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

Authors: Zhi Zeng; Chunyan Meng; Xuening Ye; Zhuo Zeng
Journal: Journal of Chemistry
 (2013)

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## Abstract

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%), 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.


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## Body

## 1. 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, α-limonene, and other volatile components [4].In the past, steam distillation (SD) [4–6] 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 [8–10]. 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 ofA. indianum (Lour.) Merr. using either SD [4–6] 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.


## 2. Experimental

### 2.1. Materials and Reagents

The dried aerial part ofA. 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 ofA. 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 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 30/50μm 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. × 0.25μm (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/z40–400. 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.


## 2.1. Materials and Reagents

The dried aerial part ofA. 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 ofA. 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 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 30/50μm 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.


## 2.2.1. Steam Distillation

Each sample (200 g) of the dried aerial part ofA. 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 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 30/50μm 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. × 0.25μm (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/z40–400. 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 fromA. 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.Table 1
The chemical compositions of volatile components fromA. indianum(Lour.) Merr. obtained by SD and HS-SPME from two regions of China.
Number
Composition
RIa
RIb
Relative content (%)c
SD
SPME
Ad
Bd
A
B
1
α-Thujene
929
930
0.05 ± 0.01
0.10 ± 0.03
0.05 ± 0.01
0.06 ± 0.02
2
α-Pinene
938
938
1.17 ± 0.12
0.92 ± 0.10
1.51 ± 0.15
1.35 ± 0.13
3
α-Fenchene
954
0.61 ± 0.07
0.57 ± 0.05
2.00 ± 0.20
0.89 ± 0.10
4
β-Pinene
989
980
—e
—
0.17 ± 0.02
0.14 ± 0.03
5
β-Myrcene
993
994
0.10 ± 0.00
0.08 ± 0.00
—
—
6
7-Octen-4-olf
1002
0.17 ± 0.01
0.07 ± 0.00
—
—
7
3-Carene
1009
1006
—
—
0.26 ± 0.30
0.24 ± 0.40
8
α-Terpinene
1021
1017
0.18 ± 0.20
0.19 ± 0.10
0.20 ± 0.30
0.16 ± 0.08
9
γ-Terpinene
1033
0.26 ± 0.10
0.33 ± 0.30
0.33 ± 0.15
0.54 ± 0.25
10
α-Limonene
1038
1030
20.59 ± 2.10
30.15 ± 3.00
26.07 ± 1.80
35.07 ± 4.38
11
β-Ocimene
1059
1050
0.14 ± 0.03
0.19 ± 0.20
0.17 ± 0.05
0.62 ± 0.30
12
Fenchone
1097
1088
31.81 ± 2.50
31.60 ± 3.80
26.44 ± 1.80
15.79 ± 1.95
13
β-Linalool
1104
1096
0.30 ± 0.04
0.22 ± 0.02
0.08 ± 0.00
0.14 ± 0.02
14
Fenchol
1121
1110
4.34 ± 0.50
3.88 ± 0.30
3.83 ± 0.25
1.50 ± 0.18
15
p-Mentha-2,8-dien-1-olf
1138
1123
—
0.25 ± 0.02
—
0.26 ± 0.31
16
Carveol
1152
0.16 ± 0.18
0.25 ± 0.38
—
—
17
Camphor
1154
1144
—
—
0.07 ± 0.00
0.07 ± 0.01
18
trans-Carveol
1201
0.22 ± 0.03
0.22 ± 0.01
0.06 ± 0.00
0.37 ± 0.05
19
p-Cymene-8-ol
1203
1187
0.48 ± 0.06
0.33 ± 0.02
0.32 ± 0.04
0.26 ± 0.01
20
α-Terpineol
1212
1193
0.35 ± 0.02
0.26 ± 0.03
0.13 ± 0.01
0.14 ± 0.01
21
Verbenone
1222
1221
0.21 ± 0.02
0.14 ± 0.01
0.06 ± 0.00
0.23 ± 0.02
22
2-Allyl-4-methylphenolf
1232
—
—
0.42 ± 0.04
0.32 ± 0.05
23
Carvone
1245
1241
0.12 ± 0.01
0.10 ± 0.00
0.07 ± 0.00
0.12 ± 0.01
24
Piperitone
1262
0.44 ± 0.02
0.32 ± 0.04
0.09 ± 0.01
0.13 ± 0.02
25
Isothymolf
1272
—
—
0.05 ± 0.00
0.08 ± 0.00
26
Thymol
1313
1290
—
—
0.05 ± 0.01
0.11 ± 0.02
27
Piperitenone
1355
2.24 ± 0.00
1.45 ± 0.21
0.74 ± 0.08
0.54 ± 0.07
28
α-Cubebene
1348
1347
—
—
0.06 ± 0.02
0.06 ± 0.01
29
Piperitenone oxide
1370
2.92 ± 0.20
1.96 ± 0.18
11.63 ± 1.80
11.46 ± 0.89
30
α-Copaene
1383
1383
0.12 ± 0.02
0.07 ± 0.00
0.09 ± 0.01
0.08 ± 0.01
31
β-Elemene
1395
1393
0.56 ± 0.05
0.35 ± 0.04
0.77 ± 0.03
0.49 ± 0.02
32
Cinerolonef
1405
—
—
0.16 ± 0.02
0.10 ± 0.00
33
β-Caryophyllene
1431
1426
10.19 ± 1.50
6.98 ± 0.82
8.11 ± 0.63
8.82 ± 0.96
34
α-Bergamotenef
1437
1433
0.12 ± 0.02
0.19 ± 0.08
0.14 ± 0.05
0.24 ± 0.03
35
α-Caryophyllene
1468
1454
10.32 ± 1.53
6.98 ± 0.60
7.82 ± 0.71
8.87 ± 0.65
36
γ-Selinene
1485
0.27 ± 0.04
0.24 ± 0.03
0.34 ± 0.05
0.49 ± 0.02
37
β-Selinene
1500
1484
1.10 ± 0.13
0.97 ± 0.02
1.06 ± 0.01
1.27 ± 0.15
38
α-Selinene
1510
1494
1.67 ± 0.14
1.32 ± 0.10
1.49 ± 0.13
1.68 ± 0.22
39
α-Farnesene
1527
0.37 ± 0.05
0.36 ± 0.02
0.34 ± 0.03
0.95 ± 0.08
40
δ-Cadinene
1533
1524
0.49 ± 0.05
0.43 ± 0.02
0.49 ± 0.03
0.62 ± 0.06
41
2,5,9-Trimethyl-4,8-cycloundecadien-1-onef
1598
0.13 ± 0.01
0.06 ± 0.00
0.04 ± 0.00
0.08 ± 0.01
42
Caryophyllene oxide
1599
1582
3.50 ± 0.41
3.55 ± 0.22
1.15 ± 0.15
1.20 ± 0.08
43
Calarene epoxidef
1620
—
—
0.26 ± 0.04
0.47 ± 0.03
44
Tetradecanolf
1628
0.77 ± 0.02
0.86 ± 0.03
—
—
45
Humulene 1,2-epoxide
1638
3.01 ± 0.17
3.35 ± 0.36
1.06 ± 0.02
1.26 ± 0.14
46
Alloaromadendrene oxidef
1666
0.37 ± 0.04
0.43 ± 0.02
0.45 ± 0.30
1.05 ± 0.11
47
Himachalenef
1697
—
—
0.41 ± 0.03
0.75 ± 0.10
48
2-Methylene-6,8,8-trimethyl-tricyclo[5.2.2.0(1,6)]undecan-3-olf
1708
—
—
0.20 ± 0.01
0.29 ± 0.02
49
2-(4a,8-Dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-yl)-prop-2-en-1-olf
1720
—
—
0.12 ± 0.01
0.14 ± 0.01
Number of compounds
36
37
44
45
aThe retention indices (RIs) were calculated using a homologous series of n-alkanes (C8–C22).
bThe retention indices (RIs) were from the literature [34–41].
cResults expressed as mean ± standard deviation (n=3).
dThe samples from two regions of China: (A) Guangxi province and (B) Guangdong province.
eNot detected.
fIdentified tentatively for the first time as a volatile constituent in A. indianum (Lour.) Merr.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 ofA. 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[5.2.2.0(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 ofA. 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[5.2.2.0(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.Figure 1
Total ion current chromatograms of volatile components fromA. indianum (Lour.) Merr. obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.Figure 2
The five major volatile components ofA. indianum(Lour.) Merr. obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.The reports published concerning the composition of the extracts fromA. 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 [4] 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. [7] 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.


## 3.1. Essential Oil by Steam Distillation

The volatile profile of the essential oil extracted by SD fromA. 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.Table 1
The chemical compositions of volatile components fromA. indianum(Lour.) Merr. obtained by SD and HS-SPME from two regions of China.
Number
Composition
RIa
RIb
Relative content (%)c
SD
SPME
Ad
Bd
A
B
1
α-Thujene
929
930
0.05 ± 0.01
0.10 ± 0.03
0.05 ± 0.01
0.06 ± 0.02
2
α-Pinene
938
938
1.17 ± 0.12
0.92 ± 0.10
1.51 ± 0.15
1.35 ± 0.13
3
α-Fenchene
954
0.61 ± 0.07
0.57 ± 0.05
2.00 ± 0.20
0.89 ± 0.10
4
β-Pinene
989
980
—e
—
0.17 ± 0.02
0.14 ± 0.03
5
β-Myrcene
993
994
0.10 ± 0.00
0.08 ± 0.00
—
—
6
7-Octen-4-olf
1002
0.17 ± 0.01
0.07 ± 0.00
—
—
7
3-Carene
1009
1006
—
—
0.26 ± 0.30
0.24 ± 0.40
8
α-Terpinene
1021
1017
0.18 ± 0.20
0.19 ± 0.10
0.20 ± 0.30
0.16 ± 0.08
9
γ-Terpinene
1033
0.26 ± 0.10
0.33 ± 0.30
0.33 ± 0.15
0.54 ± 0.25
10
α-Limonene
1038
1030
20.59 ± 2.10
30.15 ± 3.00
26.07 ± 1.80
35.07 ± 4.38
11
β-Ocimene
1059
1050
0.14 ± 0.03
0.19 ± 0.20
0.17 ± 0.05
0.62 ± 0.30
12
Fenchone
1097
1088
31.81 ± 2.50
31.60 ± 3.80
26.44 ± 1.80
15.79 ± 1.95
13
β-Linalool
1104
1096
0.30 ± 0.04
0.22 ± 0.02
0.08 ± 0.00
0.14 ± 0.02
14
Fenchol
1121
1110
4.34 ± 0.50
3.88 ± 0.30
3.83 ± 0.25
1.50 ± 0.18
15
p-Mentha-2,8-dien-1-olf
1138
1123
—
0.25 ± 0.02
—
0.26 ± 0.31
16
Carveol
1152
0.16 ± 0.18
0.25 ± 0.38
—
—
17
Camphor
1154
1144
—
—
0.07 ± 0.00
0.07 ± 0.01
18
trans-Carveol
1201
0.22 ± 0.03
0.22 ± 0.01
0.06 ± 0.00
0.37 ± 0.05
19
p-Cymene-8-ol
1203
1187
0.48 ± 0.06
0.33 ± 0.02
0.32 ± 0.04
0.26 ± 0.01
20
α-Terpineol
1212
1193
0.35 ± 0.02
0.26 ± 0.03
0.13 ± 0.01
0.14 ± 0.01
21
Verbenone
1222
1221
0.21 ± 0.02
0.14 ± 0.01
0.06 ± 0.00
0.23 ± 0.02
22
2-Allyl-4-methylphenolf
1232
—
—
0.42 ± 0.04
0.32 ± 0.05
23
Carvone
1245
1241
0.12 ± 0.01
0.10 ± 0.00
0.07 ± 0.00
0.12 ± 0.01
24
Piperitone
1262
0.44 ± 0.02
0.32 ± 0.04
0.09 ± 0.01
0.13 ± 0.02
25
Isothymolf
1272
—
—
0.05 ± 0.00
0.08 ± 0.00
26
Thymol
1313
1290
—
—
0.05 ± 0.01
0.11 ± 0.02
27
Piperitenone
1355
2.24 ± 0.00
1.45 ± 0.21
0.74 ± 0.08
0.54 ± 0.07
28
α-Cubebene
1348
1347
—
—
0.06 ± 0.02
0.06 ± 0.01
29
Piperitenone oxide
1370
2.92 ± 0.20
1.96 ± 0.18
11.63 ± 1.80
11.46 ± 0.89
30
α-Copaene
1383
1383
0.12 ± 0.02
0.07 ± 0.00
0.09 ± 0.01
0.08 ± 0.01
31
β-Elemene
1395
1393
0.56 ± 0.05
0.35 ± 0.04
0.77 ± 0.03
0.49 ± 0.02
32
Cinerolonef
1405
—
—
0.16 ± 0.02
0.10 ± 0.00
33
β-Caryophyllene
1431
1426
10.19 ± 1.50
6.98 ± 0.82
8.11 ± 0.63
8.82 ± 0.96
34
α-Bergamotenef
1437
1433
0.12 ± 0.02
0.19 ± 0.08
0.14 ± 0.05
0.24 ± 0.03
35
α-Caryophyllene
1468
1454
10.32 ± 1.53
6.98 ± 0.60
7.82 ± 0.71
8.87 ± 0.65
36
γ-Selinene
1485
0.27 ± 0.04
0.24 ± 0.03
0.34 ± 0.05
0.49 ± 0.02
37
β-Selinene
1500
1484
1.10 ± 0.13
0.97 ± 0.02
1.06 ± 0.01
1.27 ± 0.15
38
α-Selinene
1510
1494
1.67 ± 0.14
1.32 ± 0.10
1.49 ± 0.13
1.68 ± 0.22
39
α-Farnesene
1527
0.37 ± 0.05
0.36 ± 0.02
0.34 ± 0.03
0.95 ± 0.08
40
δ-Cadinene
1533
1524
0.49 ± 0.05
0.43 ± 0.02
0.49 ± 0.03
0.62 ± 0.06
41
2,5,9-Trimethyl-4,8-cycloundecadien-1-onef
1598
0.13 ± 0.01
0.06 ± 0.00
0.04 ± 0.00
0.08 ± 0.01
42
Caryophyllene oxide
1599
1582
3.50 ± 0.41
3.55 ± 0.22
1.15 ± 0.15
1.20 ± 0.08
43
Calarene epoxidef
1620
—
—
0.26 ± 0.04
0.47 ± 0.03
44
Tetradecanolf
1628
0.77 ± 0.02
0.86 ± 0.03
—
—
45
Humulene 1,2-epoxide
1638
3.01 ± 0.17
3.35 ± 0.36
1.06 ± 0.02
1.26 ± 0.14
46
Alloaromadendrene oxidef
1666
0.37 ± 0.04
0.43 ± 0.02
0.45 ± 0.30
1.05 ± 0.11
47
Himachalenef
1697
—
—
0.41 ± 0.03
0.75 ± 0.10
48
2-Methylene-6,8,8-trimethyl-tricyclo[5.2.2.0(1,6)]undecan-3-olf
1708
—
—
0.20 ± 0.01
0.29 ± 0.02
49
2-(4a,8-Dimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-yl)-prop-2-en-1-olf
1720
—
—
0.12 ± 0.01
0.14 ± 0.01
Number of compounds
36
37
44
45
aThe retention indices (RIs) were calculated using a homologous series of n-alkanes (C8–C22).
bThe retention indices (RIs) were from the literature [34–41].
cResults expressed as mean ± standard deviation (n=3).
dThe samples from two regions of China: (A) Guangxi province and (B) Guangdong province.
eNot detected.
fIdentified tentatively for the first time as a volatile constituent in A. indianum (Lour.) Merr.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 ofA. 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[5.2.2.0(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 ofA. 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[5.2.2.0(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.Figure 1
Total ion current chromatograms of volatile components fromA. indianum (Lour.) Merr. obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.Figure 2
The five major volatile components ofA. indianum(Lour.) Merr. obtained by SD and HS-SPME from regions A (Guangxi province) and B (Guangdong province), respectively.The reports published concerning the composition of the extracts fromA. 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 [4] 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. [7] 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.


## 4. Conclusions

Analysis of the extracts by SD and HS-SPME indicated thatα-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.



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*Source: 545760-2013-10-09.xml*