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Composition of Volatile Oil of the Aerial Parts, Flowers and Roots of Ferulago blancheana Post. (Apiaceae) Growing in Turkey and Determination of Their Antimicrobial Activities by Bioautography Method/Turkiye'de Yetisen Ferulago blancheana Post. (Apiaceae) Turunun Toprak Ustu, Cicek ve Koklerinden Elde Edilen Ucucu Yaglarin Iceriklerinin ve Antimikrobiyal Aktivitesinin Biyootografi Yontemiyle Tanimlanmasi.

INTRODUCTION

Ferulago W. Koch. is a perennial genus of Apiaceae and is represented by approximately 50 taxa throughout the world (1, 2). Ferulago species are known as "Caksir" or "Cagsir" in Turkey and according to recent records the genus is represented by 35 taxa in Turkey, 18 of which are endemics. This suggests that Anatolia is the gene centre of this genus (3). Ferulago species have been used since ancient times for the treatment of intestinal worms, hemorrhoids and as tonic, digestive and sedative. In addition, they are used against snake bites, ulcers, spleen diseases and headache (4). Gums obtained from the incision of the roots of some species are used as seasoning and as a carminative (5). However, the plants are mostly well known for their aphrodisiac activities in Turkey (6). Ferulago species have been found to contain coumarins, flavonoids, quinones, sesquiterpenes, coumarin esters and furanocoumarins (7, 8). Essential oil is one of the major components of genus and recently these components in different studies from several species have been showed (4, 5, 8-18). The aim of this study is to present and compare the chemical composition of the essential oils of aerial parts, flowers, and roots of F. blancheana Post. growing wild in Turkey. We performed GC and GC/MS analysis to determine the constituents of the essential oil and also determined the antimicrobial activities of these oils by TLC-bioautography thin-layer, a simple and inexpensive tool for simultaneous chemicobiological screening of natural sources. To the best of our knowledge, this is the first report on the chemical analysis and antibacterial activity of F. blancheana. Identified constituents are presented in Table 1.

MATERIALS AND METHODS

Plant material

The plant materials were collected from the below mentioned locality and identified by Prof. Dr. Hayri Duman (Gazi University, Faculty of Science, Department of Biology) and the voucher specimens are kept in AEF (Herbarium of Ankara University Faculty of Pharmacy).

Collection locality: B6: Kayseri: Between Pinarbasi-Sariz, 4 km left to Sariz, rocky slopes, 1696 m, 13.07.2014 (AEF 26672).

Isolation of the essential oil

Aerial parts (61.79 g), flowers (33.88 g) and roots (31.38 g) were subjected to hydrodistillation for 3 h using a Clevengertype apparatus according to the method recommended in the European Pharmacopoeia. Obtained oils were dried over anhydrous sodium sulfate and stored in sealed vials at +4[degrees]C in the dark until analyzed and tested. All oils were pleasant smelling, transparent with a faint yellow color. Essential oil yields of the aerial parts, flowers and roots of the studied plant are 0.032%, 0.15% and 0.032%, respectively.

GC/MS analysis

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[degrees]C for 10 min and programmed to 220[degrees]C at a rate of 4[degrees]C/min, and kept constant at 220[degrees]C for 10 min and then programmed to 240[degrees]C at a rate of 1[degrees]C/min. Split ratio was adjusted at 40:1. Injector temperature was set at 250[degrees]C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.

GC analysis

GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300[degrees]C. To obtain the same elution order with GC/MS, simultaneous autoinjection 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 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 Library) (19, 20) and in - house "Baser Library of Essential Oil Constituents" built up by genius Constituents" built up by genuine compounds and components of known oils, as well as MS literature data (21, 22), was used for the identification.

Determination of antimicrobial compounds from essential oils by TLC-Bioautography thin-layer chromatography (TLC)

Chromatography was performed on 0.2 mm silica gel 60 [F.sub.254] aluminum sheet TLC plates. 10 [micro]L essential oils were applied to the TLC plate with minicaps capillary pipettes. TLC plates were developed with toluene: ethyl acetate, 93:7, as a mobile phase. TLC plate for bioautography was prepeared in parallel. After the development, TLC plates were evaluated at UV 254 nm and 366 nm for determination of flurocescent compounds. Alcoholic vanillin--sulphuric acid reagent was used to visualize the separated compounds and heated for 3 min at 110[degrees]C.

Preparation of microorganisms and TLC bioautography method

After TLC separation, the antimicrobial activity of the essential oil was detected with direct bioautography (23, 24). Pseudomonas aeruginosa ATCC 13388, Escherichia coli NRRL, Staphylococcus aureus ATCC BAA 1026 and Candida albicans ATCC 24433 were used for bioautography. Microbial suspensions were grown overnight in double strength Mueller-Hinton broth (MHB) were standardized to [10.sup.8] CFU/mL (corresponding to McFarland no: 0.5). TLC plates were put on nutrient agar plates. And molten agar culture medium containing inocula was spread on TLC plates and incubated at 37[degrees]C for 24h. After incubation, 2, 3, 5 -triphenyl-2H-tetrazolium chloride (TTC) solution was sprayed on TLC plates. The treated plates were incubated at 37[degrees]C for 2 h. After incubation, the inhibition zones were visible as pale spots against a red background.

RESULTS

A total of fortyfive compounds representing 97.5% of the oil were identified in the essential oil of aerial parts of F. blancheana. Bornyl acetate, [beta]-caryopyllene, sabinene, spathulenol, elemol, (Z)-[beta]-ocimene were the major components, amounting to 11.7%, 10.2%, 7.2%, 6.4%, 6.2% and 6.1%, respectively. The analysis on the flowers of F. blancheana resulted in the identification of thirty-five volatile compounds representing 97.3% of the oil. Sabinene at 23.2% was the most abundant compound in the volatile oil, followed by myrcene (17.5%), [alpha]-pinene (14%), (E)-[beta]-ocimene (5.9%) and bornyl acetate (5.4%). Ten compounds were characterized in the oil of the roots of F. blancheana representing 93.8% of the oil. The major constituents were found to be (E)-2-decenal (20.3%), caryopyllene oxide (17.8%), bornyl acetate (11.5%), spathulenol (11.2%). The oils obtained from different parts of this species did not show much qualitative and quantitative similarity. Some components such as camphene, linalool, trans-verbenol, sabinyl acetate, borneol, isocaryophyllene oxide, [alpha]-muurolene, geranyl acetate, [alpha]-cadinene, geraniol, p-cymen-8-ol and neryl acetate were only found in the essential oil of the aerial parts and some components such as [alpha]-terpinene, [gamma]-curcumene, valeranone were only found in the essential oils of the flowers and some components such as octanal, (E)-2-decenal, octanoic acid and decanoic acid were only found in the essential oil of the roots. The composition of the essential oils obtained from different parts of this species and their relative percentages are given in Table 1. Antimicrobial effect was investigated using TLC-bioautographic method.

Bioautography is a laboratory technique to detect substances affecting the growth rates of test organisms in complex mixtures and matrices and the method belongs to a large group of screening methods for the detection of antimicrobial activity. It is based on the biological activity of the analyte, which can be antibacterial, antifungal, antitumour, antiprotozoae etc. Diffusion and dilution methods are also used. The main benefit of bioautography is its providing information about antimicrobial activities of substances separated from a mixture. Bioautography is proving its worth as a simple and inexpensive tool for simultaneous chemico-biological screening of natural sources. In other word, it offers the simplest mean of bioassay guided lead discovery from natural products.

TLC bioautography assay was performed against three different microorganisms, P. aeruginosa ATCC 13388, S. aureus ATCC BAA 1026 and C. albicans ATCC 24433. Essential oils have very active components againts S. aureus ATCC BAA 1026 and showed good inhibiton. Essential oils of the aerial part and roots were found active against C. albicans ATCC 24433, however inhibition zones were less visible than S. aureus. Essential oils did not give any inhibiton zone against P. aeruginosa ATCC 13388. Compared with another study carried out on a different Ferulago species (F. sandrasica Pesmen & Quezel) performed by bioautography method showed that the essential oil of F. sandrasica of the roots was active against S. aureus ATCC 6558 and C. albicans ATCC 90028 strains; however it was not active against Escherichia coli NRRL B-3008 strain. The essential oil of the aerial part and flowers showed higher antibacterial activity against S. aureus than the roots. TLC evaluation of the essential oils is reported in Figure 1.

DISCUSSION

Comparison of the main constituents of different parts of this species shows that each part has a different set of dominant compounds in Table 1. However, previous studies of the oils of different Ferulago species (5, 25) revealed that eight compounds have been detected in high percentages, namely (Z)-[beta]-ocimene, 2,3,6-trimethylbenzaldehy, cis-chrysanthenyl acetate, nonacosane, sabinene, [delta]-cadinene, [alpha]-pinene and p-cymene have also been defined as major components in many other species. Erdurak et al. reported main components of essential oil of the fruit and root of F. isaurica and the root oil of F. syriaca (8) in 2006. They were characterized as terpinolene (42.1%) and myrcene (27%) for the root oil of F. isaurica and bornyl acetate (69.4%) and terpinolene (12.5%) for the root oil of F. syriaca. Another study showed that the major components of essential oils F. pachyloba, F. platycarpa and F. longistylis were (Z)-[beta]-ocimene (25.7%), [alpha]-pinene (9.8%), sabinene (6.3%), and [delta]-cadinene (5.6%); cis-chrysanthenyl acetate (24.2%), nonacosane (7.7%) and [alpha]-pinene (4.2%); 2,3,6-trimethylbenzaldehyde (29.8%), cischrysanthenyl acetate (24.2%), nonacosane (7.7%) and [alpha]-pinene (4.2%) respectively.

Previous studies demonstrated monoterpene hydrocarbons constituted the main fraction of the essential oils of F. campestris, with [alpha]-pinene, myrcene and [gamma] -terpinene as the major components of the flowers (11); myrcene (33.4-39.7%), [alpha]-pinene (22.7-23.0%) and [gamma] -terpinene (8.1-10.9%) as the major components of the fruits, and [alpha] -pinene (58.3-75.0%) as the predominant compound in the oil from the roots (26); monoterpene hydrocarbons (78.8-80.3%), with myrcene (33.4-39.7%), [alpha]-pinene (22.7-23.0%), and [gamma]-terpinene (8.1-10.9%) as the major components of the fruits essential oils (27). Baser et al. (2002), studied twelve Ferulago (F. asparagifolia Boiss., F. aucheri Boiss. , F. confusa Velen, F. galbanifera (Mill.) W.D. J. Koch, F. humilis Boiss., F. idaea Ozhatay & Akalin, F. macrosciadia Boiss. & Balansa, F. mughlae Pesmen, F. sandrasica Pesmen & Quezel, F. silaifolia (Boiss.) Boiss., F. sylvatica (Besser) Rchb. and F. trachycarpa Boiss.) species growing in Turkey, and studies indicated that the main essential oil components are 2, 3, 6 trimethylbenzaldehyde (38.9%) and myrcene (18.2%); [alpha]-pinene (35.9%); 2,5-dimethoxy-p-cymene (63.4%); [alpha]-pinene (31.8%) and sabinene (15.8%); (Z)-[beta]-ocimene (32.4%); p-cymene (18.4%); carvacrol methyl ether (78.1%); [alpha]-pinene (25.4%); [alpha]-pinene (40.8%); trans-chrysanthenyl acetate (83.5%); p-cymene (45.8%); (Z)- [beta]-ocimene (30.7%) respectively (28). Previous studies indicated that chemical composition of the essential oils of several Ferulago species did not show much qualitative and quantitative similarity, but it can be said that major constituents of the oils of Ferulago species are [beta]-ocimene, [alpha]-pinene, [alpha]- and [beta]-phellandrene, limonene, myrcene and p-cymene.

The results obtained in this investigation suggest that this chemical diversity may be useful in taxonomical classification. In addition, bioautography method was used to evaluate the correct test organism(s) in antimicrobial screening. Agar disc diffusion technique and broth dilution methods are also used to screen plant extracts for antimicrobial activity. However we choose this method since it is efficient, simple and inexpensive. Antimicrobial activity study performed against three different microorganisms, P.aeruginosa ATCC 13388, S. aureus ATCC BAA 1026 and C. albicans ATCC 24433 showed that the essential oils ontained from the aerial parts, roots and flowers were active against S. aureus ATCC 6558 and C. albicans ATCC 24433, however inhibition zones obtained with the essential oils obtained from the aerial parts and roots were found to be less visible than the ones obtained against S. aureus. In addition, no volatile oil yielded an inhibition zone against P. aeruginosa ATCC 13388.

CONCLUSION

In summary, the present study, reported the composition of the essential oils of F.

blancheana aerial parts, flowers and roots for the first time and showed that they are mainly composed of terpenoids and benzenoid derivatives.

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Songul KARAKAYA (1*), Gamze GOGER (2), C. Sibel KILIC (1), Betul DEMIRCI (2)

(1) Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Tandogan- Ankara, TURKEY, (2) Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Tepebasi- Eskisehir, TURKEY

(*) Correspondence: E-mail: karakayas@ankara.edu.tr; Tel: +90 312 2033123113

Received: 03.12.2015

Accepted: 04.02.2016
Table 1. Composition of essential oils of aerial parts, flowers and
roots of F. blancheana

RRI    Compound                       A%     F%

1032   [alpha]-Pinene                  5.4   14.0   -
1035   [alpha]-Thujene                tr      0.2   -
1076   Camphene                        1.4   -      -
1118   [beta]-Pinene                   0.5    1.3   -
1132   Sabinene                        7.2   23.2   -
1159   [gamma]-3-Carene                2.8    3.4   -
1174   Myrcene                         3.5   17.5   -
1188   [alpha]-Terpinene              -       0.4   -
1203   Limonene                        3.4    5.4   -
1218   [beta]-Phellandrene            tr      0.8   -
1246   (Z)-[beta]-Ocimene              6.1    4.5   -
1255   [gamma]-Terpinene              tr      1.0   -
1266   (E)-[beta]-Ocimene              1.6    5.9   -
1280   p-Cymene                        1.4    0.9   -
1286   Isoterpinolene                 -       0.2   -
1290   Terpinolene                     0.2    1.7   -
1296   Octanal                        -      -       3.2
1548   (E)-2-Nonenal                  -      -      tr
1553   Linalool                        0.4   -      -
1591   Bornyl acetate                 11.7    5.4   11.5
1600   [beta]-Elemene                  0.4   tr     -
1594   trans-[beta]-Bergamotene        0.7   -      -
1602   6-Methyl-3,5-heptadien-2-one    0.1   -      -
1612   [beta]-Caryophyllene           10.2    3.0   10.5
1655   (E)-2-Decenal                  -      -      20.3
1658   Sabinyl acetate                 0.4   -      -
1683   frans-Verbenol                  0.5   -      -
1687   [alpha]-Humulene                0.7    0.3   -
1704   [gamma]-Curcumene              -       0.3   -
1719   Borneol                         0.4   -      -
1726   Germacrene D                    2.3    2.2   -
1733   Neryl acetate                   0.3   -      -
1740   [alpha]-Muurolene               0.3   -      -
1755   Bicyclogermacrene               1.8    0.6   -
1765   Geranyl acetate                 1.0   -      -
1773   [delta]-Cadinene                1.2    0.4   -
1776   [gamma]-Cadinene               tr     -      -
1786   ar-Curcumene                    0.6    0.3   -
1857   Geraniol                        0.3   -      -
1864   p-Cymen-8-ol                    0.3   -      -
2001   Isocaryophyllene oxide          0.5   -      -
2008   Caryophyllene oxide             3.2    0.5   17.8
2096   Elemol                          6.2    0.9   -
2084   Octanoic acid                  -      -       6.8
2103   Guaiol                          2.6   tr     -
2103   Guaiol                          2.6   tr     -
2144   Spathulenol                     6.4    1.0   11.2
2145   Valeranone                     -       0.3   -
2185   [gamma]-Eudesmol                1.1    0.1   -
2187   T-Cadinol                       0.8   tr     -
2202   Germacrene D-4-ol               1.9    0.6   -
2209   T-Muurolol                      1.3    0.1   -
2250   [alpha]-Eudesmol                1.6    0.1   -
2255   [alpha]-Cadinol                 4.8    0.8   12.0
2298   Decanoic acid                  -      -       0.5
       Total                          97.5   97.3   93.8

RRI: Relative retention indices calculated against n-alkanes; %
calculated from FID data
tr: Trace (< 0.1 %); A: Aerial parts, F: Flowers, R: Roots
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Title Annotation:Original article
Author:Karakaya, Songul; Goger, Gamze; Kilic, C. Sibel; DemIrcI, Betul
Publication:Turkish Journal of Pharmaceutical Sciences
Article Type:Report
Geographic Code:7TURK
Date:Aug 1, 2016
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