Microbial Transformation of Β-Caryophyllene and Longifolene by Wolfiporia Extensa

Volume 6, Issue 3, 2019 e-ISSN: 2148-9637

NATURAL VOLATILES & ESSENTIAL OILS A Quarterly Open Access Scientific Journal

NVEO Publisher: BADEBIO Ltd.

Nat. Volatiles & Essent. Oils, 2019; 6(3): 1-7 Yılmaz et al.

RESEARCH ARTICLE Composition of the Essential Oils of Scutellaria galericulata and S. tortumensis from Turkey

Gülderen Yılmaz1, *, Mehmet Çiçek2, Betül Demirci3 and K. Hüsnü Can Başer4

1 Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, Ankara, TURKEY 2 Department of Biology, Faculty of Arts and Science, Pamukkale University, Denizli, TURKEY 3 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, TURKEY 4 Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, N. Cyprus, Mersin 10, TURKEY

*Corresponding author. Email:[email protected]

Abstract The genus Scutellaria L. (Lamiaceae) is represented by 39 taxa in Turkey. The ratio of endemism in Turkey is 43.6%. The chemical compositions of the essential oils obtained by hydrodistillation of Scutellaria galericulata and S. tortumensis from Turkey were analyzed by gas chromatography and gas chromatography-mass spectrometry, simultaneously. The main components were determined as caryophyllene oxide (27.5%), β-caryophyllene (13.4%) and caryophylla-2(12),6-dien-5β-ol (4.8%) in the oil of S. galericulata. Germacrene D (29.8%), β-caryophyllene (17.1%) phytol (8.3%) and nonacosane (7.1%) were found as major components in the oil of S. tortumensis. Overall, S. galericulata and S. tortumensis essential oils representing 88.6% and 94.3% of the total, respectively.

Keywords: Scutellaria galericulata, S. tortumensis, Lamiaceae, essential oil composition

Introduction

Scutellaria L., a member of mint family, is a subcosmopolitan genus with 471 species (Paton, 1990a; WCSP, 2019) and widely spread in the tropical and southern hemisphere in the world (Paton, 1990b). The genus comprises 39 taxa, 17 of which are endemic (43.6%) in Turkey (Edmondson, 1982; Davis et al., 1988; Duman, 2000; Çiçek et al., 2011; Çiçek, 2012; Çiçek and Yaprak, 2011; Çiçek and Yaprak, 2013; Celep and Dirmenci 2016). Scutellaria, known as "Kaside" in Turkish, is not widely used in Turkey, but uses for various purposes in traditional medicines for example as a soothing, hemostatic, tonic and wound healing agent, and for constipation in Anatolian folk medicine have been reported (Baytop, 1999; Özçelik, 1990). In recent years, according to ethnobotanical studies made in Turkey, S. orientalis subsp. pichleri (Stapf) J.R. Edm. and S. orientalis subsp. virens (Boiss. & Kotschy) J.R.Edm. have been used against cancer and hemorrhoids. S. orientalis subsp. bicolor (Hochst.) J.R.Edm. and S. orientalis subsp. pichleri (Stapf) J.R.Edm. have been used as astringent in folk remedies (Çakıcıoğlu et al., 2010). It has been reported that some Scutellaria species (S. baicalensis Georgi, S. viscidula Bunge, S. amoena C.H.Wright, S. rehderiana Diels, S. ikonnikovii Juz. and S. hypericifolia H.Lév.) are used in Traditional Chinese Medicine (TCM) to treat diseases such as hepatitis, jaundice, tumor, leukemia, arteriosclerosis, diarrhea and inflammation (Huang et al., 2005; The Grand Dictionary of Chinese Herbs, 1977). The leaves of S. indica L. and S. baicalensis species in Asia are used as vegetables and also, as herbal tea (Tanaka, 1976). S. laterifolia L. (American skullcap) has been used against neurological diseases such as epilepsy, contraction, hysteria and

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insomnia (Millspaugh, 1974). The roots of S. baicalensis (Baikal skullcap) are known to be used to increase memory in TCM (Adams, 2007). Many Scutellaria species have been tested for biological activities such as spasmolytic, antidiarrheal, antifungal, antipyretic, antioxidant, anticancer, anti-HIV, antibacterial, antiviral, anti-inflammatory and anticonvulsant (Ersöz et al., 2002; Lin et al, 2009; Shang et al., 2010). The flavones isolated from the roots of Scutellaria show antioxidant, anti-viral, anti-thrombotic, anti-inflammatory, antitumor and anti- cardiovascular properties against some diseases (Kimura et al., 1997; Wang et al., 1998; Gao et al., 1999; Huang et al., 2000; Wu et al., 2001; Ma et al., 2002; Chi et al., 2003; Hwang et al., 2005; Gua et al., 2007; Wang et al., 2007). In this present study, we aimed to investigate chemical composition of essential oils of Scutellaria galericulata L. and S. tortumensis (Kit Tan & Sorger) A.P.Khokhr. from Turkey. This study is a continuation of our previous studies. Up to now, we have reported compositions of the essential oils of all subspecies of Scutellaria albida L. (Çiçek et al., 2010), S. diffusa, S. heterophylla, S. salviifolia, (Çiçek et al., 2011) and all subspecies of S. brevibracteata Stapf from Turkey (Yılmaz et al., 2019). We, also, carried out acetylcholinesterase, butyrylcholinesterase, and tyrosine’s inhibitory activities and antioxidant activities in 33 Scutellaria taxa from Turkey (Şenol et al., 2010). Here, we report on the isolation and characterization of various components present in the essential oils of S. galericulata and S. tortumensis. Materials and Methods Plant material Aerial parts of Scutellaria galericulata (voucher number: AEF 23936) and S. tortumensis (voucher number: AEF 23967) were collected from their natural habitat in Bolu and Erzurum provinces, respectively. Voucher specimens were identified by M. Çiçek and deposited at Ankara University, Herbarium of Faculty of Pharmacy (AEF). Isolation of the essential oils Air-dried aerial parts of the plants were hydrodistilled for 3 h using a Clevenger-type apparatus to produce a small amount of essential oil which was trapped in n-hexane. The analysis processes of gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were performed with reference to Demirci et al. (2017). Results and Discussion

Analyses of the hydrodistilled oils were performed on GC and GC-MS systems, simultaneously. The composition of the Scutellaria galericulata and S. tortumensis are given in Table 1, according to their relative retention indices (RRI) and with their relative percentages (%). A total of 42 compounds were characterized in the essential oil of Scutellaria galericulata, representing 88.6% of the total oil. The main components were determined as caryophyllene oxide (27.5%), β-caryophyllene (13.4%) and caryophylla-2(12),6-dien-5β-ol (4.8%). The results of analyses showed that the essential oil of S. galericulata comprised mainly oxygenated sesquiterpenes (43.2%), sesquiterpene hydrocarbons (36.8%), fatty acids (2.9%) and diterpenes (2.2%). In the oil of Scutellaria tortumensis, 35 components were characterized representing 94.3% of the total oil. Germacrene D (29.8%), β-caryophyllene (17.1%) phytol (8.3%) and nonacosane (7.1%) were found as major

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components. The essential oil composition of S. tortumensis consisted dominantly of sesquiterpenes hydrocarbons (57.6%), diterpenes (8.3%), oxygenated sesquiterpenes (7.2%) and others (19.2%). Lawrence et al. (1972) reported caryophyllene (29.4%), trans-β-farnesene (17.0%), menthone (10.4%) and 1- octen-3-ol (8.0%) as main constituents in the oil of Scutellaria galericulata in Canada. In this study, the main components in the essential oil of S. galericulata were determined as caryophyllene oxide (27.5%), β- caryophyllene (13.4%) and caryophylla-2(12),6-dien-5β-ol (4.8%).

Table 1. The essential compositions of the Scutellaria galericulata and S. tortumensis RRI Compound S. galericulata (%) S. tortumensis (%) 1400 Nonanal 0.2 - 1452 1-Octen-3-ol 1.7 - 1466 -Cubebene 1.1 0.5 1482 Longipinene 0.6 1.3 1497 -Copaene 1.1 - 1500 Pentadecane - 2.0 1535 -Bourbonene 0.2 0.5 1549 -Cubebene 2.4 - 1553 Linalool - 0.3 1589 -Ylangene - 0.2 1600 -Elemene 0.2 - 1612 -Caryophyllene 13.4 17.1 1668 (Z)--Farnesene 1.1 1.0 1671 Acetophenone 0.5 - 1687 -Humulene 3.6 1.7 1700 Heptadecane - 2.0 1704 -Muurolene - 0.2 1711 -Himachalene 2.1 - 1726 Germacrene D 2.2 29.8 1729 -Himachalene 0.6 - 1743 Eremophilene 2.1 1.6 1755 Bicyclogermacrene - 3.0 1773 -Cadinene 1.7 0.5 1776 -Cadinene tr 0.2 1800 Octadecane - 0.4 1801 -Cuprenene 0.5 - 1849 Cuparene 2.7 - 1849 Calamenene tr - 1882 -Dehydro-ar-himachalene 0.3 - 1888 ar-Himachalene 0.6 - 1900 Nonadecane - 0.7 1900 epi-Cubebol 0.8 - 1924 -Dehydro-ar-himachalene 0.3 - 1957 Cubebol 1.1 - 1958 (E)--Ionone - 0.7 2000 Eicosane - 0.6

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2001 Isocaryophyllene oxide 0.8 0.6 2008 Caryophyllene oxide 27.5 5.3 2050 (E)-Nerolidol 1.0 - 2071 Humulene epoxide-II 3.8 - 2080 Cubenol 0.4 - 2088 1-epi-Cubenol 0.5 - 2100 Heneicosane - 0.7 2131 Hexahydrofarnesyl acetone 1.1 1.8 2144 Spathulenol - 0.4 2174 Fokienol 0.7 - 2200 Docosane - 0.5 2204 Eremoligenol - - 2209 T-Muurolol - 0.4 2255 -Cadinol - 0.5 2289 Oxo--Ylangene 1.1 - 2300 Tricosane - 0.8 2324 Caryophylla-2(12),6(13)-dien-5-ol (=Caryophylladienol II) 0.7 - 2392 Caryophylla-2(12),6-dien-5-ol (=Caryophyllenol II) 4.8 - 2400 Tetracosane - 0.3 2500 Pentacosane tr 1.4 2503 Dodecanoic acid tr - 2622 Phytol 2.2 8.3 2670 Tetradecanoic acid tr - 2700 Heptacosane - 0.2 2900 Nonacosane - 7.1 2931 Hexadecanoic acid 2.9 1.7 Oxygenated Monoterpenes - 0.3 Sesquiterpene Hydrocarbons 36.8 57.6 Oxygenated Sesquiterpenes 43.2 7.2 Fatty acids 2.9 1.7 Diterpenes 2.2 8.3 Others 3.5 19.2 Total 88.6 94.3 Oil yield tr tr RRI: Relative retention indices calculated against n-alkanes. % calculated from FID data. tr: Trace (< 0.1 %)

In this study, it was shown that high percentage of caryophyllene oxide (27.5%) and germacrene D (29.8%) were the major components of the essential oils obtained from Scutellaria galericulata and S. tortumensis, respectively. β caryophyllene content was similar in both Scutellaria species.

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Table 2: The major components in the essential oil of various Scutellaria species growing naturally in Turkey Species Major Components Referances S. albida subsp. albida linalool (20.4%), hexadecanoic acid (15.6%), β-caryophyllene (Çiçek et al., 2010) (14.2%),α-terpineol (8.2%) S. albida subsp. colchica hexadecanoic acid (12.9%), (E)-nerolidol (9.1%), (Çiçek et al., 2010) hexahydrofarnesyl acetone (7.3%) S. albida subsp. condensata linalool (28.5%), (E)-nerolidol (16.8%), geraniol (8.1%), (Çiçek et al., 2010) hexadecanoic acid (6.0%) S. albida subsp. velenovskyi β-caryophyllene (20.0%), hexadecanoic acid (17.3%) and (Çiçek et al., 2010) tetradecanoic acid (10.2%) S. diffusa hexadecanoic acid (30%)- caryophyllene oxide (9%) (Çiçek et al., 2010) S. heterophylla germacrene D (21%), hexadecanoic acid (16%) caryophyllene (Çiçek et al., 2010) (11%) S. salviifolia germacrene D (40%), bicyclogermacrene (14%), - (Çiçek et al., 2010) caryophyllene (11%) S. brevibracteata subsp. brevibracteata β-caryophyllene (22.8%),- caryophyllene oxide (16.0%) (Yılmaz et al., 2019) S. brevibracteata subsp. subvelutina β-caryophyllene(28.3%), linalool(12.4,) hexadecanoic acid (Yılmaz et al., 2019) (10.8%) S. brevibracteata subsp. pannosula β-caryophyllene (36.4%), α-cadinol (9.8%), δ-cadinene (7.0%) (Yılmaz et al., 2019)

Main components of the essential oils of various Scutellaria species in growing naturally in Turkey are given in Table 2. As shown in the table, while hexadecanoic acid (30%) was found to be at the highest ratio in S. diffusa, its content in S. albida and S. heterophylla essential oils was found to be similar. High contents of β- caryophyllene and hexadecanoic acid present in the essential oil of S. brevibracteata subsp. subvelutina showed similarity to those of S. albida subsp. albida, S. albida subsp. velenovskyi and S. heterophylla. Germacrene D and β-Caryophyllene are among the main components of the essential oils of all Scutellaria species (Çiçek et al., 2010; Raju Sripathi et al.2017; Yılmaz et al. 2019). Other main components are caryophyllene oxide and linalool. Despite many traditional uses of Scutellaria, biological activity studies are limited. According to our literature search, we could see that biological activity studies were mostly conducted on S. baicalensis. Anti-RSV, Anti- HIV, anti-inflammatory, hepatoprotective, neuroprotective, anti-oxidative, antimutagenic, anti-HBV, anticonvulsant activities has been reported from the compounds isolated from S. baicalensis. Anti-tumor activity has also been demonstrated in S.barbata and S. baicalensis. Anti-feedent activity was found to be present in S. rubicunda and S. galericulata; anti-oxidant activity in S. baicalensis, S. barbata, S. immaculata & S. ramosissina; anxiolytic activity was showed in S. baicalensis and S. laterfollia (Raju Sripathi et al., 2017). Anti-microbial studies carried out on the essential oils are very scarce. Anti-microbial activity has been shown in the essential oils of S. immaculata, S. ramosiss, S. barbata, S. albida ssp. albida, S. sieberi and S. rupestris ssp. adenotrich species. Antibacterial activity studies were performed on S. grossa; antioxidant and larvicidal activity studies were performed on S. wightiana essential oils (Raju Sripathi et al., 2017). Although there have been studies of compounds isolated from many Scutellaria species and their biological activity studies, their pharmacological activities have not been fully studied. More phytochemical studies are needed due to the fact that the plants, which have a high traditional usage, are promising in the search for new drugs. There are few studies on Scutellaria species in our country. In this study we have examined the essential oils of S. galericulata and S. tortumensis. Here, we report on the isolation and characterization of various components present in essential oils. Furthermore, we plan to carry out the biological activities of the essential oil.

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Received : 14.06.2019 Accepted: 02.08.2019

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RESEARCH ARTICLE Microbial transformation of β-caryophyllene and longifolene by Wolfiporia extensa

Özge Özşen Batur1,*, İsmail Kıran1, Ralf G. Berger2 and Betül Demirci3

1 Department of Chemistry, Faculty of Science and Art, Eskişehir Osmangazi University, 26480, Eskişehir, TURKEY 2 Institue of Food Chemistry, Gottfried Wilhelm Leibniz University, 30167, Hannover, GERMANY 3 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY

*Corresponding author. Email: [email protected]

Abstract β-Caryophyllene and longifolene are sesquiterpenes with characteristic aroma properties, which are primary constituents of essential oils and important ingredients commonly used in food, perfumery, cosmetics, detergents and pharmaceuticals, which create a worldwide market. β-Caryophyllene and longifolene were converted through microbial biotransformation by using Wolfiporia extensa to a mixture of products over seven days at 25 oC to be evaluated as potential aroma and antimicrobial agents. The characterization of transformation products was carried out by comparison of their GC-MS spectra and retention indices with that of published data (Wiley, NIST and ADAMS databases). As a result, microbial transformation reactions produced various volatile compounds mainly consisting of ketones, aldehydes and alcohol-bearing derivatives of β-caryophyllene and longifolene.

Keywords: Aroma substances, GC-MS, microbial transformation, terpenoids, W. extensa

Introduction

The production of aroma or flavour substances is carried out by 90 % extraction from plants and chemical synthesis methods. Obtaining aroma compounds from plants is expensive due to tedious isolation protocols. Chemical synthesis serves as a good alternative for the synthesis of aroma substances, but they are not preferred because of sometimes harmful effects to the health and environment. This directed attention for the development of environmentally friendly processes. In recent years, the utilisation of biotechnological processes on the production of flavour compounds appeared to be the first choice. However, only less than 10 % of aroma compounds is derived from bioprocesses (Berger, 2007; Wright, 2010). β-caryophyllene, a natural sesquiterpene, is a major plant volatile found in the essential oils of many different spices and food condiments (Fitjer et al., 1995; Schmitt, Levy, & Carroll, 2016). It has been used in a wide variety of prepared foods; baked goods, frozen dairy, meat products, commercially used as a food additive and a natural flavouring compound. It has been also used in cosmetics (Andersen et al., 2010; Sharma et al., 2016; Younis & Mohamed, 2019). Longifolene is a natural tricyclic olefin and occurs to the extent of 5-10 % in the Indian turpentine oil. Longifolene produced commercially from the oleoresin of Himalayan pine, Pinus longifolia Roxb and its structure was established in 1953, but still continues to attract attention as a novel substrate for transformations, generating interesting chemistry and widely used in many different fields (Cao et al., 2019; Dev, 1981; Ford, Api, & Letizia, 1992) As already mentioned β-caryophyllene and longifolene substrates are common widespread sesquiterpenes found in essential oils. The new biotransformations derivatives formed thereof can be used as potential flavour and fragrance ingredients.

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The aim of the present study was to biotransform the substrates β-caryophyllene and longifolene with the help of the fungus Wolfiporia extensa (Peck) Ginns (syn. Poria cocos F. A. Wolf, Polyporaceae) for the production of new natural aroma compounds, which was the most promising and substrate yielding biocatalyst. Materials and Methods Microorganisms and chemicals Wolfiporia extensa (Strain no:279.55) was purchased from the Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands). β-caryophyllene and longifolene were purchased from Sigma-Aldrich, Germany. Cyclohexanol (≥ 99 %; Merck-Schuchardt, Hohenbrunn, Germany) and cyclopentanol (99 %; Sigma-Aldrich, Steinheim, Germany) were used as analytical standards in gas chromatography. Conditions of cultivation Small scale biotransformation studies with substrates were carried out by different microorganisms. Among the evaluated, the fungi W. extensa produced at least three polar compounds according to chromatographic analyses, which urged further preparative scale biotransformation studies for 7 days at 25 oC. Pre-cultures (100 mL medium in 250 mL flasks) were prepared by homogenisation a 10 × 10 mm agar plug with mycelium using an Ultra Turrax (Miccra D-9, Art, Mühlheim, Germany). Afterwards, the fungi were grown submerged in modified standard nutrient liquid medium (SNL) by mixing D-(+)-glucose monohydrate

(30 g), L-Asparagine monohydrate (4,5 g), yeast extract (3 g), KH2PO4 (1,5 g), MgSO4 (0,5 g) and trace element solution [0.08 g/L FeCl3 x 6 H2O, 0.09 g/L ZnSO4 x 7 H2O, 0.03 g/L MnSO4 x H2O, 0.005 g/L CuSO4 x 5 H2O, 0.4 g/L EDTA disodium salt dehydrate] in distilled water (1 L). Prepared medium was adjusted to pH 6.0 with 1 M NaOH and autoclaved at 121°C and 1 bar for 20 min. Flask were grown aerobically at 24 °C and 150 rpm on an orbital shaker (Multitron, Infors, Bottmingen, Switzerland). Biotransformation of the substrates Biotransformation was started 24 h after incubation by adding 15 mM of β-caryophyllene and longifolene. The structures were examined by means of gas chromatography and mass spectrometry (GC/MS). Extraction of the volatile metabolites Liquid culture was extracted in a separating funnel three times or via continuous liquid/liquid extraction with pentane/diethyl ether [1:1.12 (v/v)] (P/E). The organic layers were dried over Na2SO4 and concentrated with a Vigreux column at 40 oC, brought to a final volume of about 1 mL by fractionated distillation. External standard was cyclopentanol, whereas internal standard (determination of the recovery) was cyclohexanol. The flavour fractionated distillation extracts were analysed by means of gas chromatography (GC). Characterization of transformation products About 1 µL was injected into GC and quantification was performed according to the internal standard and external standard. Gas chromatography-mass analysis The GC/MS analysis was carried out using 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

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kept at 60 oC 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 oC at a rate of 1 oC/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. Gas chromatography-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. Identification Identification of the essential oil components in the biotransformation mixture was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (Johnston, 1989; Koenig & D. Hochmuth, 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 (Adams, 1995; Jennings & Shibamoto, 1980; Joulain & Konig, 1998; Kondjoyan & Berdagué, 1996; Rychlik et al., 1998), were also used for the identification. Results and Discussion

The biotransformations of β-caryophyllene (Scheme. 1) and longifolene (Scheme. 2) were carried out using W. extensa. Overall, 15 volatile compounds were present for two substrates in library, but three of them not identified. The spectral of the three major transformation ketones-aldehydes products, Table 1 (7), (8) and Table 2 (7) resulted in the detection of so far unknown natural compounds. Further minor oxidation products, especially the corresponding alcohol-bearing derivatives, caryophylladienol-I (4), caryophyllenol-I (5) in Table 1 and salviadienol (3), spathulenol (4), torilenol (5), cedranol (6) in Table 2 were identified. Besides, a metabolite named as “4,4,8-trimethyltricyclo [6.3.1.0(1,5)] dodecane-2,9-diol” (Winmain) was obtained from the biotransformation of β-caryophyllene. There were two similar mass spectra in the literature (Choudhary, Siddiqui, Nawaz, & Atta ur, 2006; Noma, Hashimoto, Uehara, & Asakawa, 2010). It was thought that β-caryophyllene was first converted to caryopyllene oxide and then to clovandiol without the microorganism. Therefore, no caryophyllene was detected. As a result, substrates were converted by W. extensa over 7 days at 25 oC to new metabolites. Biotransformation reactions produced various volatile compounds mainly consisting of ketones, aldehydes and alcohol-bearing derivatives of β-caryophyllene and longifolene. The formed metabolite structures were identified by comparison of their GC-MS spectra and retention indices with that of published data (Wiley, NIST and ADAMS databases). The compounds identified in the biotransformation mixture are shown in Tables 1 and 2. The results showed that the enzymatic reactions were highly regio- and enantioselective.

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Scheme 1. Biotransformation of β-caryophyllene (Heinlein & Buettner, 2012; Ishida, 2005; King & Dickinson, 2003; Oda, Fujinuma, Inoue, & Ohashi, 2011).

Scheme 2. Biotransformation of longifolene (Abraham, Hoffmann, Kieslich, Reng, & Stumpf, 1985; Asakawa, Ishida, Toyota, & Takemoto, 1986; Bhattacharyya, Prema, Dhavalikar, & Ramchandran, 1963; Devi, 1979; Ishida, 2005; Joglekar, Vora, Dhere, & Dhavilkar, 1968; Khan, Atif, & Al-Aboudi, 2014).

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Table 1. β-caryophyllene transformation products from cultures of W. extensa identified in this study Peak no: R.R.I. % Compound 1 1612 15.1 β-caryophyllene (substrate) (Mw: 204) 2 1687 1.8 α-caryopyllene= Humulene (substrate) (Mw: 204) 3 2071 tr Humulene epoxide-II (Mw: 220) 4 2316 0.8 Caryophylladienol-I (Mw: 220) 5 2389 1.0 Caryophyllenol-I (Mw: 220) 6 2563 2.0 4,4,8-trimethyltricyclo [6.3.1.0(1,5)] dodecane-2,9-diol (Mw: 238) 7 2628 6.6 M1 (220, 205, 187, 177, 162, 147, 140, 135, 128, 121, 112, 107, 98, 93, 84, 79, 72, 67, n.i* 61, 55, 43) 8 2716 9.8 M2 (220, 202, 194, 187, 179, 168, 161, 153, 147, 139, 133, 123, 117, 109, 103, 95, 89, n.i* 81, 75, 69, 55, 45, 39) RRI: Relative retention indices calculated against n-alkanes; % percentages calculated from FID data; tr: Trace (<0,1 %); n.i*: non identified.

Scheme 3. β-caryophyllene transformation products (Table 1)

Table 2. Longifolene transformation products from cultures of W. extensa identified in this study Peak no: R.R.I. % Compound 1 1553 0.28 Linalool (Mw: 154) 2 1583 0.92 Longifolene (Substrate) (Mw: 204) 3 2130 1.27 Salviadienol (Mw: 220) 4 2144 3.38 Spathulenol (Mw: 220) 5 2278 3.31 Torilenol (Mw: 220) 6 2736 5.59 Cedranol (Adams, 1995) (Mw: 222) 7 2930 14.97 M3 (236, 218, 203, 189, 181, 175, 163, 157, 147, 136, 128, 121, 115, 109, 99, 93, 82, n.i* 73, 67, 55, 41) RRI: Relative retention indices calculated against n-alkanes; % percentages calculated from FID data; n.i*: non identified.

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Scheme 4. Longifolene transformation products (Table 2.)

As conclusion, the fungi W. extensa produced also several polar compounds according to chromatographic analyses, which could not be identified in the present study. Further studies are needed to evaluate the three of metabolites which are not identified.

ACKNOWLEDGMENT Part of this work was conducted during the ERASMUS visit of Özge ÖZŞEN BATUR in Germany. Part of this work was presented at the IV. International Conference on Antimicrobial Research Congress from 29 June to 1 July, 2016 in Torremolinos-Malaga (Spain).

REFERENCES Abraham, W.R., Hoffmann, H. M., Kieslich, K., Reng, G., & Stumpf, B. (1985). Microbial transformations of some monoterpenoids and sesquiterpenoids. Ciba Found Symp, 111, 146-160. Adams, R.P. (1995). Identification of Essential Oil Components by Gas Chromatography/mass Spectrometry. Carol Stream: Allured Publishing Corporation. Andersen, F.A., Bergfeld, W.F., Belsito, D. V., Hill, R.A., Klaassen, C.D., Liebler, D.C. & Snyder P.W. (2010). Final report of the cosmetic ingredient review expert panel amended safety assessment of Calendula officinalis-derived cosmetic ingredients. Int. J. Toxicol., 29 (Suppl. 4), 221-243. Asakawa, Y., Ishida, T., Toyota, M. & Takemoto, T. (1986). Terpenoid biotransformation in mammals. IV Biotransformation of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (- )-elemol, (-)-abietic acid and (+)-dehydroabietic acid in rabbits. Xenobiotica, 16(8), 753-767. Berger, R.G. (Ed.). (2007). Flavours and fragrances: chemistry, bioprocessing and sustainability. Springer Science & Business Media. Bhattacharyya, P.K., Prema, B.R., Dhavalikar, R.S., & Ramchandran, B.V. (1963). Microbiological transformations of terpenes. IV. Structure of an anhydride obtained after fermentation of some terpenoid hydrocarbons by Aspergillus niger. Indian Journal of Chemistry, 1, 171-176.

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Cao, Y., Zhang, R., Liu, W., Zhao, G., Niu, W., Guo, J., Xian, M. & Liu, H. (2019). Manipulation of the precursor supply for high-level production of longifolene by metabolically engineered Escherichia coli. Scientific Reports, 9(1), 95. Choudhary, M.I., Siddiqui, Z.A., Nawaz, S.A. & Atta-ur-Rahman (2006). Microbial transformation and butyrylcholinesterase inhibitory activity of (−)-caryophyllene oxide and its derivatives. Journal of Natural Products, 69(10), 1429-1434. Dev, S. (1981). Aspects of longifolene chemistry. An example of another facet of natural products chemistry. Accounts of Chemical Research, 14(3), 82-88. Devi, J.R. (1979). Microbiological transformations of terpenes: Part XXVI--Microbiological transformation of caryophyllene. Indian Journal of Biochemistry & Biophysics, 16(2), 76-79. Fitjer, L., Malich, A., Paschke, C., Kluge, S., Gerke, R., Rissom, B., Weiser, J. & Noltemeyer, M. (1995). Rearrangement of (-)-β.-Caryophyllene. A Product Analysis and Force Field Study. Journal of the American Chemical Society, 117(36), 9180- 9189. Ford, R.A., Api, A.M. & Letizia, C.S. (1992). Fragrance raw materials monographs. Longifolene. Food and Chemical Toxicology, 30, 67S-68S. Heinlein, A. & Buettner, A. (2012). Monitoring of biotransformation of hop aroma compounds in an in vitro digestion model. Food & Function, 3(10), 1059-1067. Ishida, T. (2005). Biotransformation of terpenoids by mammals, microorganisms, and plant-cultured cells. Chemistry & Biodiversity, 2(5), 569-590. Jennings, W., & Shibamoto, T. (1980). Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography: Academic Press.

Joglekar, S.S., Vora, M.A., Dhere, S.G., & Dhavilkar, R.S. (1968). Microbial transformations of terpenoids: citronellal, citral, eugenol, Δ-3-carene, α-pinene, and longifolene. Indian Oil and Soap Journal, 34, 85-88. Johnston, C. (1989). The Wiley / NBS Registry of Mass Spectral Data, Volumes 1-7 (McLafferty, Fred W.; Stauffer, Douglas B.). Journal of Chemical Education, 66(10), A256. Joulain, D., & Konig, W. (1998). The Atlas of Spectral Data of Sesquiterpene Hydrocarbons: E.B.-Verlag. Khan, N.T., Atif, M., & Al-Aboudi, A. (2014). Microbial Transformation of (-)-Alloisolongifolene. Oriental Journal of Chemistry, 30(3), 941-945. King, A.J., & Dickinson, J.R. (2003). Biotransformation of hop aroma terpenoids by ale and lager yeasts. FEMS Yeast Research, 3(1), 53-62. Koenig, W.A., & D. Hochmuth, J.D.H. (2004). Terpenoids and Related Constituents of Essential Oils. MassFinder 3, Hamburg, Germany. Kondjoyan, N., & Berdagué, J.L. (1996). A compilation of relative retention indices for the analysis of aromatic compounds. INRA de Theix, France: Laboratoire Flaveur. Noma, Y., Hashimoto, T., Uehara, S., & Asakawa, Y. (2010). Microbial transformation of isopinocampheol and caryophyllene oxide. Flavour Fragrance Journal, 25(3), 161-170. Oda, S., Fujinuma, K., Inoue, A. & Ohashi, S. (2011). Synthesis of (−)-β-caryophyllene oxide via regio-and stereoselective endocyclic epoxidation of β-caryophyllene with Nemania aenea SF 10099-1 in a liquid–liquid interface bioreactor (L–L IBR). Journal of Bioscience and Bioengineering, 112(6), 561-565. Rychlik, M., Schieberle, P., Grosch, W., Deutsche Forschungsanstalt für, L., Universität, M., & Institut für Lebensmittelchemie der, T. (1998). Compilation of odor thresholds, odor qualities and retention indices of key food odorants. Garching: Deutsche Forschungsanstat für Lebensmittelchemie and Instit für Lebensmittelchemie der Technischen Universität München.

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Schmitt, D., Levy, R. & Carroll, B. (2016). Toxicological evaluation of β-caryophyllene oil: subchronic toxicity in rats. International Journal of Toxicology, 35(5), 558-567. Sharma, C., Al Kaabi, J.M., Nurulain, S.M., Goyal, S.N., Kamal, M.A. & Ojha, S. (2016). Polypharmacological Properties and Therapeutic Potential of β-Caryophyllene: A Dietary Phytocannabinoid of Pharmaceutical Promise. Current Pharmaceutical Design, 22(21), 3237-3264. Wright, J. (2010). Creating and formulating flavours. In: Food Flavour Technology, Second Edition, Taylor A.J. and Linforth R.S.T., 1-23. Younis, N. S., & Mohamed, M.E. (2019). β-caryophyllene as a potential protective agent against myocardial injury: the role of toll-like receptors. Molecules, 24(10), 1929.

Received : 28.06.2019 Accepted: 02.08.2019

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RESEARCH ARTICLE Essential oil composition of two Greek cultivated Sideritis spp.

Charalampia Kloukina, Ekaterina-Michaela Tomou, Helen Skaltsa*

Department of Pharmacognosy & Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece

*Corresponding author. Email: [email protected]

Abstract In this present work, essential oil composition of cultivated commercially available Sideritis raeseri Boiss. & Heldr. and Sideritis scardica Griseb. from three different restricted areas of Kozani (central Greece) were studied. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC-MS. Monoterpene hydrocarbons constituted the main fraction of S. raeseri from Polirraxo (Kozani) and of S. scardica from Chromio (Kozani), while sesquiterpene hydrocarbons were the main group of S. raeseri from Metamorfosis (Kozani) and S. scardica from Metamorfosis (Kozani). Despite the different cultivations, some constituents were found even in different percentages in both samples of S. scardica: α-pinene, -pinene, cis-caryophyllene, bicyclogermacrene and germacrene D. It is noteworthy that the two samples of S. raeseri have totally different main constituents, which could be related to the different cultivation conditions, as well as to the known tendency of some Sideritis species to hybridize, which suggests further research.

Keywords: Sideritis raeseri, Sideritis scardica, cultivation, composition of volatiles

Introduction

Sideritis L. (Lamiaceae) comprises more than 150 species, indigenous in Central Europe, the Mediterranean, the Balkans, the Iberian Peninsula and the west Asia (González-Burgos et al., 2011). Traditionally, the infusion of herba Sideritis (S. scardica, S. clandestina, S. raeseri, S. syriaca) has been used against the common cold, mild gastrointenstinal disorders and for cough relief (EMA, 2015). Essential oils (EOs) from the genus Sideritis L. are widely used in pharmaceutical herbal products, food additives and as important sources in aromatic and cosmetic industries. Numerous studies over the past years have elucidated a plethora of significant biological activities of the Sideritis EOs, such as anti-inflammatory, antioxidant, antimicrobial, antifungal and anti- ulcerative (González-Burgos et al., 2011). Intriguingly, the analyses of the essential oils of Sideritis spp. have been the subject of several studies and show significant diversity in their chemical compositions, mainly due to hybridism. Therefore, it seemed interesting to investigate the volatile constituents of this genus. The overall aim of this study was to examine two cultivated Sideritis species, originated from different restricted locations of Kozani (Metamorfosis, Polirraxo and Chromio), focusing on determining the effects of organic cultivations on essential oil yield and composition. The chemical analyses of the essential oils of each species were compared with the previously stated in the literature. Materials and Methods Plant material Aerial parts of four Sideritis different organic cultivated populations, commercially available, were provided as follows: S. raeseri, one sample from Metamorfosis Kozani (SR_M) and another from Polirraxo Kozani (SR_P); S. scardica, one sample from Metamorfosis Kozani (SS_M) and a second one from Chromio Kozani (SS_X). The plants were identified by Associate Prof. Th. Constantinidis (Faculty of Biology, National and

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Kapodistrian University of Athens). Voucher specimens have been deposited in the Department of Pharmacognosy and Chemistry of Natural Products (Faculty of Pharmacy, NKUA) under the following codes: ATHU Skaltsa & Tomou 002A & 002B for SR_M and SR_P, respectively; ATHU Skaltsa & Tomou 003A & 003B for SS_M and SS_X, respectively. Extraction and chemical composition of essential oils 25.0 g of air-dried aerial parts of each plant material were cut in small pieces, and the essential oil was obtained by hydrodistillation for 1.5 h in a modified Clevenger apparatus with a water-cooled oil receiver to reduce artefacts produced during distillation by over-heating. The oils, taken in 2 mL of capillary GC grade n- pentane and dried over anhydrous sodium sulphate, was subsequently analysed by GC-MS and stored at -20 °C. The composition of the volatile constituents was established by GC-MS analyses, performed on a Hewlett- Packard 6890/5973 system operating in EI mode (70eV) equipped with a split/splitless injector, using a fused silica HP-5 MS capillary column (30 m x 0.25 mm I.D., film thickness: 0.25 μm). Helium was used as a carrier gas at a flow rate of 1.0 mL/min and the oven temperature was increased from 60°C to 240°C at a rate of 10°C/min. Injection was at 280°C in a split ratio 1:5; the ion source temperature and transfer line temperature were set at 230 and 250°C, respectively. Injection volumes of each sample were 2 μL. Retention indices for all compounds were determined according to the Van den Dool approach (Van den Dool & Kratz, 1986), using n-alkanes as standards. The identification of the components was based on comparison of their mass spectra with those of Wiley and NBS Libraries (Massada, 1986) and those described by Adams (2007), as well as by comparison of their retention indices with literature data (Adams, 2007). In many cases, the essential oils were subjected to co-chromatography with authentic compounds (Fluka, Sigma). Results and Discussion Sideritis raeseri Boiss. & Heldr. The yields (v/w) of the essential oils from the aerial parts of cultivated S. raeseri from Metamorfosis Kozani (SR_M) and of cultivated S. raeseri from Polirraxo Kozani (SR_P) were 0.12% and 0.10 %, respectively. The chemical composition of the essential oil of SR_M showed 41 constituents, where the main components were germacrene D (14.8 %), 9-octadecen-1-ol (12.4%), bicyclogermacrene (11.2 %) and cis-caryophyllene (10.0 %) (Table 1). In contrast, the essential oil of SR_P was consisted by 57 compounds with -pinene (19.1 %) being the major compound followed by germacrene D (15.9 %), α-pinene (12.9 %) and limonene (8.5 %) (Table 1). Furthermore, it is important to note that the two samples demonstrated a totally different chemical load. Sesquiterpene hydrocarbons (46.9 %) were the main group of SR_M, while monoterpene hydrocarbons (45.4 %) were the main fraction of SR_P (Table 3). Comparing our results with previous data (Papageorgiou et al., 1982; Koedam, 1985; Aligiannis et al., 2001 και Kostadinova et al., 2007; Pljevljakušic et al., 2011), we observed some chemical variations between the components of the essential oils. According to Koedam (1985), the key constituents of the essential oil of S. raeseri were the monoterpene hydrocarbons α-pinene (16.5%), β-pinene (20.61%) and limonene (6.73%). This is in accordance only with the results of our sample SR_P. In contrast, Kostadinova et al. (2007) reported the sesquiterpene hydrocarbons elemol acetate (15.9%), germacrone (25.0%) and α-cadinol (8.2%) as major components. Despite the fact that sesquiterpene hydrocarbons were the main fraction in our sample (SR_M), its chemical composition differed considerably. Moreover, Pljevljakusic et al. (2011) mentioned that the sesquiterpene fraction of cultivated S. raeseri EOs was the principal group in all samples with bicyclogermacrene as main compound. Our analyses revealed that also our samples are abundant in sesquiterpenes; bicyclogermacrene was found in both samples, but in lower

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percentages. Furthermore, compared to the study of Aligiannis et al. (2001) ar-curcumene was not detected in our samples. Papageorgiou et al. (1982) has mentioned naphthalene (22.0%) as major volatile component, while in the present study, naphthalene was not detected. Sideritis scardica Griseb. The yields (v/w) of the essential oils of the aerial parts of SS_M and SS_X were 0.18 % and 0.12 %, respectively. The chemical composition of the essential oil of SS_M showed 46 constituents, where the main components were α-pinene (8.2%), β-pinene (12.8%), cis-caryophyllene (6.6%), bicyclogermacrene (6.6%) and germacrene D (6.6 %) (Table 2). In contrast, the essential oil of the SS_X was consisted of 49 compounds with α-pinene (17.8%), β-pinene (13.1 %), cis-caryophyllene (7.6 %), bicyclogermacrene (7.1 %), m-camphorene (10.3 %) and germacrene D (2.2 %) being the main compounds (Table 2). It is interesting note that the two different samples of S. scardica presented similar chemical compositions with small differences in the quantities of the individual volatile components. Monoterpene hydrocarbons were the main fraction of SS_X (Table 3). This is in accordance with the previous studies (Kokkalou, 1987; Baser et al., 1997; Todorova et al., 2000; Konstadinova et al., 2007; Tredafilova et al., 2013; Todorova & Trendafilova, 2014). According to Todorova et al. (2013), the predominant group of EOs of cultivated S. scardica could be mono- or sesquiterpene hydrocarbons which is related to the environmental conditions such as altitude. In sample SS_M, sesquiterpene hydrocarbons were the major fraction of the EO (Table 3). This difference between the major groups of the two samples could be attributed to the different altitude of the two locations. A distinguishing feature between the two samples was the presence of m-camphorene in the essential oil of SS_X. It is noteworthy to be mentioned that only in this sample, diterpene hydrocarbons were observed in a considerable amount (11.2 %). Moreover, m-camphorene has been previously reported in the essential oil of this species from Bulgaria (Tredifilova et al., 2013). Comparing our results with the literature (Kokkalou, 1987; Baser et al., 1997; Tredafilova et al., 2013), we found a significant uniformity. Todorova et al. (2000) noticed the presence of β-caryophyllene (18.8 %) and nerolidol (12.1 %) in the essential oil of S. scardica from Bulgaria. In our study, we found β-caryophyllene in lower percentages, while we didn’t identify nerolidol. The research by Kostadinova et al. (2007) supports the presence of α-cadinol and octadecenol as major components of the essential oil of S. scardica originated from south Bulgaria and North Macedonia, which were not identified in our samples. The present study revealed the chemical variations in comparison to the chemical profile of S. raeseri and S. scardica previously investigated. In the EOs of Greek Sideritis species, monoterpenes are the dominant components (Aligiannis et al., 2001; González-Burgos et al., 2011). In the present study, the samples were rich in monoterpenes or sesquiterpenes. Kirimer et al. (2000) indicated a classification of the Turkish Sideritis species based on the main components of their essential oils. Regarding to this study, our samples could be categorised as follows: the EOs of SR_P and SS_X are monoterpene-rich oils, while the EO of SR_M and SS_M are sesquiterpene-rich oils. Considering the yields and the chemical variations of our samples, we support that many factors such as hydrodistillation and location could influence the amount of the oil (Pljevljakušic et al., 2011, Todorova et al., 2013; Todorova & Trendafilova, 2014). Taking into consideration the small different altitudes of each location (Polliraxo-Kozani: 567m; Chromio-Kozani: 640m and Metamorfosi-Kozani: 700m), we observed that the monoterpene-rich samples SR_P and SS_X were both located at 567-640 m a.s.l. On the other hand, the sesquiterpene-rich samples SR_M and SS_M were originated from the same location at 700 m a.s.l.. Different distillation methods, as well as various cultivation methods could increase the yield of the essential oil of this high valuable species. Thus, we assume that the aforementioned factors

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(altitude, cultivation methods and distillation methods) have played an important role about the chemical variations of our samples. The scope of this study was to analyse samples of two Greek cultivated species of Sideritis, indicating the significant differences in the chemical composition of these essential oils even if the plants are originated from cultivations. In this present study it was underscored also that the EOs of Sideritis raeseri showed totally different chemical constituents compared to the already studied Greek Sideritis raeseri, therefore more detailed studies should be conducted to examine and clarify its high tendency of hybridism with other species.

Table 1. Chemical composition of the essential oils from samples SR_M and SR_P. Compound KI RIa Composition (%) SR_M SR_P α-thujene 924 924 - 1.1 α-pinene 932 930 - 12.9 allylbenzene 934 932 - 0.3 camphene 946 945 - 0.2 β-pinene 974 970 - 19.1 myrcene 988 982 - 1.2 α-phellandrene 1002 998 - 0.4 α-terpinene 1014 1009 - 0.1 p-cymene 1020 1016 - 0.1 limonene 1024 1021 - 8.5 cis-ocimene 1032 1028 - 0.4 γ-terpinene 1054 1048 - 0.2 octanol 1063 1064 - 0.3 terpinolene 1086 1078 - 0.9 linalool 1095 1091 - 0.2 nonanal 1100 1095 - 0.6 α-campholenal 1122 1116 - 0.2 trans-pinocarveol 1135 1127 - 0.2 trans-verbenol 1140 1133 - 0.2 pinocarvone 1160 1150 - 0.4 terpinen-4-ol 1162 1165 - 0.2 α-terpineol 1186 1180 - 0.1 myrtenal 1195 1193 - 0.7 bornyl acetate 1284 1282 - 0.5 δ-elemene 1335 1335 - 0.1 bicycloelemene 1336 1335 0.2 - eugenol 1356 1354 - 0.1 α-copaene 1374 1372 0.4 1.3 β-bourbonene 1387 1385 0.3 0.5 β-cubebene 1387 1385 - 0.1 β-elemene 1389 1382 0.5 0.5 benzyl isovalerate 1396 1390 - 0.2 cis-caryophyllene 1408 1401 10.0 -

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trans-β-caryophyllene 1417 1412 - 3.8 β -copaene 1428 1428 - 0.1 1-butanol, 3-methyl-benzoate 1430 1427 - 0.3 β-humulene 1433 1433 - 0.2 α-humulene 1452 1450 0.3 - neryl acetone 1453 1451 0.3 - trans-β-farnesene 1454 1454 6.0 2.4 dodecanal 1465 1463 0.3 - germacrene D 1484 1484 14.8 15.9 β-ionone 1487 1486 0.6 0.2 bicyclogermacrene 1500 1499 11.2 4.6 β-bisabolene 1505 1500 1.5 0.9 germacrene A 1508 1506 - 0.2 2,4-bis (1,1- - 1513 1512 1.4 dimethylethyl)phenol phenol, 2,5-bis(1,1- 0.3 1517 1515 - dimethylethyl) δ-cadinene 1522 1520 1.7 2.3 spathulenol 1577 1575 4.1 1.0 hexyl benzoate 1579 1579 - 0.2 caryophyllene oxide 1582 1580 4.5 0.8 viridiflorol 1592 1591 0.2 0.1 hexadecane 1600 1500 0.4 - t-cadinol 1632 1629 - 0.2 α-cadinol 1638 1636 - 0.2 isospathulenol 1639 1635 0.2 - valeranone 1656 1656 - 0.2 6(Z), 9(E)-heptadecadiene 1668 1666 0.3 - α-bisabolol 1685 1680 1.6 - pentadecanal 1711 1708 1.7 0.2 benzyl benzoate 1759 1755 2.3 5.6 4-methyl phenyl ester octanoic - 1777 1769 0.8 acid octadecane 1800 1796 0.3 - hexadecanal 1822 1819 0.3 - 6,10,14-trimethyl-2- 0.1 1847 1842 1.7 pentadecanone heptadecanal 1920 1915 - 0.2 ent-pimara-8(14),15-diene 1939 1932 0.7 - (3Z)-cemdrene A 1965 1958 - 2.6 kaur-15-ene 1997 1990 0.8 0.1 n-eicosane 2000 1995 0.4 - 13-epi-manoyl oxide 2009 2001 0.4 - 9-octadecen-1-ol 2077 2074 12.4 1.5 heneicosane 2100 2095 0.6 - phytol 2111 2108 0.6 - linoleic acid 2132 2129 0.2 -

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iso-dextro-pimaraldehyde 2184 2181 1.4 - docosane 2200 2197 0.3 - tricosane 2300 2298 1.1 - tetracosane 2400 2398 0.3 - pentacosane 2500 2499 0.9 - TOTAL 88.0 96.0 a Retention Index calculated against C9-C24 n-alkanes on the HP 5MS column capillary column; SR_M: S. raeseri from Metamorfosis Kozani; SR_P: S. raeseri from Polirraxo Kozani

Table 2. Chemical composition of the essential oil s from samples SS_M and SS_X.

Compound KI RIa Composition (%) SS_M SS_X α-thujene 924 924 0.4 0.2 α-pinene 932 930 8.2 17.8 verbenene 961 960 - 0.2 β-pinene 974 970 12.8 13.1 myrcene 988 982 0.6 0.7 α-phellandrene 1002 997 0.5 - δ-3-carene 1002 1003 0.5 - p-cymene 1020 1016 0.4 0.1 limonene 1024 1019 3.4 2.7 trans-β-ocimene 1032 1028 - 2.3 cis-β-ocimene 1044 1038 0.4 0.4 γ-terpinene 1054 1048 0.2 1.2 α-terpinolene 1086 1082 0.3 0.3 linalool 1095 1090 0.4 0.3 nonanal 1100 1095 0.8 0.7 α-campholenal 1122 1115 0.2 0.7 trans-pinocarveol 1135 1132 0.5 0.3 trans verbenol 1140 1138 0.3 0.4 pinocarvone 1160 1160 0.5 0.7 myrtenal 1195 1189 0.6 1.0 bornyl acetate 1284 1284 0.2 0.2 δ-elemene 1335 1333 - 0.2 eugenol 1356 1353 - 0.3 α-copaene 1374 1368 2.2 1.1 β-bourbonene 1387 1384 0.6 0.4 β-elemene 1389 1385 0.4 - cis-β-caryophyllene 1408 1401 7.9 7.6 α-humulene 1452 1448 0.2 0.3 geranyl acetone 1453 1450 - 0.2 trans-β-farnesene 1454 1456 - 1.6 cis-β-farnesene 1440 1440 1.5 - germacrene D 1484 1480 7.7 2.9 β-ionone 1487 1485 0.3 0.5 bicyclogermacrene 1500 1499 6.6 7.1

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β-bisabolene 1505 1501 1.1 0.8 γ-cadinene 1513 1513 0.7 - δ-cadinene 1522 1524 4.0 1.9 spathulenol 1577 1575 2.1 2.7 caryophyllene oxide 1582 1580 2.4 3.8 viridiflorol 1592 1590 1.4 0.4 hexadecane 1600 1600 - 1.0 t-cadinol 1632 1630 - 0.6 t-muurolol 1644 1644 4.2 tetradecanol 1671 1668 - 1.9 valeranone 1674 1674 0.9 - α-bisabolol 1685 1685 1.7 0.5 heptadecane 1700 1700 - 0.4 pentadecanal 1711 1709 0.5 1.2 benzyl benzoate 1759 1756 1.5 2.3 6,10,14-trimethyl-2- 0.6 1845 1842 - pentadecanone 6,10,14-trimethyl-pentan-2-one 1847 1842 0.9 - ent-pimara-8(14),15-diene 1948 1942 0.4 0.4 m-camphorene 1944 1949 - 10.3 kaur-15-ene 1997 1995 0.8 0.5 13-epi-manoyl oxide 2009 2001 0.2 - 9-octadecen-1-ol 2077 2077 3.0 0.3 heneicosane 2100 2100 0.3 - tricosane 2300 2299 0.4 - TOTAL 85.1 95.9 aRetention Index calculated against C9-C24 n-alkanes on the HP 5MS column capillary column; SS_M: S. scardica from Metamorfosis Kozani; SS_X: S. scardica from Chromio Kozani

Table 3. Grouped Components (% v/v) of each sample.

Group Components SR_M SR_P SS_M SS_X Monoterpene hydrocarbons - 45.4 27.7 39.0 Oxygenated monoterpenes - 3.4 3.5 4.6 Sesquiterpene hydrocarbons 46.9 32.9 32.9 23.9 Oxygenated sesquiterpenes 11.5 2.7 13.0 8.7 Diterpenes 1.9 2.7 1.4 11.2 Oxygenated diterpenes 0.6 - - - Hydrocarbons 4.6 - 0.7 2.2 Oxygenated hydrocarbons 22.5 8.9 5.9 6.3 SR_M: S. raeseri from Metamorfosis Kozani; SR_P: S. raeseri from Polirraxo Kozani; SS_M: S. scardica from Metamorfosis Kozani; SS_X: S. scardica from Chromio Kozani

ACKNOWLEDGMENT The authors wish to thank Associate Prof. Th. Constantinidis (Department of Ecology & Systematics, Faculty of Biology, NKUA) for the identification of the plant materials.

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REFERENCES

Adams R.P. (2007). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. 4th edn. Allured Publ. Corp., Carol Stream, IL.

Aligiannis, N., Kalpoutzakis, E., Chinou, I.B., Mitakou, S., Gikas, E., Tsarbopoulos, A. (2001). Composition and Antimicrobial Activity of the Essential Oils of Five Taxa of Sideritis from Greece. Journal of Agricultural and Food Chemistry, 49, 811–815. Baser, K.H.C., Kirimer, N., Tümen, G. (1997). Essential Oil of Sideritis scardica Griseb. subsp. scardica. Journal of Essential Oil Research, 9, 205–207. EMA/HMPC/39455/2015. Assessment report on Sideritis scardica Griseb.; Sideritis clandestina (Bory & Chaub.) Hayek; Sideritis raeseri Boiss. & Heldr.; Sideritis syriaca L., herba. González-Burgos, E., Carretero, M.E., Gómez-Serranillos, M.P. (2011). Sideritis spp.: Uses, chemical composition and pharmacological activities-A review. Journal of Ethnopharmacology, 135, 209-225. Kırımer, N., Tabanca, N., Özek, T., Tümen, G., & K.H.C. Başer, K.H. (2000). Essential oils of annual Sideritis species growing in Turkey. Pharmaceutical Biology, 38, 106-111. Koedam, A. (1986). Volatile oil composition of greek mountain tea (Sideritis spp.). Journal of Science and Food Agriculture, 37, 681–684. Kokkalou, E. (1987). Constituents Entrainables A la Vapeur D’eau de Sideritis scardica Gris. ssp. scardica. Plantes Médicinales et Phytothérapie, 21, 262-266. Kostadinova, E., Nikolova, D., Alipieva, K., Stefova, M., Stefkov, G., Evstatieva, L.V. & Bankova, V. (2007). Chemical constituents of the essential oils of Sideritis scardica Griseb. and Sideritis raeseri Boiss and Heldr. from Bulgaria and Macedonia. Natural Product Research, 21, 819–823. Massada, Y. (1976). Analysis of Essential oil by GC/MS. J. Wiley & Sons, N. York. Papageorgiou, V.P., Kokkini, S. & Argyriadou, N. (1982). Chemotaxonomy of the Greek species of Sideritis. 1. Components of the volatile fraction of Sideritis raeseri ssp. raeseri. In: Aromatic Plants Basic and Applied Aspects (World Crops:Production, Utilizafion, Description. Vol. 7) (Margaris, N.; Koedam, A,; Vokou, D., Eds) Martinus Nijhoff Publishers, The Hague, 1211-220. Pljevljakušić, D., Šavikin, K., Janković, T., Zdunić, G., Ristić, M., Godjevac, D., Konić-Ristić, A. (2011). Chemical properties of the cultivated Sideritis raeseri Boiss. & Heldr. subsp. raeseri. Food Chemistry, 124, 226–233. Todorova, M., Christov, R.C., Evstatieva, L.N. (2000). Essential Oil Composition of Three Sideritis Species from Bulgaria. Journal of Essential Oil Research, 12, 418–420. Todorova, M, Trendafilova, A., Evstatieva, L.N., Antonova, D.V. (2013). Volatile components in Sideritis scardica Griseb. cultivar. Comptes rendus de l'Académie bulgare des sciences: sciences mathématiques et naturelles, 66, 507-512. Todorova, M., Trendafilova, A. (2014). Sideritis scardica Griseb., an endemic species of Balkan peninsula: Traditional uses, cultivation, chemical composition, biological activity. Journal of Ethnopharmacology, 152, 256–265. Trendafilova, A.B., Todorova, M.N., Evstatieva, L.N., Antonova, D.V. (2013). Variability in the Essential‐Oil Composition of Sideritis scardica Griseb. from Native Bulgarian Populations. Chemistry & Biodiversity, 10, 484-492. Van den Dool H., Kratz P.D. (1963). A generalization of the Retention Index System including linear temperature programmed Gas Liquid partition Chromatography. Journal of Chromatography, 11, 463-471.

Received : 09.09.2019 Accepted: 25.11.2019

Nat. Volatiles & Essent. Oils, 2019; 6(3): 24-27 Köse et al.

RESEARCH ARTICLE Volatile Constituents and Antimicrobial Activity of Hirtellina lobelii (DC.) Dittrich

Yavuz Bülent Köse1,*, Gökalp İşcan2 and Betül Demirci2

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

*Corresponding author. Email: [email protected]

Abstract The volatiles obtained by hydrodistillation of Hirtellina lobelii (DC.) Dittrich (Asteraceae) collected from Turkey was analyzed by gas chromatography and gas chromatography/mass spectrometry (GC/MS), simultaneously. Main constituents were identified as fokienol (% 7.4), cadinol (% 6.8), and β-caryophyllene (% 6.7), respectively. The H. lobelii volatiles were screened for antimicrobial properties against various pathogenic bacteria. The volatile showed a relatively good inhibitory concentration with 0.06 mg/mL against Staphylococcus aureus.

Keywords: Hirtellina lobelii, Antimicrobial, Volatile

Introduction

Many Asteraceae species, widespread in the world and Anatolia exert biological and pharmacological activity. The phytochemistry of the family mainly consists of diterpenes, flavonoids as well as sesquiterpene lactone type secondary metabolites which show biological activities such as antibacterial, antifungal, anthelminthic, anti-inflammatory, insecticide, antitumor among many others (Shing et al, 2002). Hirtellina (Cass.) Cass. (Asteraceae), is represented by 4 species in the world (Davis, 1975). Hirtellina lobelii (DC.) Dittrich is the only species found in Turkey. This species is recorded within the genus Staehelina L. in Flora of Turkey. Later, its status was changed to Hirtellina (Dittrich, 1996). There is only one very recent report on the phytochemical composition and bioactivity on this species (Khoury et al., 2019). To the best of our knowledge, this is the first report on the chemistry of the volatiles and its antibacterial evaluation. Materials and Methods Plant material and isolation of essential oil The plant sample was collected from Antalya, İbradi, Altınbeşik cave in May, 2018 in Turkey. Voucher specimen is kept in the Faculty of Pharmacy Herbarium, Anadolu University, Eskişehir, Turkey. The air-dried aerial parts were hydrodistilled for 3 h using a Clevenger-type apparatus to produce a small amount of essential oil, which was trapped in n-hexane. It wasthen analysed by GC-FID and GC/MS.

GC/MS analysis The analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 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

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60 oC for 10 min and programmed to 220 oC at a rate of 4 oC/min, and kept constant at 220 oC 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 oC. 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 oC. 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 1. Identification of the components 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) (1,2) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils were conducted. Antibacterial activity Antibacterial effect of the volatiles of H. lobelii was screened by using partly modified CLSI (formerly NCCLS) microdilution broth methods M7-A7 standard protocol (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically) (CLSI (NCCLS) M7-A7, 2006). Standard strains such as Escherichia coli NRRL B-3008, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, Serratia marcescens NRRL B-2544 and Klebsiella pneumoniae NCTC 9633 were used as test bacteria. Ampicillin and chloramphenicol were used as standard antibacterial agents. n-Hexane and water were used also as controls. The experiments were repeated in dublicates, and MIC values were reported as mean. Results and Discussion Volatile Composition 48 compounds representing 89.5% of the essential oil trapped in hexane were characterized. Fokienol (% 7.4), cadinol (% 6.8), and β-caryophyllene (% 6.7) were found as main components. α-Bisabolol (34.5%), fokienol (12%) and T-muurolol (6.8%) were found as main constituents in the only previously published report (Khoury et al., 2019). Fokienol was a major constituent in both oils.

Table 1. The Volatile Composition of H. lobelii RRI Compound % 1225 (Z)-3-Hexenal 5.9 1400 Nonanal 0.5 1466 α-Cubebene 0.8 1495 Bicycloelemene 0.2 1497 α-Copaene 1.0 1535 β-Bourbonene 0.8 1553 Linalool 1.8

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1589 β-Ylangene 0.3 1600 β-Elemene 0.3 1597 β-Copaene 0.4 1612 β-Caryophyllene 6.7 1638 β-Cyclocitral 0.5 1639 Cadina-3,5-diene 0.6 1677 epi-Zonarene 0.6 1681 (Z)-3-Hexenyl tiglate 5.5 1687 α-Humulene 1.1 1704 γ-Muurolene 1.1 1706 α-Terpineol 1.0 1722 Bicyclosesquiphellandrene 0.8 1726 Germacrene D 1.8 1740 α-Muurolene 2.0 1742 β-Selinene 2.8 1755 Bicyclogermacrene 0.6 1773 δ -Cadinene 5.9 1776 γ -Cadinene 4.4 1798 Methyl salicylate 0.4 1799 Cadina-1,4-diene (=Cubenene) 0.5 1807 α-Cadinene 0.8 1849 Calamenene 1.0 1900 epi-Cubebol 1.0 1941 α-Calacorene 1.0 1957 Cubebol 1.0 1958 (E)-β-Ionone 1.4 1984 γ-Calacorene 0.5 2008 Caryophyllene oxide 1.0 2050 (E)-Nerolidol 0.8 2057 Ledol 0.8 2080 Cubenol 1.6 2104 Viridiflorol 0.9 2148 (Z)-3-Hexen-1-yl benzoate 1.4 2161 Muurola-4,10(14)-dien-1-ol 0.9 2174 Fokienol 7.4 2187 T-Cadinol 6.1 2209 T-Muurolol 3.4 2219 δ-Cadinol (=alpha-muurolol) 1.9 2255 α-Cadinol 6.8 2300 Tricosane 1.0 2568 14-Hydroxy-α-muurolene 0.5 Total 89.5 RRI: Relative retention indices calculated against n-alkanes. % calculated from FID data.

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Table 2. Antibacterial effects of H. lobelii essential oil (MIC, mg/mL) Bacteria Source H. lobelii Ampicillin Chloramphenicol Escherichia coli NRRL B-3008 0.5 0.002 0.001 Staphylococcus aureus ATCC 6538 0.06 0,001 0,008 Pseudomonas aeruginosa ATCC 27853 >1 0.128 0.064 Salmonella typhimurium ATCC 13311 0.25 0.001 0.001 Serratia marcescens NRRL B-2544 0.12 0.016 0.004 Klebsiella pneumoniae NCTC 9633 0.25 0.001 0.004 MIC: Minimum inhibitoy concentration The essential oil showed weak to moderate inhibitory effects against the tested pathogens between the concentration of 0.5 to 0.06 mg/mL (MIC). The oil was rather inactive towards the growth of P. aeruginosa at the maximum test concentration of 1.0 mg/mL. S. aureus was susceptible with a MIC value of 60 µg/mL as shown in Table 2. Supporting our study, Khuory et al. (2019) showed the antibacterial effects of the α- bisabolol-rich H. lobelia essential oil, where S. aureus was inhibited at the concentration of 32 µg/mL (MBC; 64 µg/mL), and the E. coli was inhibited at > 500 µg/mL, in agreement with our findings. The initial findings on the chemistry and biological activity of H. lobelia suggest further evaluations and experimentations.

ACKNOWLEDGMENT This study was supported by the Anadolu University Scientific Research Committee (Project No. 1705S180). REFERENCES

Shing, B., Sahu, P.M., Sharma, M.K. (2002). Anti-inflammatory and antimicrobial activities of triterpenoids from Strobilanthes callosus Ness. Phytomedicine 9:355–9 Davis, P.H. (1975). The Flora of Turkey and East Aegean Islands, Vol 5., Edinburgh University Press. Dittrich, M. (1996). Die bedeutung morphologischer und anatomischer achänen-merkmale für die systematik der tribus Echinopeae Cass. und Carlineae Cass., Boissiera, Vol. 51, pp. 9–102 Khoury, M., El Beyrouthy, M., Ouaini, N., Eparvier, V., & Stien, D. (2019). Hirtellina lobelii DC. essential oil, its constituents, its combination with antimicrobial drugs and its mode of action. Fitoterapia, 133, 130-136. McLafferty, F.W., Stauffer, D.B. (1989). The Wiley/NBS Registry of Mass Spectral Data, J Wiley and Sons: New York. Hochmuth, D. H. (2008). MassFinder 4.0, Hochmuth Scientific Consulting, Hamburg, Germany. CLSI (NCCLS) M7-A7 (2006).Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, Seventh Edition.

Received : 10.11.2019 Accepted: 03.12.2019