Analysis of Volatile Components of Adenosma indianum ( Lour . ) Merr . by Steam Distillation and Headspace Solid-Phase Microextraction

The essential oil ofAdenosma 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%), βcaryophyllene (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 ofA. indianum (Lour.) Merr. from two regions of China were similar but varied with different extractionmethods.These results were indicative of the suitability of HS-SPMEmethod for simple, rapid, and solvent-free analysis of the volatile components of the medicinal plants.


Introduction
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 [1].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, -limonene, and other volatile components [4].
In the past, steam distillation (SD) [4][5][6] and supercritical fluid extraction (SFE) [7] 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][9][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][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][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][5][6] or SFE [7] 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.Anhydrous sodium sulphate and diethyl ether were purchased from Siyou Company (Tianjin, China).

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) [31].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 L.

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 m 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.

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 m (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 N 2 at a flow rate of 50 mL min −1 , the H 2 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).

Gas Chromatography-Mass Spectrometry. GC-MS anal-
yses were conducted on a FINNIGAN TRACE DSQ GCmass 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.

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][35][36][37][38][39][40][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.

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 -limonene (26.07%), piperitenone oxide (11.63%), -caryophyllene (8.11%), and -caryophyllene (7.82%) compared to -limonene (35.07%), fenchone (15.79%), piperitenone oxide (11.46%), -caryophyllene (8.87%), and -caryophyllene (8.82%) in region B (Table 1).

Conclusions
Analysis of the extracts by SD and HS-SPME indicated that limonene, fenchone, -caryophyllene, -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 solventfree method for the determination of the volatile components in medicinal plants.

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.

Figure 1 :
Figure 1: Total ion current chromatograms of volatile components from A. indianum (Lour.)Merr.obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.

Figure 2 :
Figure 2: The five major volatile components of A. indianum (Lour.)Merr.obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.

Table 1 :
The chemical compositions of volatile components from A. indianum (Lour.)Merr.obtained by SD and HS-SPME from two regions of China.