (8.7%), camphene (7.7%), and terpinen-4-ol (5.4%), respectively.

Keywords: Thymus integer, microdistillation, essential oil, GC-FID, GC-MS

Introduction The genus Thymus L. is represented in Cyprus by two species, T. capitatus L. and T. integer Griseb. (Meikle, 1985). Traditionally, both are used to treat respiratory and digestive disorders (González-Tejero et al, 2008). T. integer is locally known as “thymari” and endemic in Cyprus (Bellomaria et al., 1994). It grows at 80-1700 m altitude, and flowering stage is from February to June. T. integer infusion prepared from aerial parts is internally used as spasmolytic, carminative, stomachic, and against cough and cold. Externally, the extracts are used as incense against headaches. Moreover, due to antiseptic and astringent properties, compresses prepared from this plant are used for the treatment of burns and external wounds (Arnold, 1985). The essential oils of the genus Thymus have been well reviewed (Stahl-Biskup, 1991; Başer, 1992; Bellomaria et al., 1994; Başer & Kırımer, 2006; Özkum Yavuz et al., 2017; Başer & Kırımer, 2018). The aim of this study was to characterize the essential oil composition of Thymus integer Griseb. endemic in Cyprus obtained by microdistillation. Materials and Methods Plant material Aerial parts of the plant material, growing wild in Cyprus, was collected during the flowering stage at Madari in Southern Cyprus. A voucher specimen is kept at the Herbarium of the Near East University, Turkish Republic of Northern Cyprus (NEUN6905).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 51-54 Hanoğlu et al. DOI: 10.37929/nveo.767913

Microdistillation The volatiles were obtained after microdistillation of the dried plant material (1 g) using an Eppendorf MicroDistiller® containing 10 mL of distilled water per sample vial. The sample vial was heated to 108°C at a rate of 20°C/min for 90 min followed by heating at 112°C at the rate of 20°C/min for 30 min. The sample was subjected to a final post-run for 2 min under the same conditions. The collecting vial, containing a solution of NaCl (2.5 g) and water (0.5 ml) plus n-hexane (350 µl) to trap volatile components, which were cooled to –5°C during distillation. Thereafter, the organic layer in the collection vial was separated and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) simultaneously. GC-MS analysis The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25 mm, 0.25 m film thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was kept at 60C for 10 min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10 min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. GC analysis

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300C. To obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The analysis results are given in Table I. Identification of the essential oil components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder Software 4.0) (McLafferty & Stauffer, 1989; Koenig, Joulain & Hochmuth, 2004) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils. Results and Discussion The essential oil of T. integer obtained by microdistillation and analysed by GC-FID and GC-MS yielded 49 compounds representing 99.3% of essential oil. The major components were identified as borneol (22%), p- cymene (10.1%), -terpinene (9.6%), -pinene (8.7%), camphene (7.7%) and terpinen-4-ol (5.4%). Thymol content was determined as 2.9% while the carvacrol content was 0.3% (Table 1.).

Table 1. The Essential Oil Composition of Thymus integer

RRI Compound % IM

1014 Tricyclene 0.4 tR, MS

1032 -Pinene 8.7 tR, MS

1035 -Thujene 0.8 tR, MS

1076 Camphene 7.7 tR, MS

1118 -Pinene 2.9 tR, MS

1132 Sabinene 1.5 tR, MS

1174 Myrcene 0.7 tR, MS

1176 -Phellandrene 0.5 tR, MS

1188 -Terpinene 2.0 tR, MS

Nat. Volatiles & Essent. Oils, 2020; 7(3): 51-54 Hanoğlu et al. DOI: 10.37929/nveo.767913

1203 Limonene 2.0 tR, MS

1218 -Phellandrene 1.4 tR, MS

1255 -Terpinene 9.6 tR, MS

1280 p-Cymene 10.1 tR, MS

1290 Terpinolene 0.8 tR, MS 1452 1-Octen-3-ol 0.5 MS 1474 trans-Sabinene hydrate 0.2 MS 1479 (E,Z)-2,4-Heptadienal tr MS 1499 -Campholene aldehyde tr MS 1507 (E,E)-2,4-Heptadienal 0.1 MS

1532 Camphor 1.1 tR, MS

1553 Linalool 1.6 tR, MS 1556 cis-Sabinene hydrate 0.2 MS

1562 Octanol 0.1 tR, MS 1571 trans-p-Menth-2-en-1-ol 0.2 MS

1591 Bornyl acetate 2.7 tR, MS

1611 Terpinen-4-ol 5.4 tR, MS

1612 -Caryophyllene 2.5 tR, MS 1638 cis-p-Menth-2-en-1-ol 0.1 MS

1683 trans-Verbenol 0.2 tR, MS

1687 -Humulene 0.4 tR, MS

1706 -Terpineol 1.4 tR, MS

1719 Borneol 22.0 tR, MS 1726 Germacrene D 2.1 MS

1751 Carvone 0.2 tR, MS 1755 Bicyclogermacrene 0.7 MS 1758 (E,E)--Farnesene 0.2 MS 1773 -Cadinene 0.6 MS

1845 trans-Carveol tr tR, MS

1857 Geraniol 0.2 tR, MS

1864 p-Cymen-8-ol 0.3 tR, MS

2008 Caryophyllene oxide 1.5 tR, MS 2144 Spathulenol 0.7 MS 2187 T-Cadinol 0.2 MS

2198 Thymol 2.9 tR, MS

2239 Carvacrol 0.3 tR, MS 2255 -Cadinol 0.5 MS 2316 Caryophylla-2(12),6(13)-dien-5-ol (=Caryophylladienol I) 0.2 MS 2324 Caryophylla-2(12),6(13)-dien-5-ol (=Caryophylladienol II) 0.6 MS 2392 Caryophylla-2(12),6-dien-5-ol (=Caryophyllenol II) 0.3 MS Total 99.3 RRI: Relative retention indices calculated against n-alkanes, % calculated from FID data, tr: Trace (< 0.1 %), IM: Identification method, tR, identification based on the retention times of genuine compounds on the HP Innowax column; MS, identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data.

Nat. Volatiles & Essent. Oils, 2020; 7(3): 51-54 Hanoğlu et al. DOI: 10.37929/nveo.767913

Bellomaria et al. (1994) reported the major compounds of T. integer essential oil as p-cymene (15.7-25%), borneol (18.7-23.8%), -terpinene (9.2-12.3%), thymol (3.1-8.4%) and carvacrol (0.5-1.6%), respectively. The authors had collected plant materials from six locations in Southern Cyprus studying leaf, flower and fruit oils. They reported the oil yields in the range of 0.2-0.9%. Our material was collected from a different location. We had also found borneol, p-cymene and -terpinene as main components. The contents of thymol and carvacrol which are typical thyme/ oregano oil constituents were low in our sample as well. This study confirms the previous study in characterizing the oil composition of Thymus integer, which is endemic in Cyprus.

ACKNOWLEDGMENT

This work was supported by the Near East University, Experimental Health Sciences Research Center [SAG-2017-1-053]. REFERENCES

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Nat. Volatiles & Essent. Oils, 2020; 7(3): 55-60 Türkmenoğlu & Demirci DOI: 10.37929/nveo.784531

RESEARCH ARTICLE Characterization of the endemic Achillea teretifolia Willd. essential oil Fatma Pınar Türkmenoğlu1,* and Betül Demirci2

1Department of Pharmaceutical Botany, Anadolu University, 26470, Eskişehir, TURKEY 2Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY

*Corresponding author. Email: [email protected] Submitted: 26.07.2020; Accepted: 02.09.2020

Abstract The genus Achillea L. (Asteraceae), which is commonly known as “yarrow”, is mainly distributed in the Northern hemisphere. Turkey is one of the most important centers of diversity for the genus in the world. Many species of the genus Achillea have been known since the times of Dioscorides and used in the folk medicine to treat various illness and disorders in our country. In this study, the aerial parts of the endemic Achillea teretifolia Willd. collected from Afyon were investigated for essential oil composition. Hydrodistilled essential oil obtained from the aerial parts of the plant was analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Forty-eight compounds were identified representing 71.5% of the total oil, 1,8-cineole (16.1 %), camphor (12.7 %), p-cymene (10.6 %) and terpinen-4-ol (6.1 %) were characterized as the main constituents.

Keywords: Achillea teretifolia, Asteraceae, essential oil, GC-MS

Introduction The genus Achillea L. (Asteraceae) constitutes of approximately 140 perennial herbaceous species worldwide. The genus is distributed in Europe and temperate areas of Asia and in North America and Middle east particularly in the Northern hemisphere (Nemeth, 2010). In Turkey, it is represented by 47 species, 24 of which are endemic (Huber-Morath, 1975; Duman, 2000; Arabacı & Budak, 2009). In Anatolia, Achillea species are usually known as “civanperçemi” and various species of the genus are used as diuretic, emmenagogue, appetizer; carminative; in wound healing, abdominal pain, stomachache, symptomatic relief of colds, ulcer; and against diarrhea and flatulence in Turkish folk medicine (Honda et al., 1996; Baytop, 1999; Tuzlacı & Erol, 1999; Sezik et. al., 2001; Ezer & Arısan, 2006). Today, some therapeutic applications, such as antinociceptive, antiinflammatory wound healing, antispasmodic and hepatoprotective uses, are approved for several Achillea species by scientific experimental results (Agar et al., 2015). In addition to medical importance, Achillea species are also have economic value as they are consumed as vegetables, spices, beverages and additives in the food and cosmetic industry and in horticulture (Turkmenoglu et. al., 2015) Numerous studies performed on various species have showed that the genus Achillea is rich in terpenoids and phenolics such as flavonoids and phenolic acids (Nemeth, 2005; Si et al., 2006; Saedina et al., 2011; Turkmenoglu et al., 2015; Agar et al., 2015), which are possible bioactive compounds. Monoterpenes were reported to be the major constituents of essential oil of the genus although high levels of sesquiterpenes were also quantified (Si et al., 2006; Saedina et al., 2011; Turkmenoglu et al., 2015; Başer, 2016). Two main centers of diversity of Achillea occur in S.E. Europe and S.W. Asia. Diversified essential oil compositions from the Balkan Peninsula have been extensively studied and reported (Radulovic et al., 2007). However, report on essential oils of Achillea species growing in Turkey, which is one of the main centers of diversity, is still very limited. As a part of our ongoing studies of Achillea species, this paper represents the chemical compositions of the essential oil obtained by hydrodistillation from the aerial part of an endemic

Nat. Volatiles & Essent. Oils, 2020; 7(3): 55-60 Türkmenoğlu & Demirci DOI: 10.37929/nveo.784531

species A. teretifolia and analysed simultaneously by gas chromatography and gas chromatography-mass spectrometry. Materials and Methods Plant material The aerial parts of A. teretifolia was collected during the flowering stage (July 2014) in Afyon, Şuhut, vicinity of Tekke Village, 1340 m. The collected samples were identified and the voucher specimens were deposited in the Herbarium of Hacettepe University, Faculty of Pharmacy (HUEF 14055). Isolation of the essential oil The air-dried aerial parts of the plant material were hydrodistilled for 3 h using a Clevenger-type apparatus to produce a small amount (<0.01%) of volatiles, which was trapped in n-hexane. Sample was stored at 4°C in the dark until analysed. Gas chromatography analysis The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 °C. To obtain the same elution order with GC/MS, simultaneous auto injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms.

Gas chromatography-mass spectrometry analysis The GC-MS analysis was carried out with an Agilent 5975 GC/MSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60 °C for 10 min and programmed to 220 °C at a rate of 4 °C/min, and kept constant at 220 °C for 10 min and then programmed to 240 °C at a rate of 1 °C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 °C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.

Identification of components Identification of the oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley and MassFinder 3) (McLafferty & Stauffer, 1989; König et al., 2004) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data (Joulain & König, 1998) were also used for the identification. Results and Discussion Essential oil of A. teretifolia was obtained by hydrodistillation from air dried aerial parts and subsequently analyzed by GC and GC-MS systems. Forty-eight compounds representing 71.5% of the essential oil of A. teretifolia were characterized. Identified compounds with their relative percentages are listed in Table 1. The main constituents of A. teretifolia were 1,8-cineole (16.1 %), camphor (12.7 %), p-cymene (10.6 %) and terpinen-4-ol (6.1 %). In the genus Achillea, composition of the essential oil is highly variable because of some biotic and abiotic factors, such as ontogenic and morphogenic differentiations, environmental factors as well as applied method of oil extraction (Kindlovits & Nemeth, 2012). Previous investigations demonstrated that, 1,8-

Nat. Volatiles & Essent. Oils, 2020; 7(3): 55-60 Türkmenoğlu & Demirci DOI: 10.37929/nveo.784531

cineole, found in almost every essential oil, was reported to be the most frequently identified component, ranging from trace levels to 47.7% in essential oils of Balkan Achillea, while camphor and borneol were the second and third repeatedly detected compounds, respectively. Moreover, caryophyllene oxide and β- caryophyllene were reported to be frequently identified sesquiterpenoids (Nemeth, 2005; Radulovic et al., 2007). The review on essential oil compositions of Achillea species growing in Turkey (Başer, 2016) indicated that 1,8-cineole and camphor were the most abundant constituents. In addition to these compounds, piperitone, ascaridol, cis-piperitol, p-cymene rich oils were also reported from Turkey.

Table 1. Main components (%) of the essential oil of A. teretifolia

No RRIa Compounds %b IMc 1. 1014 Tricyclene 0.1 MS 2. 1032 -Pinene 1.3 RRI, MS 3. 1035 -Thujene 0.2 MS 4. 1076 Camphene 2.3 RRI, MS 5. 1118 -Pinene 0,5 RRI, MS 6. 1132 Sabinene 0,4 RRI, MS 7. 1188 -Terpinene 1.1 RRI, MS 8. 1203 Limonene 0.2 RRI, MS 9. 1213 1,8-Cineole 16.1 RRI, MS 10. 1255 -Terpinene 0.3 RRI, MS 11. 1280 p-Cymene 10.6 RRI, MS 12. 1445 Filifolone 0.3 MS 13. 1451 -Thujone 0.2 MS 14. 1522 Chrysanthenone 4.3 MS 15. 1532 Camphor 12.7 RRI, MS 16. 1553 Linalool 0.3 RRI, MS 17. 1556 cis-Sabinene hydrate 0.3 MS 18. 1571 trans-p-Menth-2-en-1-ol 1.0 MS 19. 1582 cis-Chrysanthenyl acetate 0.2 MS 20. 1586 Pinocarvone 0.3 RRI, MS 21. 1611 Terpinen-4-ol 6.1 RRI, MS 22. 1612 -Caryophyllene 0.1 RRI, MS 23. 1638 cis-p-Menth-2-en-1-ol 0.4 MS 24. 1661 Alloaromadendrene 0.2 MS 25. 1670 trans-Pinocarveol 0.1 RRI, MS 26. 1682 -Terpineol 0.3 MS 27. 1689 trans-piperitol 0.3 MS 28. 1706 -Terpineol 0.8 RRI, MS 29. 1719 Borneol 0.6 RRI, MS 30. 1748 Piperitone 0.2 RRI, MS 31. 1758 cis-Piperitol 0.3 MS 32. 1764 cis-Chrysanthenol 0.9 MS 33. 1776 -Cadinene 0.1 MS 34. 1786 ar-Curcumene 0.2 MS 35. 1804 Myrtenol 0.1 MS

Nat. Volatiles & Essent. Oils, 2020; 7(3): 55-60 Türkmenoğlu & Demirci DOI: 10.37929/nveo.784531

36. 1969 cis-Jasmone 0.2 MS 37. 2008 Caryophyllene oxide 0.4 RRI, MS 38. 2071 Humulene epoxide-II 0.1 MS 39. 2074 Caryophylla-2(12),6(13)-dien-5-one 0.1 MS 40. 2144 Spathulenol 0.2 MS 41. 2186 Eugenol 0.1 RRI, MS 42. 2187 T-Cadinol 1.0 MS 43. 2257 -Eudesmol 3.2 RRI, MS 44. 2260 15-Hexadecanolide 0.3 MS 45. 2300 Tricosane 0.4 RRI, MS 46. 2500 Pentacosane 1.1 RRI, MS 47. 2670 Tetradecanoic acid 0.3 RRI, MS 48. 2931 Hexadecanoic acid 0.7 RRI, MS Monoterpene Hydrocarbons 17.0 Oxygenated Monoterpenes 45.9 Sesquiterpene Hydrocarbons 0.6 Oxygenated Sesquiterpenes 5.0 Fatty acid 1.0 Others 2.0 Total 71.5 a Relative retention indices calculated against n-alkanes; b % calculated from TIC data. c IM: Identification method based on the relative retention indices (RRI) of authentic compounds on the HP Innowax column; MS, identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data Ünlü et al. (2002) previously reported 28 compounds in the essential oil of A. teretifolia collected from Sivas while main components of which were 1,8-cineole (19.9 %), camphor (11.1 %), borneol (11.9 %). In another study on A. teretifolia collected from Sivas, 1,8-cineole (15.9 %), borneol (8.1 %) and camphor (7.0 %) were the major identified compounds (Polatoğlu et al., 2013). According to the report on A. teretifolia collected from Konya-Beyşehir, the main components of essential oil were 1,8-cineole (34 %), camphor (11 %), terpinen-4-ol (8 %), -thujone (5 %) (Demirci et al., 2009). 1,8-cineole (34.3-54.5 %), camphor (14.2-19.8 %) and -thujone (3.5-6.7 %) were reported to be the major identified compounds in essential oil of this plant collected from Isparta-Keçiborlu (Yaşar & Fakir 2016). Kose et al. (2017) reported 1,8-cineole (18.8 %), camphor (17.6 %), p-cymene (13.7 %) and chrysanthenone (10.2 %) as the main constituents of essential oil of A. teretifolia collected from Afyon-Emirdağ. Completely different oil compositions were also published. Aslan et al. (2009), identified 37 compounds in A. teretifolia essential oil and reported piperitone (21.37 %), linalool (18.99 %) as major compounds. However, location of the plant was not reported in the article. In another study, 3-cyclohexen-1-one (21.6 %), linalool (14,3 %), 1,8-cineole (12.7 %), chrysanthenone (8.6 %), trans-chrysanthenol (7.8 %), -cadinene (4.0 %) were reported as main constituents (Kocak et al., 2010). According to Başer (2016) as 3-cyclohexen-1-one is not found in nature, these data requires reconfirmation.

ACKNOWLEDGMENT

Present work was supported by a grant from Hacettepe University Scientific Research Projects Coordination Unit (Project No: 014 D06 301 003-666).

Nat. Volatiles & Essent. Oils, 2020; 7(3): 55-60 Türkmenoğlu & Demirci DOI: 10.37929/nveo.784531

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