C. Mahidol, N. Chimnoi and D. Chokchaichamnankit S. Techasakul. Faculty of Science

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1 Identification of Volatile Constituents in Artabotrys hexapetalus Flowers Using Simple Headspace Solvent-Trapping Technique in Combination with Gas Chromatography-Mass Spectrometry and Retention C. Mahidol, N. Chimnoi and D. Chokchaichamnankit S. Techasakul Chulabhorn Research Institute Department of Chemistry Bangkok Faculty of Science Thailand Kasetsart University Bangkok Thailand Keywords: esters, odor, solid-phase microextraction, hydrodistillation, solvent extraction Abstract The compositions of the volatiles from Artabotrys hexapetalus flowers were investigated. These volatiles were obtained by simple headspace-solvent trapping technique with dichloromethane as the trapping solvent. The investigation was performed in combination of gas chromatography-mass spectrometry (EI and PCI) and retention indices. Seventeen components were identified with ethyl acetate (47.3%), ethyl isobutanoate (9.2%), isobutyl acetate (26.8%) and ethyl butanoate (9.7%) as the major components. The extract was also compared with those obtained by solid-phase microextraction (SPME), solvent extraction and hydrodistillation. The odor of the solution from this technique was similar to that of the fresh flowers and differed from that obtained by solvent extraction and hydrodistillation. INTRODUCTION Artabotrys hexapetalus is widely distributed throughout the southern part of China and also in the southern part of Asia. In China, its roots and fruits are used for treating malaria and scrofula, respectively (Wong and Brown, 2002). The odor from flowers is sweet and fresh; however, in our knowledge, this is the first report to investigate the volatile components in this flower. The volatile components from this flower are released only in the morning (~5-8 a.m.) and in the evening (~6-8 p.m.); therefore, the on-site sampling and preconcentration method are required. The objective of this work was to develop the method for extraction and identification of the volatile components from this flower. MATERIALS AND METHODS Artabotrys hexapetalus flowers were sampled about 5-6 a.m. and extracted as soon as possible. In the simple headspace-solvent trapping technique, approximately 30 g of fresh flowers were placed in a 500 ml erlenmyer flask. Air was flushed through the flask inlet with the aid of an aquarium pump. Then the headspace vapor was passed through a round-bottom flask contained 20 ml of dichloromethane (J.T.Baker, USA) as shown in Fig. 1. The sampling peroid was 3 h. After that, the solution volume was reduced to 4 ml with the aid of rotary evaporator. In the hydrodistillation, approximately 100 g of fresh flowers were hydrodistilled for 4 h in a simple distillation apparatus. Then, the distillate was extracted with 200 x 3 ml of hexane (J.T.Baker, USA), dry over anhydrous sodium sulfate (Fluka, Switzerland) and concentrated to 4 ml with the aid of rotary evaporator. In the solvent extraction, approximately 10 g of fresh flowers were extracted with 50 ml of hexane (J.T.Baker, USA) for 30 min under ultrasonication and left at room temperature for 3 h. The extract was dried over anhydrous sodium sulfate (J.T.Baker, Switzerland) and concentrated to 1 ml with the aid of rotary evaporator. In solid-phase microextraction, approximately 18 g of fresh flowers were placed in the glass bottle with a rubber septum. The extraction was performed on 100 µm layer of Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS

2 polydimethyl siloxane fiber (PDMS) (Supelco, USA) for 1 h. Then the fiber was desorbed in the GC injector at 220 C for 10 min. The identification was performed on the GC-MS system (Varian 3400GC, USA and ITS40 Mass spectrometer, Finnigan MAT, USA) with electron impact at 70 ev and positive chemical ionization (PCI) by using methane (>99.97%, AIRCO, USA) as the reagent gas. DB-5 (30 m x 0.25 mm I.D., 0.25 µm film thickness, J&W Scientific, USA) was used for separation. The injection volume was 0.5 µl in split mode (1:10). The temperature program was used as follows: the initial temperature was 35 C for 3 min, which was increased to 260 C at 4 C min -1 and held at this temperature for 10 min. The injector and transferline temperature was set at 220 C. Helium (99.999%, Praxair, Thailand) was used as the carrier gas with 15 psi. head pressure. The volatile components were analyzed by using both NIST Mass Spectral Search Program (National Institute of Standards and Technology, USA) and retention indices. The retention indices were calculated according to equation 1 (van den Dool and Kratz, 1963) I = 100Z + [(t R(x) t R(z) )/(t R(z+1) t R(z) )] x 100 (1) I = retention indices, t R = retention time, x = substance of interest and z, z+1 = n-alkanes with z and z+1 carbon atom emerging before and after substance x, respectively. RESULTS AND DISCUSSION The identification of the volatile components was performed by using both mass spectral library and retention indices (RI). Identification using mass spectral library alone is not always possible since some structural molecular fragment cannot be distinguished solely on the basis of MS data, for instance differences in the number or position of carbon skeleton branchings, isomeric systems, etc. Therefore, to increase the reliability of the analytical results, it is necessary to utilize both MS data and retention indices identities as the identification criteria (Shellie et al., 2002). To confirm the molecular weight of the components, positive chemical ionization (PCI) was performed. Four different extraction methods were chosen to extract the volatile components from Artabotrys hexapetalus flowers i.e. hydrodistillation, solvent extraction, solid-phase microextraction and simple headspace solvent-trapping technique. The compositions and relative amounts for each extraction method are reported in Table 1 while GC-MS chromatograms are shown in Fig. 2. Hydrodistillation and solvent extraction are common methods for extraction of volatile components from plants. In hydrodistillation, thirty one components were identified together with five unidentified ones. The major components were β- caryophyllene (10.1%), β-gurjunene (30.0%) and globulol (13.8%). In solvent extraction, thirty one components were identified together with two unidentified ones. The major components were 3-methylbutanol (5.7%), 2-methylbutanol (3.9%), ethyl butanoate (3.1%), isopentyl acetate (12.6%), 2-methylbutyl acetate (7.7%), limonene (5.7%), linalool (7.7%), benzeneethanol (5.3%) and two unidentified components (13.9%). Two alternative extraction methods, simple headspace solvent-trapping technique and SPME, were also performed, both methods based on headspace extraction. In the simple headspace solvent-trapping extraction technique, the headspace vapor of the fresh flowers was flushed with air and collected in dichloromethane. Seventeen components were identified with ethyl acetate (47.3%), ethyl isobutanoate (9.2%), isobutyl acetate (26.8%) and ethyl butanoate (9.7%) as the major components. In the SPME, the headspace vapor of the fresh flowers was absorbed on the viscous liquid, polydimethyl siloxane (PDMS) and directly injected into the GC-MS. Thirtynine components were identified together with three unidentified ones. Ethyl acetate (12.8%), ethyl isobutanoate (4.9%), isobutyl acetate (39.5%), ethyl 2-methyl butanoate (2.3%), ethyl isovalerate (11.3%), ethyl 3-methyl-2-butenoate (6.8%), isobutyl isovalerate (2.1%) and β- caryophyllene (5.4%) were found to be the major components. 44

3 Comparison of the identified components in the four extraction methods, we found that sesquiterpenes and sesquiterpenic alcohols were the major components in hydrodistillation. Alcohols, esters, monoterpenes, monoterpenic alcohols and aromatic alcohol were the major components in solvent extraction while esters were the major components in headspace solvent-trapping technique and SPME. The odor from two extraction methods, hydrodistillation and solvent extraction, were very green which different very much from that of the fresh flowers. It is likely that the enzymatic processes that were used for the odor formation and release were denatured by means of the high temperature in hydrodistillation and the organic solvent used in solvent extraction process. Therefore, the components which gave the significant odor could not be accumulated so the strong green odor was obtained instead together with the relative high boiling point components, sesquiterpenes and sesquiterpenic alcohols (Tava et al., 2000). In contrast, the headspace methods, the simple headspace solvent-trapping technique and SPME, were operated under the mild conditions, without using high temperature and organic solvent, which have less effect on the enzymatic processes. Therefore, the relative low boiling point components, esters, were obtained as the major components in both methods. In addition, the odor of the solution from the headspace solvent-trapping technique was similar to that of the fresh flowers. Although esters were obtained as the major components from the both methods, some difference in the relative amount of each component could be observed. This may be because the difference in the absorption selectivity between dichloromethane and PDMS fiber and/or the sampling time. CONCLUSIONS The use of simple headspace solvent-trapping technique in combination with GC- MS and retention indices allowed us to identify the compositions of the volatile components from Artabotrys hexapetalus flowers and the odor of the obtaining solution was similar to that of the fresh flowers. These results support the advantage of the headspace extraction, the compositions from this method is more representative because it is closer to that of the atmosphere around the flowers as mentioned in the previous works (Ishizaka et al., 2002; Tava et al., 2000). Furthermore, the simplicity and the use of just only few pieces of apparatus and small amount of organic solvent allow us to sampling on-sites easily. These are considered as the positive features supporting the choice of the simple headspace solventtrapping technique. Literature Cited Acree, T.E. June Adams, R.P Identification of Essential Oils by Ion Trap Mass Spectroscopy, Academic Press, San Diego. van Den Dool, H. and Kratz, P.D A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. 11: Ishizaka, H., Yamada, H. and Sasaki, K Volatile compounds in the flowers of Cyclamen persicum, C. purpurascens and their hybrids. Ser. Hort. 94: Shellie, R., Mondello, L., Marriott, P. and Dugo, G Characterization of lavender essential oils by using gas chromatography-mass spectrometry with correlation of linear retention indices and comparison with comprehensive two-dimensional gas chromatography. J. Chromatogr. A 970: Tava, A., Pecetti, L., Povolo, M. and Contarini, G A Comparison between two systems of Volatile Sampling in Flowers of Alfalfa (Medicago sativa L.). Phytochem. Anal. 11: Wang, Y., Yao, X., Zhang, X., Zhang, R., Liu, M. Hu, Z. and Fan, B The prediction for gas chromatographic retention indices of saturated esters on stationary 45

4 phases of different polarity. Talanta 57: Wong, H. and Brown, G.D Beta-Methoxy-gamma-methylene-alpha,betaunsaturated-gamma-butyrolactones from Artabotrys hexapetalus. Phytochemistry 59(1): Xiangmin, Z. and Peichang, L Unified equation between kováts indices on different stationary phases for select types of compounds. J. Chromatogr. A 731:

5 Tables Table 1. Compositions of the volatile components from Artabotrys hexapetalus flowers. No. Components Retention (RI) References Retention (RI ref ) Hydro distillation Peak area of the GC-MS chromatogram (%) Solvent Simple headspace extraction solvent-trapping Solid-phase micro extraction (SPME) technique 1 Ethyl acetate Methyl isobutanoate ND Ethyl propanoate Propyl acetate Methyl butanoate Methyl butanol Methyl butanol Ethyl isobutanoate Isobutyl acetate Methyl isovalerate ND 11 Ethyl methacrylate Methylallyl acetate Ethyl butanoate Methyl 3-methyl-2-butenoate Ethyl 2-butenoate Ethyl 2-methyl butanoate Ethyl isovalerate Isobutyl propanoate Methyl 3-hydroxy methylbutanoate 20 Isopentyl acetate Methylbutyl acetate Isobutyl isobutanoate Ethyl 3-methyl-2-butenoate

6 No. Components Retention (RI) References Retention (RI ref ) Hydro distillation Peak area of the GC-MS chromatogram (%) Solvent Simple headspace Solid-phase extraction solvent-trapping micro extraction technique (SPME) 24 α-pinene Ethyl 3-hydroxybutanoate Ethyl 3-hydroxy methylbutanoate 27 Benzaldehyde Isobutyl butanoate Sabinene (5H)-Furanone,3-methyl β-myrcene Ethyl hexanoate Unidentified compound Isobutyl isovalerate Limonene ,8-Cineole E-Ocimene Methyl,2-butenoic acid,isopropyl ester 39 Linalool Benzeneethanol α-terpineol α-cubebene Unidentified compound α-copaene β-elemene Unidentified compound Unidentified compound

7 No. Componenrs Retention (RI) References retention indices (RI ref ) Hydro distillation Peak area of the GC-MS chromatogram (%) Solvent Simple headspace extraction solvent-trapping Solid-phase micro extraction (SPME) technique 48 α-gurjunene β-caryophyllene β-gurjunene Unidentified compound α-caryrophyllene Unidentified compound γ-muurolene α-curcumene Germacrene D Germacrene B Pentadecane α-muurolene γ-cadinene δ-cadinene Unidentified compound Unidentified compound Spathulenol Globulol Unidentified compound Cubenol Unidentified compound tau-muurolol δ-cadinol α-cadinol ND = not detect; 1 (Acree, 2002); 2 (Zhang and Lu, 1996); 3 (Adams, 1989); 4 Retention index was calculated according to the equation I = AI + BN + C (Zhang and Lu, 1996) on the basis of DB-1 retention index (Wang et al., 2002). 49

8 Figures Fig. 1. Schematic of the simple solvent-trapping device. Fig. 2. Typical GC-MS Total Ion Chromatogram of the volatile components from Artabotrys hexapetalus flowers. The numbers refer to those in Table 1. A, B, C and D are the chromatograms obtained from hydrodistillation, solvent extraction, simple headspace solvent-trapping technique and solid-phase microextraction, respectively. 50