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Journal of Chemistry
Volume 2013 (2013), Article ID 545760, 7 pages
Analysis of Volatile Components of Adenosma indianum (Lour.) Merr. by Steam Distillation and Headspace Solid-Phase Microextraction
School of Chemistry and Environment, South China Normal University, Guangzhou 510631, China
Received 2 May 2013; Revised 24 August 2013; Accepted 2 September 2013
Academic Editor: Zenilda L. Cardeal
Copyright © 2013 Zhi Zeng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The essential oil of Adenosma indianum (Lour.) Merr. plays an important role in its antibacterial and antiphlogistic activities. In this work, the volatile components were extracted by steam distillation (SD) and headspace solid-phase microextraction (HS-SPME) and analysed by gas chromatography-mass spectrometry (GC-MS). A total of 49 volatile components were identified by GC-MS, and the major volatile components were α-limonene (20.59–35.07%), fenchone (15.79–31.81%), α-caryophyllene (6.98–10.32%), β-caryophyllene (6.98–10.19%), and piperitenone oxide (1.96–11.63%). The comparison of the volatile components from A. indianum (Lour.) Merr. grown in two regions of China was reported. Also, the comparison of the volatile components by SD and HS-SPME was discussed. The results showed that the major volatile components of A. indianum (Lour.) Merr. from two regions of China were similar but varied with different extraction methods. These results were indicative of the suitability of HS-SPME method for simple, rapid, and solvent-free analysis of the volatile components of the medicinal plants.
Traditional Chinese medicines (TCMs) are invaluable drug resources. Because of their high pharmacological activity, low toxicity, and rare complications, they have been used in clinical therapy of many diseases for a thousand years in China . The dried aerial part of Adenosma indianum (Lour.) Merr. is commonly used to treat pyrexia, dyspepsia, and headaches [2, 3]. This plant grows in provinces of Southern China such as Guangxi and Guangdong. The antibacterial and antiphlogistic properties of its essential oil are mainly due to the presence of fenchone, linalool, α-limonene, and other volatile components .
In the past, steam distillation (SD) [4–6] and supercritical fluid extraction (SFE)  were used to extract the essential oil from A. indianum (Lour.) Merr. Steam distillation (SD) is the most common extraction technique used to obtain the volatile components from the plant materials, but it is time-consuming and needs large amounts of sample as well as losses of low-boiling-point volatile compounds during solvent removal. Alternatively, headspace solid-phase microextraction (HS-SPME) is a promising technique for the extraction and enrichment of volatile compounds from different sample matrices [8–10]. It uses a fine rod with a polymeric coating to extract organic compounds from their matrix and directly transfer them into the injector of a gas chromatograph for thermal desorption and analysis. It is a growing sample preparation technique and an attractive alternative to conventional extraction methods such as SD and SFE, which reduces solvent usage and exposure, disposal costs, and extraction time for sample separation and concentration purposes. This technique has been used to extract volatile compounds from a variety of natural products and is now considered a mature extraction technique [11–30]. However, the extraction depends on the characteristics of the SPME fibres used and the properties of the volatile compounds. Therefore, the volatile profile may not exactly reflect the proportion of volatile components from the medicinal plant by HS-SPME sampling.
A few studies have analysed the essential oil of A. indianum (Lour.) Merr. using either SD [4–6] or SFE  methods. The objectives of this research were to identify the chemical compositions of volatile oil obtained from A. indianum (Lour.) Merr. grown in two regions of China (Guangxi and Guangdong provinces) using HS-SPME and to compare the extraction with SD from the same plant materials.
2.1. Materials and Reagents
The dried aerial part of A. indianum (Lour.) Merr. grown in two regions of China (A) Guangxi province and (B) Guangdong province, respectively, was purchased from a local drug store in Guangzhou (Guangdong, China). The plants were identified by Professor J. Bin at the College of Life Science, South China Normal University. The voucher specimens have been deposited at Guangdong Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University.
Anhydrous sodium sulphate and diethyl ether were purchased from Siyou Company (Tianjin, China).
2.2. Extraction of the Volatile Components
2.2.1. Steam Distillation
Each sample (200 g) of the dried aerial part of A. indianum (Lour.) Merr. was milled into crude powder. The essential oils were extracted from the powder during a period of 7 h by using the SD method described in the Chinese Pharmacopoeia (2010) . These oils were collected followed by extraction using diethyl ether and then dried over anhydrous sodium sulphate and careful removal of the solvent. The yields of the essential oils were 0.29% w/w (region A) and 0.24% w/w (region B), respectively, based on the dried plant weight. The oil samples were stored at 4°C until they were analysed. Before injection, these oil samples were diluted 1 : 10 in dichloromethane, and the injection volume of the solution was 1 μL.
2.2.2. Headspace Solid-Phase Microextraction
Extraction conditions such as time and temperature were optimized. Extraction and enrichment or concentration of volatile components were performed using an SPME fibre (Supelco, USA) 1 cm in length, coated with triple-phase 30/50 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) that had been preconditioned in an SPME fibre conditioner (GL Sciences) at 250°C for 1 h before the first measurement. All extractions were performed in 15 mL glass vial equipped with screw caps and PTFE/silicone septa, using 0.5 g of powdered A. indianum (Lour.) Merr. The vial was immersed to a depth of 5 mm in a thermostatically controlled bath at 80°C for 30 min before sampling. The SPME fibre was maintained 0.5 cm above the powder sample at the same temperature for 30 min. After the extraction, the SPME fibre was thermally desorbed for 5 min at 250°C in the injector of gas chromatograph.
2.3. Gas Chromatography
Gas chromatography (GC) is comprised of a GC-2010 gas chromatograph (Shimadzu, Japan) equipped with a flame ionization detector (FID). The GC-2010 is equipped with a split/splitless injector. Desorption time was 5 min in the splitless mode in the injection port at 250°C. A column, DB-5, 30 m × 0.32 mm i.d. × 0.25 μm (stationary phase thickness) (J & W Scientific, USA) was applied. GC was temperature-programmed at 40°C for 2 min and then increased to 230°C at a rate of 4°C min−1 and maintained at 230°C for 4.5 min. The carrier gas was helium, and the column head pressure was 114.6 kPa at a constant linear velocity of 35 cm sec−1. The FID temperature was 250°C. The following gases and flow rates were used for the FID system: the makeup gas was N2 at a flow rate of 50 mL min−1, the H2 flow rate was 50 mL min−1, and the air flow rate was 400 mL min−1. Data were collected by GC Solution software (Shimadzu, Japan).
2.4. Gas Chromatography-Mass Spectrometry
GC-MS analyses were conducted on a FINNIGAN TRACE DSQ GC-mass spectrometer (FINNIGAN, USA) with Xcalibur Data System and FINNIGAN TRACE DSQ Upgrade MS software. Desorption time was 5 min in the injection port at 250°C, with a split ratio of 10 : 1. The same column, injection conditions, and oven temperature programming as for GC analyses were used. It means that “those” has been deleted. The carrier gas was helium, which was delivered at a linear velocity of 2 mL min−1. The mass selective detector was operated in an electron impact ionization mode at 70 eV, in a scan range of m/z 40–400. The interface temperature was 230°C. Retention time of each volatile was converted to the retention index (RI) using C8-C22 n-alkanes (Supelco, USA) as the references.
2.5. Component Identification
The volatile compounds were tentatively identified by either matching both their mass spectra and RI values or only their mass spectra with the spectra of reference compounds in the Wiley mass spectral library (6th edition) and the NIST 147 mass spectral database and verified on the basis of mass spectra reported in the literature [32, 33]. The identification was confirmed by comparison of their RI values on DB-5 column with those reported in the literature [34–41]. This retention index (RI) was determined by comparison with a standard mixture of C8-C22 n-alkanes (Supelco, USA) under the same chromatographic conditions. All experiments were performed in triplicate.
3. Results and Discussion
3.1. Essential Oil by Steam Distillation
The volatile profile of the essential oil extracted by SD from A. indianum (Lour.) Merr. grown in region A (Guangxi province) was found to be in good agreement with that from region B (Guangdong province). The analysis of essential oil components of the samples from regions A and B allowed the identification of 36 and 37 compounds. As shown in Table 1, fenchone (31.81%, 31.60%), α-limonene (20.59%, 30.15%), α-caryophyllene (10.32%, 6.98%), and β-caryophyllene (10.19%, 6.98%) were the major volatile components of the samples from regions A and B, respectively. A relative similarity was noted for these oils showing a homogenous qualitative composition; in contrast, the quantitative composition varied depending on the samples.
Of the components identified by using SD/GC-MS method, 7-octen-4-ol, p-mentha-2,8-dien-1-ol, α-bergamotene, 2,5,9-trimethyl-4,8-cycloundecadien-1-one, tetradecanol, and alloaromadendrene oxide were tentatively identified for the first time in the volatile oils of A. indianum (Lour.) Merr. based on the literature [4–7].
3.2. Volatile Components by Headspace Solid-Phase Microextraction
The volatile components of A. indianum (Lour.) Merr. from two regions of China were extracted using HS-SPME under optimized parameters. The optimization of HS-SPME sampling parameters was carried out using the sample grown in region A based on the sum of total peak areas obtained by GC-FID. The amounts of volatile components varied with extraction temperature by HS-SPME. For analysis of the volatile components of A. indianum (Lour.) Merr., the use of a water bath at 80°C was chosen as the optimum temperature, with an equilibrium time of 30 min for the sample. The optimum extraction time was found to be 30 min. Under these conditions, 44 and 45 compounds were identified from A. indianum (Lour.) Merr. In the HS-fractions obtained from A. indianum (Lour.) Merr. grown in region A, fenchone (26.44%) was the major component, followed by α-limonene (26.07%), piperitenone oxide (11.63%), β-caryophyllene (8.11%), and α-caryophyllene (7.82%) compared to α-limonene (35.07%), fenchone (15.79%), piperitenone oxide (11.46%), α-caryophyllene (8.87%), and β-caryophyllene (8.82%) in region B (Table 1).
Of the components identified by using HS-SPME/GC-MS method, p-mentha-2,8-dien-1-ol,2-allyl-4-methylphenol, isothymol, cinerolone, α-bergamotene, 2,5,9-trimethyl-4,8-cycloundecadien-1-one, calarene epoxide, alloaromadendrene oxide, himachalene, 2-methylene-6,8,8-trimethyl-tricyclo[220.127.116.11(1,6)]undecan-3-ol, and 2-(4a,8-dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-yl)-prop-2-en-1-ol were tentatively identified for the first time in the volatile components of A. indianum (Lour.) Merr. based on the literature [4, 5, 7].
3.3. Comparison of Steam Distillation and Headspace Solid-Phase Microextraction Methods
The composition of volatile components of A. indianum (Lour.) Merr. extracted by HS-SPME is largely consistent with that of the SD extracts, but the relative contents of each component were significantly different. Total ion current chromatograms of volatile components from A. indianum (Lour.) Merr. obtained by SD and HS-SPME from regions A and B, respectively, were presented in Figure 1. Extracts obtained with HS-SPME technique allowed for GC-MS identification of the higher number of volatiles, 45 compounds compared to 37 for the SD extracts. The major volatile components of A. indianum (Lour.) Merr. from two regions of China by SD and HS-SPME were α-limonene (20.59–35.07%), fenchone (15.79–31.81%), α-caryophyllene (6.98–10.32%), β-caryophyllene (6.98–10.19%), and piperitenone oxide (1.96–11.63%). Figure 2 showed the five major volatile components of A. indianum (Lour.) Merr. obtained by SD and HS-SPME from regions A and B. A total of 12 compounds, namely, β-pinene, 3-carene, camphor, 2-allyl-4-methylphenol, thymol, isothymol, α-cubebene, cinerolone, calarene epoxide, himachalene, 2-methylene-6,8,8-trimethyl-tricyclo[18.104.22.168(1,6)]undecan-3-ol, and 2-(4a,8-dimethyl-1,2,3,4,4a, 5,6,7-octahydronaphthalen-2-yl)-prop-2-en-1-ol, were identified in HS-SPME extractions but were not identified from SD extracts. Only 4 compounds, namely, β-myrcene, 7-octen-4-ol, carveol, and tetradecanol, were not identified from SPME extractions but identified in SD extracts.
The reports published concerning the composition of the extracts from A. indianum (Lour.) Merr. [4–7] found that the compositions of the essential oils from A. indianum (Lour.) Merr. by SD and SFE varied. Ji and Pu  reported that fenchone (13.90%), p-cymene + cineol (12.79%), limonene (12.36%), and linalool (7.92%) were the major volatile components of A. indianum (Lour.) Merr. by SD, while Wu et al.  reported that 6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran (9.99%), 2H-1-benzopyran-2-one (6.56%), and caryophyllene oxide (5.40%) were significant in SFE oils. However, p-cymene, cineol, 6,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran, and 2H-1-benzopyran-2-one were not found in our study.
Analysis of the extracts by SD and HS-SPME indicated that α-limonene, fenchone, α-caryophyllene, β-caryophyllene, and piperitenone oxide were the major volatile components of A. indianum (Lour.) Merr. grown in two regions of China. Only quantitative differences of some components could be observed in both volatile profiles, while qualitatively both volatile mixtures were rather similar. This work provides the first report of the analysis of the volatile components from A. indianum (Lour.) Merr. by HS-SPME. Compared with extraction by SD, HS-SPME is a simple, sensitive, and solvent-free method for the determination of the volatile components in medicinal plants.
Conflict of Interests
The authors declare that they have no conflict of interests.
The authors gratefully acknowledge the support of the National Natural Science Foundation of China (21272080), Department of Science and Technology, Guangdong, China (2010A020507001-76, 5300410, FIPL-05-003), and The Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China.
- K. C. Wen, C. Y. Huang, and F. L. Lu, “Determination of baicalin and puerarin in traditional Chinese medicinal preparations by high-performance liquid chromatography,” Journal of Chromatography, vol. 631, no. 1-2, pp. 241–250, 1993.
- National Bureau of Traditional Chinese Medicine Editorial Board of Chinese Materia Medica, Chinese Meteria Medica, Shanghai Scientific & Technical Publishers, Shanghai, China, 2002.
- Jiangsu New Medical College, Great Dictionary of Chinese Materia Medica, Shanghai Scientific & Technical Publishers, Shanghai, China, 1997.
- X. D. Ji and Q. L. Pu, “Studies on the components of the essential oil from Adenosma ndianum (Lour.),” Acta Botanica Sinica, vol. 27, pp. 80–83, 1985.
- Q. K. Ya, W. J. Lu, J. Y. Chen, and X. Tan, “GC-MS analysis of the chemical constituent of volatile oil from Zhuang drug Adenosma indianum (Lour.) Merr,” Chinese Journal of Pharmaceutical Analysis, vol. 31, pp. 544–546, 2011.
- Y. Huang, H. E. Wu, Z. Y. Wei, Y. F. Xiao, and X. L. Yu, “Chemical constituents and anti-bacterial activity of essential oil form Adenosma indianum,” Chinese Journal of Experimental Traditional Medical Formulae, vol. 17, pp. 79–82, 2011.
- H. E. Wu, C. Y. Liang, Y. H. Li, X. Q. Huang, and X. Y. Zhu, “GC-MS analysis of chemical constituents of the essential oil from Adenosma indianum (Lour.) Merr. by different extraction methods,” Chinese Journal of Pharmaceutical Analysis, vol. 30, pp. 1941–1946, 2010.
- Z. G. Li, M. R. Lee, and D. L. Shen, “Analysis of volatile compounds emitted from fresh Syringa oblata flowers in different florescence by headspace solid-phase microextraction-gas chromatography-mass spectrometry,” Analytica Chimica Acta, vol. 576, no. 1, pp. 43–49, 2006.
- F. Belliardo, C. Bicchi, C. Cordero, et al., “Headspace-solid-phase microextraction in the analysis of the volatile fraction of aromatic and medicinal plants,” Journal of Chromatographic Science, vol. 44, no. 7, pp. 416–429, 2006.
- Z. B. Xiao, J. C. Zhu, T. Feng et al., “Comparison of volatile components in Chinese traditional pickled peppers using HS-SPME-GC-MS, GC-O and multivariate analysis,” Natural Product Research, vol. 24, no. 20, pp. 1939–1953, 2010.
- C. Zhang, M. L. Qi, Q. L. Shao, S. Zhou, and R. N. Fu, “Analysis of the volatile compounds in Ligusticum chuanxiong Hort. using HS-SPME-GC-MS,” Journal of Pharmaceutical and Biomedical Analysis, vol. 44, no. 2, pp. 464–470, 2007.
- X. Di, R. A. Shellie, P. J. Marriott, and C. W. Huie, “Application of headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography (GC × GC) for the chemical profiling of volatile oils in complex herbal mixtures,” Journal of Separation Science, vol. 27, no. 5-6, pp. 451–458, 2004.
- R. Bozalongo, J. D. Carrillo, M. Á. F. Torroba, and M. T. Tena, “Analysis of French and American oak chips with different toasting degrees by headspace solid-phase microextraction-gas chromatography-mass spectrometry,” Journal of Chromatography A, vol. 1173, no. 1-2, pp. 10–17, 2007.
- J. Pawliszyn, Solid Phase Microextraction: Theory and Practice, Wiley-VCH Press, New York, NY, USA, 1997.
- J. Pawliszyn, Applications of Solid Phase Microextraction, The Royal Society of Chemistry Press, Cambridge, UK, 1999.
- B. Zygmunt, A. Jastrzȩbska, and J. Namieśnik, “Solid phase microextraction: a convenient tool for the determination of organic pollutants in environmental matrices,” Critical Reviews in Analytical Chemistry, vol. 31, no. 1, pp. 1–18, 2001.
- Z. Zeng, R. Xie, T. Zhang, H. Zhang, and J. Y. Chen, “Analysis of volatile compositions of Magnolia biondii pamp by steam distillation and headspace solid phase micro-extraction,” Journal of Oleo Science, vol. 60, no. 12, pp. 591–596, 2011.
- C. A. Zini, T. F. de Assis, E. B. Ledford Jr. et al., “Correlations between pulp properties of eucalyptus clones and leaf volatiles using automated solid-phase microextraction,” Journal of Agricultural and Food Chemistry, vol. 51, no. 27, pp. 7848–7853, 2003.
- J. M. Vaz, “Screening direct analysis of PAHs in atmospheric particulate matter with SPME,” Talanta, vol. 60, no. 4, pp. 687–693, 2003.
- W. M. Mullett, K. Levsen, J. Borlak, J. C. Wu, and J. Pawliszyn, “Automated in-tube solid-phase microextraction coupled with HPLC for the determination of N-nitrosamines in cell cultures,” Analytical Chemistry, vol. 74, no. 7, pp. 1695–1701, 2002.
- C. A. Zini, H. Lord, E. Christensen, T. F. de Assis, E. B. Caramão, and J. Pawliszyn, “Automation of solid-phase microextraction-gas chromatography-mass spectrometry extraction of eucalyptus volatiles,” Journal of Chromatographic Science, vol. 40, no. 3, pp. 140–146, 2002.
- P. J. Watkins, G. Rose, R. D. Warner, F. R. Dunshea, and D. W. Pethick, “A comparison of solid-phase microextraction (SPME) with simultaneous distillation-extraction (SDE) for the analysis of volatile compounds in heated beef and sheep fats,” Meat Science, vol. 91, no. 2, pp. 99–107, 2012.
- E. Moreno, A. Fita, M. C. González-Mas, and A. Rodríguez-Burruezo, “HS-SPME study of the volatile fraction of Capsicum accessions and hybrids in different parts of the fruit,” Scientia Horticulturae, vol. 135, pp. 87–97, 2012.
- G. Basaglia and M. C. Pietrogrande, “Optimization of a SPME/GC/MS method for the simultaneous determination of pharmaceuticals and personal care products in waters,” Chromatographia, vol. 75, no. 7-8, pp. 361–370, 2012.
- J. B. Lin, X. J. Shi, H. L. Liu, and K. Yuan, “Analysis of chemical constituents of the volatile oil in the different parts of okra by SPME-GC/MS,” Asian Journal of Chemistry, vol. 24, no. 3, pp. 1309–1312, 2012.
- I. Gokbulut and I. Karabulut, “SPME-GC-MS detection of volatile compounds in apricot varieties,” Food Chemistry, vol. 132, no. 2, pp. 1098–1102, 2012.
- M. Khani and S. Imani, “Development of HS-SPME/GC-MS method for analysis of fenitrothion residues in wheat,” Annals of Biological Research, vol. 3, pp. 236–239, 2012.
- M. A. Rather, B. A. Dar, S. N. Sofi et al., “Headspace solid phase microextraction (HS-SPME ) gas chromatography mass spectrometric analysis of the volatile constituents of Cannabis sativa L. from Kashmir,” Journal of Pharmacy Research, vol. 4, pp. 2651–2653, 2011.
- O. Mastrogianni, G. Theodoridis, K. Spagou et al., “Determination of venlafaxine in post-mortem whole blood by HS-SPME and GC-NPD,” Forensic Science International, vol. 215, no. 1–3, pp. 105–109, 2012.
- G. Vas and K. Vékey, “Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis,” Journal of Mass Spectrometry, vol. 39, no. 3, pp. 233–254, 2004.
- National Committee of Pharmacopoeia, Pharmacopoeia of the People's Republic of China, appendix 63, Chinese Medical Science and Technology Press, Beijing, China, 2010.
- L. S. Cai, J. A. Koziel, J. Davis, Y. C. Lo, and H. W. Xin, “Characterization of volatile organic compounds and odors by in-vivo sampling of beef cattle rumen gas, by solid-phase microextraction, and gas chromatography-mass spectrometry-olfactometry,” Analytical and Bioanalytical Chemistry, vol. 386, no. 6, pp. 1791–1802, 2006.
- R. P. Adams, Identification of Essential Oils by Ion Trap Mass Spectroscopy, Academic Press, New York, NY, USA, 1989.
- S. Hamm, J. Bleton, J. Connan, and A. Tchapla, “A chemical investigation by headspace SPME and GC-MS of volatile and semi-volatile terpenes in various olibanum samples,” Phytochemistry, vol. 66, no. 12, pp. 1499–1514, 2005.
- F. Sefidkon and R. Kalvandi, “Chemical composition of the essential oil of Micromeria persica Boiss. from Iran,” Flavour and Fragrance Journal, vol. 20, no. 5, pp. 539–541, 2005.
- Z. Zeng, H. Zhang, T. Zhang, S. Tamogami, and J. Y. Chen, “Screening for γ-nonalactone in the headspace of freshly cooked non-scented rice using SPME/GC-O and SPME/GC-MS,” Molecules, vol. 14, no. 8, pp. 2927–2934, 2009.
- S. Zrira, A. Elamrani, and B. Benjilali, “Chemical composition of the essential oil of Pistacia lentiscus L. from Morocco—a seasonal variation,” Flavour and Fragrance Journal, vol. 18, no. 6, pp. 475–480, 2003.
- Z. Zeng, H. Zhang, J. Y. Chen, et al., “Direct extraction of volatiles of rice during cooking using solid-phase microextraction,” Cereal Chemistry, vol. 84, no. 5, pp. 423–427, 2007.
- Z. Zeng, H. Zhang, T. Zhang, S. Tamogami, and J. Y. Chen, “Analysis of flavor volatiles of glutinous rice during cooking by combined gas chromatography-mass spectrometry with modified headspace solid-phase micro-extraction method,” Journal of Food Composition and Analysis, vol. 22, no. 4, pp. 347–353, 2009.
- F. Sefidkon and Z. Jamzad, “Essential oil analysis of Iranian Satureja edmondi and S. isophylla,” Flavour and Fragrance Journal, vol. 21, no. 2, pp. 230–233, 2006.
- F. Askari and F. Sefidkon, “Essential oil composition of Pimpinella affinis Ledeb. from two localities in Iran,” Flavour and Fragrance Journal, vol. 21, no. 5, pp. 754–756, 2006.