Studies on Betula essential oils

Essential oils were obtained from leaf, branch and buds of Betula species: B. pendula Roth, B. browicziana A.Güner, B. litwinowii Doluch., B. recurvata V. Vassil., and B. medwediewii Regel naturally growing in various parts of Turkey. Also buds of the common birch B. pendula essential oil from Germany and two species native to Finland namely, Betula pubescens ssp. czerepanovii (Orlova) Hämet-Ahti and Betula pubescens ssp. pubescens Erhr. were investigated. Betula essential oils were obtained by different distillation techniques such as hydrodistillation, microdistillation and Likens-Nickerson simultaneous distillation-extraction method (SDE). The resulting volatile compositions were elucidated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. Known and new sesquiterpenes were isolated from Betula essential oils using column chromatographic techniques. Structure determination of each isolated compound was carried out using 1D and 2D NMR spectroscopic techniques supported by MS, UV and GC FITR. Biological activities were determined both for essential oils and pure compounds isolated from the oils of Betula species. Antifungal, antibacterial and antioxidant activity results were carried out using various in vitro techniques.


Introduction
Well-known as birch tree, the genus Betula of the family Betulaceae, has a wide distribution in the northern hemisphere from Canada to Japan. 1 Five Betula species, namely B. browicziana A. Güner, B. litwinowii Doluch., B. medwediewii Regel, B. pendula Roth and B. recurvata V. Vassil are naturally growing in eastern and northern Turkey, at high altitudes.Only B. browicziana is endemic to Turkey. 2,3he Birch tree has a long history of medicinal use in different countries and cultures to cure skin diseases especially eczema, infections, inflammations, rheumatism and urinary disorders.
][6][7] Birch bark contains betulin, betulinol and a betuloside.The young leaves are rich in saponins and contain a diuretic flavonoid derivative (hyperoside), sesquiterpenes and tannins.The buds are rich in volatile oil.Birch tar contains creosol and guaiacol. 5,8][9][10][11][12] Betulenol, the main component of the oil, was isolated and reported from Betula buds and named first as betulol by Soden and Elze in 1905.Its structure was tentatively elucidated as a bicyclic primary sesquiterpene alcohol.Further investigations on this molecule were conducted by Triebs and other researchers as compiled in a work of Guenther. 5Afterwards Treibs and Lossner reported αand β-betulenol, their acetates and α-betulenal by means of synthesis to support the chemical structures present in the essential oil of Betula lenta. 8In contrast, Holub reported the occurrence of αand β-betulenol, as well as α-betulenol acetate with different structures as the previous investigators, also isolated from Betula species. 9Dhar et al. reviewed the chemistry of the birch tree including the essential oil which appeared to support Holubs' previous work. 10Hiltunen and co-workers reconfirmed by means of chemical reactions and gas chromatography / mass spectrometry (GC -MS), the occurrence of the main compounds as αand β-betulenol and their relevant acetates in the bud oil of B. pubescens Ehrh., supporting Treibs' work. 11ssential oil components of B. pendula were analyzed by Stepen and co-workers using GC where the main components were identified as α-betulenol acetate, caryophyllene and derivatives including low amounts of αand β-betulenol. 12Kaneko et al. reported betulenols and their acetates in the essential oils isolated from the buds of nineteen Betula species. 7][15][16][17][18] This present work covers the essential oil chemistry as well as biological activities of the main components isolated from various Betula species investigated by our group.

Results and Discussion
]23 The buds of B. pendula, B. litwinowii and B. medwediewii collected from various parts of Turkey were hydrodistilled while B. browicziana and B. recurvata buds were subjected to simultaneous distillation-extraction method (SDE) using a Likens-Nickerson apparatus due to limited plant material.The bud oils were analysed by GC-MS.The main component was isolated by Medium Pressure Liquid Chromatography (MPLC) in high purity. 13,16Literature search and comparison with spectral data 23 confirmed the identity of this compound as 14-hydroxy-βcaryophyllene (1), which was found 20.5-37.5% in all investigated oils.
Compound 2 and 4 were isolated individually from the reaction mixture followed by structures confirmation by 1 H and 13 C NMR.This information supported that 14-hydroxy-isocaryophyllene (4) is synonymous with β-betulenol and β-betulenal (2) with isocaryophyllen-14-al, when compared with previous investigations. 8To ensure the proposal, 2 was subjected to a mild reduction with NaBH 4 , resulting in β-betulenol (4).These compounds were also detected in the Betula bud essential oils by GC-MS.Content of β-betulenol and β-betulenal, in the oils of investigated Betula species were found from trace to 1.2% and 2.0-5.2%,respectively (Table 1).Manns and Hartmann reported (4E)-isocaryophyllen-14-al (=β-betulenal) in Cunila spicata Benth.(Lamiacea). 26Barrero et al. 27 and Hiede et al. 28 reported the presence of betulenal in Juniperus oxycedrus and J. virginiana essential oils without its configuration.Kaiser and Lamparsky 29 assigned the structure of the aldehyde, formed in the reaction mixture by direct oxidation of caryophyllene with SeO 2 , as caryophyllen-14-al (=α-betulenal), which was also detected in lavender oil.
Various phytopathogenic fungi were evaluated by agar tube dilution method 35 to test the antifungal activities of the leaf essential oils of B. pendula, B. browicziana, B. medwediewii, B. recurvata and B. litwinowii at 400 µg/mL concentration.Cephalosporium aphidicola, Drechslera sorokiniana, Fusarium solani, Rhizoctonia cerealis, were inhibited, whereas weak activity or no inhibition was observed against Aspergillus quadrilieneatus, A. flavus, Gibberella fujikuroi, Trichoderma harzianum and Trichothecium roseum. 14Previous studies demonstrated the antifungal activity of some Betula species; B. alba 36 , B. lenta 37 , B. nigra 38 , B. papyrifera 39 and B. plathyphylla var.japonica. 40n the course of our research into Betula species, we isolated essential oils from the buds of Betula pubescens ssp.pubescens and B. pubescens ssp.czerepanovii naturaly growing in Finland which were analyzed both by GC and GC-MS.14-Acetoxy-β-caryophyllene (1a) was determined as the main component in both oils (32.5 and 30.0%, respectively).The essential oil was subjected to column chromatography and a bicyclic aldehyde; birkenal (11) and a tricyclic lactone; hushinone (12) were isolated as new compounds.Birkenal (11) was subjected to a mild reduction with NaBH 4 to result in birkenol (13).This compound was shown to be naturally present in both essential oils (0.4-0.6%) with the aid of GC-MS (Table 2).The acetate of this alcohol; birkenyl acetate (13a) was shown to be naturally present at low concentrations (0.1%) in both essential oils investigated, as a new natural compound.6-Hydroxycaryophyllene ( 14) was also isolated from the oils.Acetylation of this compound resulted in the formation of 6acetoxycaryophyllene (14a) (Figure 2).The new acetate was also detected in the essential oils and identified as such by co-elution by means of TLC and GC-MS. 17Recently, Domrachev and Tkachev assigned the absolute configuration of birkenal by chemical correlation with known caryophyllene-type derivatives. 41he air-dried buds were hydrodistilled for 3 h using a Clevenger-type apparatus to yield 5.0% (A) and 7.8% (B) of essential oils on a dry-weight basis.

Conclusions
Betula species display an important resource for sesquiterpenes in particular for caryophyllene derivatives.Their biological activity is also worthwhile to investigate as in in vitro pre-screens we have observed antimicrobial activity against various human and plant pathogens.Another interesting aspect of sesquiterpenes and caryophyllenes is the potential use in flavour and fragrance industries, however, the mentioned secondary metabolites need to be investigated further from this aspect.

Experimental Section
Plant material.Leaf The plant materials were either hydrodistilled using a Clevenger type apparatus or were subjected to Likens-Nickerson simultaneous distillation-extraction (SDE) method and Microdistillation method when the plant material amounts were insufficient.The essential oils were analysed by GC and GC-MS.

Isolation of the essential oils
Hydrodistillation.The plant materials were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus to produce the essential oils.The percentage (%) yields were calculated on dry weight basis after drying over anhydrous Na 2 SO 4 .
Likens-Nickerson distillation-extraction method.B. browicziana and B. recurvata (1.0 g of buds) were subjected to SDE for 1 hour using a Likens-Nickerson apparatus with 1 ml of nhexane as solvent.
Microdistillation.The plant material (~200 mg) was placed in the sample vial of the MicroDistiller® (Eppendorf, Germany) system together with 10 ml of distilled water.NaCl (2.5 g) and water (0.5 ml) were added into the collection vial to break any possible emulsion formation.n-Hexane (300 µl) was also added into the collecting vial to trap the volatile components.The sample vial was heated to 100ºC at a rate of 20ºC/min and then kept at 100ºC for 15 min.It was then heated to 112ºC at a rate of 20ºC/min and kept at this temperature for 35 min.Later, the sample was subjected to post-run for 2 min under the same conditions.The collecting vial was cooled to -5ºC during the distillation.After the distillation was completed the n-hexane-trapped volatiles were analyzed by both by GC and GC-MS.The plant material was hydrodistilled for 3 h using a Clevenger type apparatus.The essential oil yield was calculated on dry weight basis corresponding to 0.5%.

Analysis of the essential oils
Gas chromatography (GC).Betula essential oils were analyzed by GC using a Hewlett Packard 6890 system and an HP Innowax FSC column (60 m x 0.25 mm ∅, with 0.25 µm film thickness) was used with nitrogen at 1 mL/min.Initial oven temperature was 60°C for 10 min, and increased at 4°C/min to 220°C, then constant at 220°C for 10 min and increased at 1°C/min to 240°C.Injector temperature was set at 250°C.Percentage composition of the individual components were obtained from electronic integration using flame ionization detection (FID) at 250°C.n-Alkanes were used as reference points in the calculation of relative retention indices (RRI).Relative percentages of the characterized components were as cited in Table 1-3.

Gas chromatography-mass spectrometry (GC-MS). GC-MS analysis was performed with a
Hewlett-Packard GCD, system and Innowax FSC column (60 m x 0.25 mm ∅, 0.25 µm film thickness) was used with Helium.GC oven temperature conditions were as described above, split flow was adjusted at 50 mL/min, the injector temperature was at 250°C.Mass spectra were recorded at 70 eV.Mass range was from m/z 35 to 425.Identification of components.Identification of the essential oil components were carried out by comparison of individual 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 2.1) and in-house "Baser Library of Essential Oil Constituents" libraries made up by genuine compounds and components of known oils, as well as MS literature data was also used for the identification.

Table 1 .
Main components of the different parts of Betula species growing in Turkey

Table 2 .
Essential oil compositions the buds of B. pubescens ssp.pubescens (A) and B. pubescens ssp.czerepanovii (B) , branch and buds of B. pendula, B. browicziana, B. litwinowii, B. recurvata and B. medwediewii were collected from different localities in North Eastern region of Turkey.Voucher specimens are kept at the Herbarium of the Faculty of Pharmacy of Anadolu University in Eskişehir, Turkey (ESSE).Detailed information on the plant materials used are given in Table 4. Buds of B. pubescens ssp.czerepanovii and B. pubescens ssp.pubescens were collected in April 2002 from the Botanical Garden of the University of Turku (SW Finland).Voucher specimens of the buds have been deposited in the Turku University Herbarium under numbers TUR 573172 and TUR 573171, respectively.Buds of B. pendula growing in Germany, were collected from Maxhütte, Regensburg in April 2002.

Table 3 .
Main components of Betula pendula buds growing in Germany

Table 4 .
Information on the Betula species growing in Turkey and essential oils # *Yields are given on moisture free basis ** Due to the poor yield of oil, it was dissolved in n-hexane.# Likens-Nickerson SDE