International Journal of Analytical Chemistry
Volume 2020, Issue 1 4575030
Research Article
Open Access

Chemical Constituents and Anti-Inflammatory Effect of Incense Smoke from Agarwood Determined by GC-MS

De-Qian Peng, 

De-Qian Peng

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

School of Pharmacy, Hainan Medical University, Haikou 571199, China hainmc.edu.cn

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Zhang-Xin Yu, 

Zhang-Xin Yu

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

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Can-Hong Wang, 

Can-Hong Wang

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

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Bao Gong, 

Bao Gong

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

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Yang-Yang Liu, 

Yang-Yang Liu

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

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Jian-He Wei, 

Corresponding Author

Jian-He Wei

Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Haikou 570311, China cacms.ac.cn

National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China cacms.ac.cn

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First published: 01 August 2020
https://doi.org/10.1155/2020/4575030
Citations: 9
Academic Editor: Kevin Honeychurch

Abstract

Agarwood is generally used to make incense sticks in China and Southeast Asia. It emits smoke with a pleasant odor when burned. There are few reports on the chemical components of smoke generated by burning or heating agarwood. The agarwoods were produced by the whole-tree agarwood-inducing technique (AWIT), agarwood induced by axe wounds (AAW), burning-chisel-drilling agarwood (BCDA), wood of Aquilaria sinensis trees (AS), respectively. Herein, we used GC-MS to analyze the chemical constituents of incense smoke generated from AWIT, AAW, BCDA, AS, and the extracts of sticks from agarwood produced by the whole-tree agarwood-inducing technique (EAWIT), and 484 compounds were identified. A total of 61 chemical constituents were shared among AWIT, AAW, and BCDA. The experimental data showed that aromatic compounds were the main chemical constituents in agarwood smoke and that some chromone derivatives could be cracked into low-molecular-weight aromatic compounds (LACs) at high temperature. Furthermore, agarwood incense smoke showed anti-inflammatory activities by inhibiting lipopolysaccharide- (LPS-) induced TNF-α and IL-1α release in RAW264.7 cells.

1. Introduction

Agarwood, called chen-xiang in China, is a valuable resinous wood from Aquilaria spp. or Gyrinops spp. trees [1–3]. It has been applied in medicine and shown obvious medicinal effects, such as sedative, carminative, and antiemetic effects [4, 5]. Agarwood does not form until a tree has been affected by factors such as lightning strike, animal grazing, insect attack, and fungi [6, 7]. Moreover, it takes a long time (years or even decades) to form in the wild. Natural agarwood is considered to be the finest source of incense and has been applied in cultural, religious, and medicinal uses for centuries. The market demand for agarwood is increasing daily. As a result, the supply of wild agarwood is not enough to meet the market demand. Many Aquilaria plantations have been established in some Southeast Asian countries, such as Indonesia, Cambodia, Laos, Thailand, Vietnam, and Malaysia. Aquilaria trees have been planted in South China, for example, in Hainan, Guangdong, and Yunnan provinces [8]. Some artificial technologies designed to rapidly induce agarwood formation have been demonstrated to make A. sinensis (AS) trees produce agarwood [7, 9–11]. In 2009, Blanchette and Heuveling developed cultivated agarwood kits (CA-Kits) [12]. In 2013, Liu et al. developed a whole-tree agarwood-inducing technique (Agar-Wit) [11]. Recently, Peng et al. also developed a similar technology to induce agarwood formation [13]. The above methods induce agarwood formation simply and effectively.

Presently, agarwood and its volatile components are seen as important and efficient natural substances that can be used to produce valuable products such as perfumes and incense because of their fragrance characteristics. Many teams have researched the chemical constituents of agarwood [1, 14–16]. The chemical constituents of agarwood essential oil or solvent extracts have been studied by column chromatography, spectroscopic techniques, gas chromatography (GC), and multidimensional GC analysis. Many studies have reported the use of GC-MS to analyze the volatile components in agarwood smoke obtained by heating. For example, in 1993, Ishihara et al. analyzed the volatile constituents in agarwood smoke and identified 53 chemical compounds from Vietnamese agarwood [17]. Nurlaila et al. identified 8 significant compounds from agarwood smoke by Z-score analysis [18]. Recently, Zhou et al. used glass fiber pads to absorb volatile constituents of agarwood smoke from different kinds of agarwood from different countries and extracted the samples with dichloromethane (CH2Cl2) for GC-MS analysis [19, 20]. Kao et al. analyzed agarwood smoke from Kynam agarwood by headspace (HS) preheating with gas chromatography-mass spectrometry (HS GC-MS) and identified 40 compounds [21]. However, there are no reports on agarwood smoke produced by Agar-Wit. Herein, we analyzed the chemical constituents of agarwood smoke produced by Agar-Wit and identified 484 compounds.

2. Experimental Setup

2.1. Chemicals and Reagents

All chemicals were purchased from J&K Scientific (Beijing, China), unless otherwise indicated.

The agarwood samples were identified by Prof. Jianhe Wei (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, China). Incense sticks of AWIT, AAW, BCDA, and AS were made by Bao Gong (Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, China).

2.2. Sample Preparation

Each stick was placed in a gas washing bottle (250 mL) fitted with an air inlet/outlet tube. The smoke components were collected by bubbling through a 30 mL amount dichloromethane during the 10 min burn time. In addition, sticks made from AWIT (3.0 g) were pulverized and extracted with CH2Cl2 (30 mL).

2.3. Sample Analysis

Chromatographic separation of the resulting mixture (1.0 μL) was undertaken on an Agilent 7890 A GC coupled to a 5975C quadrupole mass spectrometer and an automated 7683B sample injector system (Agilent Technologies, Santa Clara, California, USA). Chromatography was performed on a HP-5MS capillary column (30 m × 250 μm ID, 0.25 μm film thickness, 5% diphenyl methyl siloxane (Agilent Technologies, USA). Helium was used as carrier gas at a constant flow rate of 1.0 mL/min. The injections (1.0 μL) were performed in splitless injection mode (10 : 1) at 240°C. The operating parameters included the following temperature program: 40°C for 3 min, increase from 40°C to 140°C at a rate of 2.5°C/min, hold at 140°C for 5 min, increase from 140°C to 170°C at a rate of 1.5°C/min, hold at 170°C for 5 min, and increase to 280°C at a rate of 4°C/min. The total run time was 100.5 min as shown in Figure 1. The mass selective detector was operated with electron energy of 70 eV in electron ionization mode. The ion source and quadrupole temperatures were 230°C and 150°C, respectively. The scan range was 40–500 amu in full scan mode. Peak identification was completed by comparing mass spectra with those stored in the NIST 11 database and MSD ChemStation, or by comparing fragmentation patterns with those published by the Dai group [22]. Table 1 shows the representative data.

Details are in the caption following the image
Figure 1
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Gas chromatograph mass spectrometry (GC-MS) chromatograms of incense smoke (AWIT: whole-tree agarwood-inducing technique, AAW: agarwood produced by axe wounds, BCDA: burning-chisel-drilling agarwood, EAWIT: extract of stick from agarwood produced by whole-tree agarwood-inducing technique, AS: A. sinensis).
Table 1. Chemical constituents and relative amounts of AAW, BCDA, AWIT, AS, and EAWIT.
No. Name RT (min) Relative content (%)
AAW BCDA AWIT AS EAWIT
1 Ethylbenzene 6.774 6.215 (B) 3.296 (B) 6.384 (B) 1.313 (B) —
2 Furfuryl alcohol 6.872 — — — 2.212 —
3 1,4-Xylene 7.080 3.709 (B) — — 1.765 (B) —
4 1,3-Xylene 7.086 — — 2.354 (B) — —
5 1,2-Xylene 7.098 — 1.326 (B) — 0.778 (B) —
6 Phenylacetylene 7.392 1.387 (B) 0.698 (B) 1.612 (B) 1.013 (B) —
7 Phenyl carbamate 7.646 — — — 0.308 (B) —
8 Phenylethylene 7.901 8.596 (B) 3.413 (B) 7.973 (B) 5.132 (B) —
9 2-Methyl-2-cyclopenten-1-one 8.680 — — — 0.400 —
10 4,4-Dimethyl-2-cyclopenten-1-one 8.952 — — — 0.302 —
11 2,5-Dimethyl-2,4-hexadiene 9.056 — — — 0.408 —
12 Anisole 9.096 0.239 (B) — 0.21 (B) — —
13 2(5H)-Furanone 9.200 0.202 0.132 0.299 0.981 —
14 3,4-Dihydro-H-pyran 9.356 0.202 0.202 — 0.381 —
15 Methyl-2-oxo-1-pyrrolidineacetate 9.605 — 0.222 — — —
16 Tetrahydro-22-desoxy-tomatillidine 9.610 — — — 0.211 —
17 1-Methylene-2-vinylcyclopentane 10.962 — — — 0.113 —
18 Benzaldehyde 11.118 0.671 (B) 0.28 (B) 1.008 (B) 1.146 (B) —
19 5-Methyl-furfural 11.407 — — — 0.508 —
20 2,3-Dihydroxystearic acid 11.875 — — — 0.209 —
21 2-Chloro-2,2-difluoro-acetonitrile 11.950 — — — 0.157 —
22 2-Methyl-2-pentenal 12.192 — — — 0.104 —
23 Benzonitrile 12.308 — — — 0.359 (B) —
24 2,2,4,6,6-Pentamethylheptane 12.510 0.902 0.753 1.152 — 0.126
25 Benzofuran 12.793 — — — 0.541 (B) —
26 Phenol 12.839 1.225 (B) 0.558 (B) 0.592 (B) 0.852 (B) —
27 2,2-Diethyl-3-methyl-oxazolidine 13.105 0.751 0.785 0.808 — —
28 (2S, 3S)-2,3-Dimethoxy-N1,N1,N1,N4-tetramethyl-1,4-butanediamine 13.111 — — — 0.846 —
29 5-Norbornene-2-carboxaldehyde 13.654 0.169 — 0.189 — —
30 4-Methylanisole 14.185 0.305 (B) 0.102 (B) 0.135 (B) 0.159 (B) —
31 2-Azido-2,4,4,6,6-pentamethylheptane 14.445 0.255 0.187 0.220 — —
32 2,3-Dioxabicyclo[2.2.2]oct-5-ene 14.671 0.424 0.189 0.175 — —
33 3-Methyl-1,2-cyclopentanedione 14.717 — — — 0.369 —
34 2-Methyl-3-furanthiol 15.918 0.106 0.201 — — —
35 3-Methyl-phenol 16.635 0.219 (B) 0.176 (B) 0.12 (B) — —
36 2-Methyl-phenol 16.669 — — — 0.226 (B) —
37 3-Methyl-bicyclo[3.3.0]oct-2-en-8-one 16.918 — — — 0.129 —
38 2,2,2-Bicyclo-2-octene 16.935 0.496 0.183 (B) 0.453 (B) — —
39 p-Cresol 17.801 0.73 (B) 0.473 (B) 0.321 (B) 0.205 (B) —
40 Guaiacol 17.998 0.877 (B) 0.51 (B) 0.608 (B) 1.362 (B) —
41 2-t-Butylamino-acrylonitrile 18.165 0.421 0.224 0.240 — —
42 3,5-Dimethyl-4H-pyran-4-one 18.182 — — — 0.107 —
43 3-Hydroxy-2-methyl-4-pyrone 19.338 — 0.169 — 0.147 —
44 2,5-Dimethylphenol 21.677 0.223 (B) 0.129 (B) — 0.165 (B) —
45 2,3-Dihydroxybenzaldehyde 22.064 — — — 0.148 (B) —
46 5-Ethyl-3-(3-methyl-5-phenylpyrazol-1-yl)-1,2,4-triazol-4-amine 22.215 0.152 (B) — — — —
47 Ethyl disulfide 22.220 — — 0.094 — —
48 1-Methylene-1H-indene, 22.740 0.166 (B) — — — —
49 2-Ethylphenol 22.913 0.129 (B) — — — —
50 Trehalose 22.914 — 0.324 — — —
51 2-Isopropyl-5-methyl-1-heptanol 23.197 — — 0.059 — —
52 3-Methyl-2-butene-1-thiol 23.208 0.120 — — — —
53 2-Methylbutyl pentanoic acid ester 23.225 — 0.251 — — —
54 4-Methoxy-1,3-benzenediamine 23.300 — — — 0.216 (B) —
55 2-Methoxy-3-methyl-phenol 23.306 0.236 (B) — — — —
56 2-Methoxy-4-methylphenol 23.676 0.444 (B) 0.283 (B) 0.271 (B) 0.309 (B) —
57 2-Methoxy-5-methylphenol 23.688 — 0.327 (B) — — —
58 3,6-Dimethyl-2,6-octadiene-4,5-diol 24.144 — — — 0.129 —
59 1,4 : 3,6-Dianhydro-α-d-glucopyranose 24.600 0.148 0.158 0.172 0.120 —
60 trans-Cinnamaldehyde 24.866 — — — 0.821 (B) —
61 (1α, 2β, 5β, 6α)-Tricyclo[4.2.1.1(2,5)]deca-3,7-diene-9,10-dione 24.883 0.446 0.325 — — —
62 α-Methylene-benzeneacetaldehyde 24.895 — — 0.247 (B) — —
63 2,4-Cyclopentadiene-1-ethanamine 25.045 0.128 — 0.230 — —
64 1,11-Dibromo-undecane 25.062 — 0.101 — — —
65 Pyrocatechol 25.357 0.125 (B) 0.287 — 0.93 (B) —
66 2,3-Anhydro-d-galactosan 25.357 — — 0.204 (B) — —
67 2,3-Dihydrobenzofuran 25.727 0.369 (B) 0.382 (B) 0.299 (B) 0.255 (B) —
68 1-Methyl-1H-pyrrole-2(5H)-one 26.131 — — 0.203 — —
69 2-Isopropoxyphenol 26.183 — — — 0.226 (B) —
70 3-Methoxyphenol 26.206 0.263 (B) — — — —
71 5-Hydroxymethylfurfural 26.229 — 0.252 — 0.389 —
72 4-Phenyl-2-butanone 26.339 0.77 (B) 1.13 (B) 1.06 (B) — 0.177 (B)
73 1-Methyl-4-amino-4,5(1H)-dihydro-1,2,4-triazole-5-one 26.847 — — — 0.259 —
74 Anisic aldehyde 26.905 0.306 (B) 0.301 (B) 0.389 (B) — —
75 2-Oxohexamethylenimine 27.032 — 0.185 — — —
76 3-Methoxy-2-benzenediol 27.454 0.401 (B) 0.365 (B) 0.251 (B) 0.582 (B) —
77 3-Methoxybenzenethiol 27.731 0.131 (B) — — — —
78 1-Indanone 28.043 0.279 (B) — — 0.148 (B) —
80 2-Isopropyl-3-methoxypyrazine 28.291 — — — 0.177 —
79 4-Ethyl-2-methoxyphenol 28.291 0.292 (B) 0.104 (B) 0.071 (B) — —
81 3-(4-Methylphenyl)-2-propenal 28.441 — — — 0.206 (B) —
82 2,3-Dihydro-2-methyl-1H-inden-1-one 28.447 0.223 — 0.056 (B) — —
83 α-Methylcinnamaldehyde 28.557 — — — 0.127 (B) —
84 2-Methylnaphthalene 28.621 0.285 (B) — — — —
85 1-Azabicyclo[2.2.2]octane-4-methanol 29.527 — 0.158 — — —
86 (E)-2,4,4,7-Tetramethyl-5,7-octadien-3-ol 29.666 — — — 0.179 —
87 4-Hydroxy-3-methoxystyrene 30.099 2.343 (B) 1.799 (B) 1.512 (B) 2.254 (B) —
88 o-tert-Butyl phenol 30.417 — — — 0.131 (B) —
89 3-Hydroxybenzaldehyde 30.844 — — — 0.252 (B) —
90 2-Methoxybenzyl alcohol 31.081 — — — 0.106 (B) —
91 trans-3-Hexenedioic acid-bis(trimethylsilyl) ester 31.411 0.127 — — — —
92 2-exo-Chlorobicyclo[2.2.1]heptane-1-carbonyl chloride 31.942 0.282 — 0.285 — —
93 2-Ethyl-1H-pyrrolo[2,3-b]pyridine 32.000 — — — 0.157 —
94 2,6-Dimethoxyphenol 32.115 3.013 (B) 2.939 (B) 2.178 (B) — —
95 cis-4,5-Diethyl-1,2-dimethyl-cyclohexene 32.190 — — — 4.085 —
96 Eugenol 32.358 0.517 (B) 0.303 (B) 0.262 (B) — —
97 3-Allyl-6-methoxyphenol 32.375 — — — 0.413 (B) —
98 3,4-Dimethoxyphenol 32.566 — 0.112 (B) — 0.219 (B) —
99 3-Ethenyl-4-methyl-1H-pyrrole-2,5-dione 32.849 0.205 — — 0.149 —
100 3-Cyclohexene-1-acetaldehyde 33.247 0.191 — 0.136 — —
101 11-Methylene-tricyclo[4.3.1.1(2,5)]undecane 33.247 — — — — —
102 2-Propyl-phenol 33.848 — — — 0.1 (B) —
103 4-Hydroxybenzaldehyde 33.975 — — — 0.225 (B) —
104 Dichlorophenylsilane 34.010 — — 0.321 (B) — —
105 Phenylboronic acid 34.016 0.406 (B) 0.143 (B) — — —
106 Vanillin 34.408 0.8 (B) 0.83 (B) 0.708 (B) 1.842 (B) —
107 4-(Methylthio)-benzaldehyde 34.593 0.254 (B) — — — —
108 (E)-isoeugenol 34.899 0.341 (B) 0.18 (B) 0.133 (B) 0.31 (B) —
109 o-Methoxy-benzenethiol 35.084 0.125 (B) — — 0.197 (B) —
110 2-Methoxy-1,4-benzenediol 35.217 — — — 0.209 (B) —
111 2-Benzylidenemalonaldehyde 35.217 0.204 (B) — — — —
112 2-Vinylnaphthalene 35.442 0.115 (B) — — — —
113 3-Hydroxy-2-methyl-5-(1-methylethyl)-2,5-cyclohexadiene-1,4-dione 36.083 — — — 0.237 —
114 Biphenylene 36.234 0.105 (B) — — — —
115 2-Methoxy-4-(1-propen-1-yl)-phenol 36.892 2.962 (B) 2.126 (B) 2.379 (B) 3.254 (B) —
116 4-Hydroxy-2-methoxybenzaldehyde 37.227 0.243 0.142 — 0.135 (B) —
117 2-Methoxy-4-propyl-phenol 37.429 0.268 (B) 0.242 (B) 0.257 (B) 0.454 (B) —
118 1,7-Dimethylpentacyclo[5.5.0(4,11).0(5,9).0(8,12)]dodecane-2,6-dione 37.689 — — 0.126 (B) — —
119 2-Methoxy-6-[(4H-1,2,4-triazol-4-ylamino)methyl]-phenol 37.689 0.107 — — — —
120 4-Hydroxybenzylidene acetone 37.707 — 0.101 — — —
121 N-Phenylthioformamide 37.712 — — — 0.126 (B) —
122 7-Ethylbenzo[b]thiophene 38.053 — — — 0.153 —
123 5,6-Dimethyl-2-benzimidazolinone 38.382 — — — 0.164 (B) —
124 Cyclohexylmethylbenzene 38.440 0.145 (B) — 0.161 (B) — —
125 3,4-Dimethoxy-benzaldehyde 38.573 — — — 0.712 (B) —
126 4′-(Methylthio)acetophenone 38.660 0.370 0.298 (B) 0.256 (B) — —
127 4-(4-Methoxyphenyl)-2-butanone 39.232 1.067 (B) 1.161 (B) 1.124 (B) 0.137 —
128 Pentadecane 39.434 — — 0.168 — —
129 Dibenzofuran 39.451 0.186 (B) 0.145 (B) — — —
130 2′,6′-Dihydroxyacetophenone, bis(trimethylsilyl) ether 39.850 0.207 (B) — — — —
131 1-Methyl-1-phenylmethoxy-1-silacyclohexane 39.873 — 0.104 (B) 0.111 (B) — —
132 1,3,3-Trimethyl-2-(1-methylbut-1-en-3-on-1-yl)-1-cyclohexene 40.173 0.273 — — — —
133 1,2-Dimethoxy-4-(methoxymethyl)benzene 40.196 — 0.178 (B) — — —
134 2,4-Di-tert-butylphenol 40.202 — — — 0.287 (B) —
135 2-(2-Hydroxyhex-1-enyl)-3-methyl-5,6-dihydropyrazine 40.260 0.138 — — — —
136 5-(1,1-Dimethylethyl)-1,2,3-benzenetriol 40.653 0.578 (B) 0.34 (B) 0.212 (B) 0.684 (B) —
137 Homovanillyl alcohol 40.780 0.594 (B) 0.507 (B) 0.417 (B) 1.119 (B) —
138 [4-(1,1-Dimethylethyl)phenoxy]-acetate-methanol 41.912 — — — — 0.159 (B)
139 (S)-4,5,6,7,8,8a-Hexahydro-8aα-methylazulen-2(1H)-one 42.137 0.109 — — — —
140 Acetic acid-2-propylphenyl ester 42.160 — 0.114 (B) — — —
141 3-Nitrobenzaldehyde-(O-methyl oxime) 42.420 — — — — —
142 2,3,5,6-Tetrafluoroanisole 42.443 4.532 (B) 3.741 (B) 4.13 (B) — —
143 3-tert-Butyl-4-hydroxyanisole 42.547 — — — 5.641 (B) —
144 2,5-Dihydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one 42.819 — — — 0.329 —
145 α-Santalol 42.848 — — — — 0.511 (S)
146 2,3-Dihydro-2,2-dimethyl-3,7-benzofurandiol 42.888 0.359 (B) 0.39 (B) 0.394 (B) — —
147 7-(1,1-Dimethylethyl)-3,4-dihydro-1(2H)-naphthalenone 43.119 — — — — 0.105 (B)
148 3-Ethoxy-4-methoxybenzaldehyde 43.183 0.324 (B) — 0.221 (B) — —
149 3-Hydroxy-4-methoxybenzoic acid-methyl ester 43.200 — 0.318 (B) — — —
150 Ethyl vanillate 43.211 — — — 0.279 (B) —
151 α-Amino-3′-hydroxy-4′-methoxyacetophenone 43.443 0.126 (B) 0.143 (B) — — —
152 (3S, 4R, 5R, 6R)-4,5-Bis(hydroxymethyl)-3,6-dimethylcyclohexene 43.610 — — 0.202 — —
153 Carbonic acid-2,3-dimethylphenyl methyl ester 43.628 0.41 (B) — — — —
154 3-(4-Methoxyphenyl)propionic Acid 43.656 — 0.431 (B) — — —
155 Hexadecane 44.078 0.329 0.351 0.321 0.127 —
156 2,6-Dimethoxy-4-(2-propen-1-yl)-phenol 44.274 0.785 (B) 0.619 (B) 0.521 (B) 1.029 (B) —
157 2,6-Dimethyl-4-nitrophenol 44.586 0.256 (B) — — — —
158 [1S-(1α, 4α, 7α)]-1,2,3,4,5,6,7,8-Octahydro-1,4,9,9-tetramethyl-4,7-methanoazulene 44.592 — — — — 0.406 (S)
159 2-Ethyl-4-methyl-4,6-bis(1-methylethyl)-4H-1,3,2-dioxaborin 44.592 — — — 0.139 —
160 8-Epi-γ-eudesmol 44.633 — 0.261 (S) 0.219 (S) — —
161 Methyl-2,6,6-trimethyl-3-oxo-1-cyclohexene-1-acrylate 44.737 — — 0.155 — —
162 2′,6′-Dimethylacetanilide 44.771 — 0.187 — — —
163 [1S-(1α, 4aβ, 8aα)]-1,2,4a,5,8,8a-Hexahydro-4,7-dimethyl-1-(1-methylethyl)-naphthalene 45.152 — — — — 0.141 (S)
164 [1R-(1α, 3aα, 7aα)]-1,2,3,6,7,7a-Hexahydro-2,2,4,7a-tetramethyl-1,3a-ethano-3aH-indene 45.326 — 0.241 (B) — — 0.149 (S)
165 Agarospirol 45.551 — 0.238 (S) 0.195 (S) — 0.413 (S)
166 Methyl 3-(bicyclo[2.2.1]hept-1-yl)-propenoate 45.586 — — — 0.116 —
167 Hinesol 45.719 0.126 — — — 0.251 (S)
168 (1α, 6α, 7α)-1,5,5-Trimethyl-2-methylene-bicyclo[4.1.0]heptane-7-methanol 45.736 0.121 (S) — — — —
169 (1R, 3aR, 4R, 7R)-1,2,3,3a,4,5,6,7-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-azulene 45.748 — — 0.191 (S) — —
170 [1S-(1α, 4α, 7α)]-1,2,3,4,5,6,7,8-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-azule 45.776 — 0.222 (B) — — —
171 Longifolene 45.990 — — — — 0.565 (S)
172 10S,11S-Himachala-3(12),4-diene 46.007 0.155 (S) — — — —
173 Neoisolongifolene 46.042 — 0.262 (S) 0.248 (S) — —
174 Ledol 46.279 — — 0.219 (S) — 0.519 (S)
175 β-Eudesmol 46.325 0.150 0.343 (S) — — —
176 Guaiol 46.498 — — 0.939 (S) — —
177 γ-Selinene 46.504 0.589 (S) 1.164 (S) — — 1.868 (S)
178 1-Bromooctadecane 46.712 — — — — —
179 7,9-Dimethyl-hexadecane 46.729 0.169 — 0.219 — —
180 (4-Methoxyphenyl)glycolic acid 46.752 — — — 0.212 —
181 3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid 46.880 0.629 (B) 0.509 (B) 0.517 (B) 0.814 (B) —
182 Dehydroaromadendrene 47.301 — — — — 0.442 (S)
183 3,5-Dimethoxy-4-hydroxybenzaldehyde 47.353 1.599 (B) 1.78 (B) 1.891 (B) 2.92 (B) —
184 2,4,6-Trimethyl-pyridine 47.428 — — — — 0.383
185 Camphene 47.561 — — — — 0.600
186 Ethyl (3-pyridyl)carbamate N-oxide 47.608 — 0.753 — — —
187 Hexamethyl-benzene 47.654 0.503 — 0.646 (B) — —
188 1-(1,3a,4,5,6,7-Hexahydro-4-hydroxy-3,8-dimethyl-5-azulenyl)-ethanone 47.896 — — — — 0.142 (S)
189 2-Allyl-1,4-dimethoxy-3-methyl-benzene 48.029 0.249 (B) — 0.33 (B) — —
190 2,5-Dibutyl-furan 48.150 — — — 1.392 —
191 Vanillylacetone 48.208 1.036 (B) 0.967 (B) 0.425 (B) — —
192 [1S-(1α, 7α, 8aβ)]-1,2,3,5,6,7,8,8a-Octahydro-1,4-dimethyl-7-(1-methylethenyl)-azulene 48.480 — — — — 0.55 (S)
193 Dehydro-cyclolongifolene oxide 48.509 — 0.717 (S) 0.473 (B) — —
194 4-Methoxymethyl-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-ylamine 48.572 — — — 0.283 —
195 1-Cyclohexyl-2-methoxy-benzene 48.601 — — 0.384 (B) — 0.72 (B)
196 N,N-Diethyl-2-benzoxazolamine 48.613 0.781 (B) — — — —
197 Octahydro-2-(1-methylethylidene)-4,7-methano-1H-indene 49.161 — — — — 0.361 (B)
198 4,6,6-Trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo[5.1.0.0(2,4)]octane 49.167 — — 0.172 — —
199 4-Methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-cycloheptane 49.219 0.121 0.181 — — —
200 3-Phenoxy-phenol 49.467 0.325 0.154 (B) 0.134 (B) 0.248 (B) —
201 (Z)-3,7-Dimethyl-1,3,6-octatriene 49.537 — — — — 0.406
202 1,7-Dimethyl-7-(4-methyl-3-pentenyl)-tricyclo[2.2.1.0(2,6)]heptane 49.612 — 0.2 (S) 0.192 (S) — —
203 2-Acetate-1,3-dimethoxy-5-(1-propenyl)-benzene 50.057 3.206 (B) 2.309 (B) — — —
204 2,5-Dimethoxyterephthalic acid 50.178 — — 2.516 (B) 3.780 —
205 2-(2-Furanylmethylene)-6-methyl-cyclohexanone 50.270 — — — — 0.255
206 4-Propylbiphenyl 50.380 0.704 (B) 0.711 (B) — — —
207 1-Ethyl-3-(phenylmethyl)-benzene 50.386 — — 0.745 (B) — —
208 N,N,S-Trimethyl-3-aminothiophenol 50.415 — — — 0.684 (B) —
209 Neocurdione 50.438 — — — — 0.161 (S)
210 endo-Borneol 51.056 — — — — 0.611
211 9-Fluorenone 51.073 0.205 (B) — — — —
212 [1S-(1α, 3aβ, 4α, 8aβ, 9R∗)]-Decahydro-4,8,8-trimethyl-1,4-methanoazulene-9-methanol 51.108 — 0.488 (S) 0.295 (S) — —
213 Methyl α-hydroxy-4-methoxy-benzeneacetate 51.605 — — — 0.101 (B) —
214 2,2′-Methylenebis[5-methyl-furan 51.743 — — — — 0.136
215 4-Hydroxy-2-methoxycinnamaldehyde 51.882 0.937 (B) 0.897 (B) 0.78 (B) — —
216 3-(4-Hydroxy-3-methoxyphenyl)-2-propenal 52.072 — — — 2.664 (B) —
217 Acetosyringone 52.096 0.967 (B) 0.829 (B) 0.88 (B) 1.499 (B) —
218 syn-3,3,5,6,8,8-Hexamethyl-tricyclo[5.1.0.0(2,4)]oct-5-ene, 52.333 1.530 1.979 1.762 — 0.624
219 2-Phenylethyl-1,1,2,2-d4-amine 52.541 — — 1.184 (B) — —
220 2-Methyl-5-(1-methylethyl)-phenol 52.552 — — — — 1.377 (B)
221 2′-Hydroxy-3,3-dimethyl-3-phenylpropanal 52.558 0.762 (B) — — — —
222 2-[3-Methoxyphenyl]-propionic acid 52.581 — 1.048 (B) — — —
223 3-(2-Pentenyl)-1,2,4-cyclopentanetrione 52.679 — — — 6.688 (B) —
224 7-(1,3-Dimethylbuta-1,3-dienyl)-1,6,6-trimethyl-3,8-dioxatricyclo[5.1.0.0(2,4)]octane 52.951 0.340 0.621 0.732 — 0.491
225 o-Mentha-1(7),8-dien-3-ol 53.262 — — — — 0.206
226 1,10b(2H)-Dihydropyrano[3,4,5-jk]fluorene 53.488 0.153 (B) — — — —
227 Anthracene 53.499 — — — 0.124 (B) —
228 (1S, 6R, 9S)-5,5,9,10-Tetramethyltricyclo[7.3.0.0(1,6)]dodec-10(11)-ene 53.609 0.121 0.457 0.414 — —
229 (Z)-3-Methyl-2-(2,4-pentadienyl)-2-cyclopenten-1-one 53.632 — — — — 0.559
230 1-Methoxy-4-methyl-2-(1-methylethyl)-benzene 54.019 — — — — 0.565 (B)
231 (1Z, 3aα, 7aβ)-1H-1-Ethylideneoctahydro-7a-methyl-indene 54.089 0.466 (B) 0.773 (B) 0.531 (B) — —
232 3-(Phenylmethoxy)-1-propanol 54.233 — — — — 0.238 (B)
233 trans-1,10-Dimethyl-2-methylene-decalin 54.510 — — — — 0.113
234 3,5-Dimethoxy-4-hydroxyphenylacetic acid 54.649 1.383 (B) 1.359 (B) 1.391 (B) 2.15 (B) —
235 (1aR, 4S, 4aR, 7S, 7aR, 7bS)-Decahydro-1,1,4,7-tetramethyl-1H-cycloprop[e]azulene 54.672 — — — — 0.177 (S)
236 2,7-Dimethyl-5-(1-methylethenyl)-1,8-nonadiene 55.036 — — — — 0.126
237 Longifolenaldehyde 55.261 — 0.12 (S) — — —
238 (2R-cis)-1,2,3,4,4a,5,6,7-Octahydro-α,α,4a,8-tetramethyl-2-naphthalenemethanol 55.377 — — — — 0.409 (B)
239 (4aR, 5S)-4,4a,5,6,7,8-Hexahydro-4a,5-dimethyl-3-(1-methylethylidene)-2(3H)-naphthalenone 55.850 0.459 (S) 0.535 (S) 0.609 (S) — 0.611 (S)
240 1,2,4-Triethyl-benzene 56.130 — — — — 0.301 (B)
241 Isoaromadendrene epoxide 56.145 0.114 (S) — 0.143 (S) — —
242 2,3,4,5-Tetramethyl-tricyclo[3.2.1.02,7]oct-3-ene 56.405 — — — — 0.659
243 Globulol 56.584 — — — — 0.344 (S)
244 Octadecane 56.624 0.285 0.331 0.393 0.179 —
245 2,3-Dihydro-2,2-dimethyl-7-benzofuranol 56.803 — — — — 0.258 (B)
246 1β, 2α-Dimethyl-3α, 5β-bis(1-methylethenyl)cyclohexane 56.844 — — 0.285 — —
247 4,6-Dimethoxy-1-naphthaldehyde 56.896 0.214 (B) — — — —
248 2-Bromo-1,3-dimethoxy-benzene 56.919 — 0.305 (B) — — —
249 1,3,5-Triethyl-benzene 57.040 — — — — 0.252 (B)
250 7-Methyl-pentadecane 57.144 — — 0.189 — —
251 2-Methyl-dodecane 57.150 0.154 — — — —
252 Corymbolone 57.225 — — — — 0.164 (S)
253 2,6,10,14-tetramethyl-Hexadecane 57.225 0.130 0.285 0.151 0.298 —
254 1-(2-Thienyl)-1-heptanone 57.520 — — 0.500 — —
255 1-(2-Thienyl)-1-hexanone 57.543 0.391 — — — —
256 2,4-Dimethylcyclopentane-1,3-dione 57.601 — 0.475 — — —
257 1-(2-Thienyl)-ethanone 57.612 — — — — 1.207
258 1-(2,6-Dihydroxy-4-methoxyphenyl)-ethanone 57.716 — — — 0.162 (B) —
259 5-(2-Thienyl)-4-pyrimidinamine 57.832 1.086 1.513 2.402 — —
260 1,3-Benzenedicarboxylic acid-4-methyl-1,3-dimethyl ester 57.941 — — — — 5.197 (B)
261 3,4-Dimethoxy-benzaldehyde oxime 58.051 — — 0.196 (B) — —
262 1,2-Dimethoxy-4-(1,2-dimethoxyethyl)benzene 58.063 0.295 (B) 0.233 (B) — — —
263 5-Methoxy-[1,2,4]triazolo[4,3-a]pyridine-3-thiol 58.149 — — — 0.335 —
264 Methyl-3-amino-4-methoxybenzoate 58.248 0.192 (B) 0.304 (B) 0.29 (B) — —
265 Isomaltol 58.271 — — — — 0.623
266 9-Methyl-9-azabicyclo[4.2.1]nona-2,4-diene 58.721 0.103 — — — 0.970
267 Chlordimeform 58.791 — 0.309 (B) — — —
268 Nootkatone 59.229 — — — — 0.118 (S)
269 4-(3-Methyl-2-butenyl)-phenol 59.541 — — — — 0.772 (B)
270 [2R-(2α, 4aα, 8aβ)]-1,2,3,4,4a,5,6,8a-Octahydro-4a,8-dimethyl-2-(1-methylethenyl)-naphthalene 59.663 — 0.125 (S) — — —
271 [1R-(1R∗, 4Z, 9S∗)]-4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene 59.952 — — — — 0.193 (S)
272 2,3,4,5,6-Pentamethyl-benzoic acid 60.200 0.221 (B) 0.151 (B) — — —
273 [1aR-(1aα, 7α, 7aα, 7bα)]-1a,2,3,5,6,7,7a,7b-Octahydro-1,1,7,7a-tetramethyl-1H-cyclopropa[a]naphthalene 60.206 — — — — 0.167 (S)
274 4,5-Dihydro-6-(4-fluorophenyl)-pyridazin-3(2H)-one 60.235 — — — — —
275 N(1)-[(3-Methoxyphenyl)methyl]-1H-1,2,3,4-tetrazole-1,5-diamine 60.367 — — 0.17 (B) — —
276 1-Ethenyl-1-methyl-2-(1-methylethenyl)-4-(1-methylethylidene)-cyclohexane 60.396 — — — — 0.601 (S)
277 (E,E)-1,5-Dimethyl-8-(1-methylethylidene)-1,5-cyclodecadiene 60.443 — 0.165 (S) — — —
278 6,7-Dimethyl-8-(1-methylethyl)-2,4(1H,3H)-pteridinedione 60.708 — 0.105 — — —
279 trans-Z-α-Bisabolene epoxide 60.737 — — — — 0.42 (S)
280 Aromadendrene oxide-(1) 61.066 — — 0.814 (S) — —
281 Cedrol 61.072 0.376 (S) — — — —
282 1-Hydroxy-6-(3-isopropenyl-cycloprop-1-enyl)-6-methyl-heptan-2-one 61.153 — 1.008 — — —
283 (1R, 7R, 8aS)-1,2,3,5,6,7,8,8a-Octahydro-1,8a-dimethyl-7-(1-methylethenyl)-naphthalene 61.170 — — — — 2.925 (S)
284 α-Farnesene 61.546 — — — — 0.429
285 2-Methylene-6,8,8-trimethyl-tricyclo[5.2.2.0(1,6)]undecan-3-ol 61.592 — 0.156 — — —
286 Thiocyanic acid-4-(dimethylamino)phenyl ester 61.702 — — — — 0.354
287 [4aR-(4aα, 7α, 8aβ)]-Decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-naphthalene 61.892 — — — — 0.198 (S)
288 Decahydro-2,2,4,8-tetramethyl-4,8-methanoazulen-9-ol stereoisomer 62.060 — — — — 0.306 (S)
289 Diphenylmethane 62.551 — — — 0.27 (B) —
290 [1aR-(1aα, 4β, 4aβ, 7α, 7aβ, 7bα)]-Decahydro-1,1,4,7-tetramethyl-1H-cycloprop[e]azulen-4-ol 62.568 — — — — 0.205 (S)
291 Spiro(tricyclo[6.2.1.0(2,7)]undeca-2,4,6,9-tetraene-11,1′-cyclopropane 62.568 0.198 (B) — — — —
292 4-Methyl-1-[2,6,6-trimethyl-2-cyclohexen-1-yl]-1-penten-3-one, 62.603 — 0.149 — — —
293 [1S-(1α, 7α, 8aα)]-1,2,3,5,6,7,8,8a-Octahydro-1,8a-dimethyl-7-(1-methylethenyl)-naphthalene 62.788 — — — — 0.248 (S)
294 Caryophyllene 63.007 — — — — 0.172 (S)
295 Nonadecane 63.059 0.129 0.134 0.125 0.145 —
296 7-Methoxy-3,4-dihydro-2[1H]-quinoxalinone 63.233 0.241 (B) 0.27 (B) 0.367 (B) — —
297 2-Allyl-1,4-dimethoxybenzene 63.296 — — — — 1.347 (B)
298 Dehydroxy-isocalamendiol 63.493 — — 0.241 (S) — —
299 3,4,5,6-Tetramethyl-2,5-octadiene 63.504 0.133 (S) — — — —
300 Megastigmatrienone 63.539 — — — — 0.914
301 6-(1-Hydroxymethylvinyl)-4,8a-dimethyl-3,5,6,7,8,8a-hexahydro-1H-naphthalen-2-one 63.573 — 0.286 (B) — — —
302 α-Ethyl-benzeneacetamide 63.729 — — — — 0.442 (B)
303 8-Ethenyl-3,4,4a,5,6,7,8,8a-octahydro-5-methylene-2-naphthalenecarboxylic acid 63.747 — 0.175 (B) — — —
304 [1S-(1α, 2β, 4β)]-1-Ethenyl-1-methyl-2,4-bis(1-methylethenyl)-cyclohexane 63.989 — — — — 0.666 (S)
305 1-Methylphenazine 5-oxide 63.989 — — — 0.945 (B) —
306 3-Phenylbicyclo(3.2.2)nona-3,6-dien-2-one 63.995 — — 0.217 (B) — —
307 1-(1-Hydroxybutyl)-2,5-dimethoxybenzene 64.035 0.423 (B) 0.49 (B) — — —
308 Methyleugenol 64.538 0.123 (B) 0.315 (B) 0.23 (B) — —
309 2-Ethyl-3,4-dihydro-2H-1-benzothiopyran 64.630 — — — — 1.177 (B)
310 Acetic acid-cyano-hydroxyimino-methyl ester 65.006 — — — — 0.484
311 N-Dimethylaminomethylene-anthranilic acid 65.012 0.252 (S) 0.26 (S) 0.165 (S) — —
312 2-Methyl-9-(prop-1-en-3-ol-2-yl)-bicyclo[4.4.0]dec-2-ene-4-ol 65.243 — — 1.25 (S) — —
313 (1aR, 4aR, 7R, 7aR, 7bS)-Decahydro-1,1,7-trimethyl-4-methylene-1H-cycloprop[e]azulene 65.254 0.514 (B) 1.064 (B) — — —
314 [4aR-(4aα, 5α, 8aα)]-4a,5,6,7,8,8a-Hexahydro-3,4a,5-trimethyl-naphtho[2,3-b]furan-9(4H)-one 65.381 — — — — 4.963 (S)
315 7,7,8,8-Tetracyanoquinodimethane 65.716 0.137 (B) — — — —
316 4-(1,3,3-Trimethyl-bicyclo[4.1.0]hept-2-yl)-but-3-en-2-one 65.780 — 0.233 — — —
317 Alloaromadendrene oxide 65.820 — — — — 0.543 (S)
318 γ-Elemene 66.207 — — — — 0.835 (S)
319 1-(2,4,6-Trimethylphenyl)-3-(2-propynyl)-thiourea 66.698 0.68 (B) 1.52 (B) 1.68 (B) — 5.466 (B)
320 3,4-Dimethylphenyl trifluoro-acetate 66.883 — — 0.209 (B) — —
321 12-Azabicyclo[9.2.2]pentadeca-1(13),11,14-trien-13-ylamine 66.901 0.205 0.276 — — 1.177
322 1-Butyl-1H-pyrrole 67.062 0.213 0.288 0.174 — —
323 (3aα, 8β, 8aα)-5,6-1,2,3,3a,8,8a-Hexahydro-2,2,8-trimethyl-azulenedimethanol 67.259 — — — — 0.631 (S)
324 (2R, 5S, 10R)-6,10-Dimethyl-2-(1-methylethenyl)-spiro[4.5]dec-6-en-8-one 67.386 0.326 (S) — — — —
325 N-Salicylidene-N′-salicyloylhydrazine 67.496 — 0.597 (B) — — —
326 3,5-Dimethyl-benzenamine 67.507 — — — — 0.633 (B)
327 2-Allyl-3-ethoxy-4-methoxyphenol 67.709 — — 1.709 (B) — —
328 1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene 67.732 1.572 (B) — — — —
329 Levomenol 67.778 — — — — 0.305 (S)
330 n-Hexadecanoic acid 67.807 — — — 1.701 —
331 2,2,8,8-Tetramethyl-3,6-nonadien-5-one 67.958 — — — — 0.428
332 3,5-Dimethoxy-4-hydroxycinnamaldehyde 68.119 0.123 (B) 1.723 (B) — 3.472 (B) —
333 1-Hydroxy-6-methylphenazine 68.529 1.19 (B) 1.897 (B) 1.555 (B) — —
334 Desaspidinol 68.847 — — — 3.559 (B) —
335 2-Chloro-4-cyclohexyl-phenol 68.882 0.177 (B) — 0.302 (B) — —
336 5-Ethyl-1,2,3,4-tetrahydro-naphthalene 68.899 — — — — 0.779 (B)
337 Heptadecane 69.361 — — — 0.195 —
338 Humulane-1,6-dien-3-ol 69.384 — — — — 0.784 (S)
339 8,8-Dimethyl-9-methylene-1,5-cycloundecadiene 69.777 — — — — 0.465 (S)
340 2,4-Dichloro-1-nitrobenzene 70.083 — — — — 0.429 (B)
341 1-(2-Benzyloxyethyl)cyclohexene 70.453 — — 0.369 (B) — —
342 Caryophyllene oxide 70.476 — — — — 2.082 (S)
343 Aromadendrene oxide-(2) 70.476 0.272 (S) — — — —
344 Diepicedrene-1-oxide 70.545 — 0.368 (S) — — —
345 3,4-Dihydro-3,3,6,8-tetramethylnaphthalen-1(2H)-one 70.580 — — — 0.115 (B) —
346 1-Methyl-2,4-bis(1-methylethenyl)-cyclohexane 71.031 — — — — 0.248 (S)
347 [1aR-(1aα, 4aα, 7β, 7aβ, 7bα)]-Decahydro-1,1,7-trimethyl-4-methylene-1H-cycloprop[e]azulen-7-ol 71.140 — — — — 0.512 (S)
348 3-Hydroxy-2-methyl-4-[4-t-butyl]-butanal 72.481 0.242 — — — —
349 1-(1-Hydroxy-3-methoxy-2-naphthyl)ethanone 72.498 — — — — 1.586 (B)
350 2-(Butenyl)-5-(1,1-dimethylethyl)-1,3-dimethyl-benzene 72.544 — 0.346 (B) — — —
351 (1R, 2R, 6S, 7S, 8S) -1-Methyl-8-(1-methylethyl)-tricyclo[4.4.0.02,7]dec-3-ene-3-methanol 72.885 — — 0.359 (S) — —
352 4-Hydroxy-4a,5-dimethyl-3-methylene-3a,4,4a,5,6,7,9,9a-octahydro-3H-naphtho[2,3-b]furan-2-one 72.896 0.194 (B) — — — —
353 1,2,3,4-Tetrahydro-6-nitronaphthalene 72.931 — — — — 0.888 (B)
354 1-(3,3-Dimethyl-1-yl)-2,2-dimethylcyclopropane-3-carboxylic acid 72.960 — 0.324 — — —
355 N-(p-Methoxy-trans-styryl)-formamide 73.272 — — — — 0.546 (B)
356 8,9-Dehydro-9-formyl-cycloisolongifolene 73.705 0.548 (S) — 0.691 (S) — —
357 2-tert-Butyl-quinoxaline 4-oxide 73.745 — — — — 1.237 (B)
358 β-Vatirenene 73.763 — 0.803 (S) — — —
359 Octadecanal 73.919 — — — 0.144 —
360 [1S-(1α, 3aβ, 4α, 7aβ)]-Octahydro-1,7a-dimethyl-4-(1-methylethenyl)-1,4-methano-1H-indene 74.872 — — — — 0.368 (S)
361 1-(1-Hydroxyethyl)-1-(diethylphosphonyl)-2-methylene-cyclopropane 74.878 — 0.109 — — —
362 1-Nonadecene 75.265 — — — 0.149 —
363 (E,E)-3,7-Dimethyl-10-(1-methylethylidene)-3,7-cyclodecadien-1-one 75.692 — — — — 0.232 (S)
364 Alloaromadendrene 76.195 — — — — 0.488 (S)
365 Heneicosane 76.235 — 0.148 0.177 0.127 —
366 2,3-Dihydro-7-hydroxy-2,2-dimethyl-4H-1-benzopyran-4-one 76.859 — — — — 0.277 (B)
367 2-Butyl-5-hexyloctahydro-1H-indene 77.344 — — — — 0.253
368 2,2′ : 5′,2″-Terthiophene 77.777 — — — — 0.228
369 1-Methyl-4-(2-methyloxiranyl)-7-oxabicyclo[4.1.0]heptane 78.355 — — — — 0.248 (B)
370 4,4-Dimethyl-1-phenyl-1-penten-3-one 78.563 — — — — 0.199 (B)
371 1,5-Diphenyl-1-penten-3-one 78.603 0.16 (B) 0.354 (B) 0.139 (B) — —
372 (1-Methylbutyl)-benzene 79.424 — — — — 0.266 (B)
373 Stearic acid 79.504 — — — 0.198 —
374 [1S-(1α, 2α, 3aβ, 4α, 8aβ, 9R∗)]-Decahydro-1,5,5,8a-tetramethyl-1,2,4-methenoazulene 79.666 — — — — 0.186 (S)
375 Z-8-Methyl-9-tetradecenoic acid 79.799 — 0.105 — — —
376 1,2,3,4-Tetrahydro-1,5,7-trimethylnaphthalene 79.903 — — — — 0.426 (B)
377 N-Phenyl-2-naphthylamine 80.746 0.121 (B) — 0.186 (B) — —
378 Z-5-Nonadecene 81.024 0.196 — 0.181 0.230 —
379 Cyclopentadecane 81.047 — 0.188 — — —
380 Diaveridine 81.105 — — — — 0.108 (B)
381 (Z)-3-Tridecen-1-yne 81.428 — — — — 0.322
382 Ambrosin 82.277 — — — — 0.102 (S)
383 2-Decanone O-methyl oxime 82.329 0.104 0.115 0.092 — —
384 N,N-Dimethyldecanamide 82.352 — — — 0.140 —
385 [1aR-(1aα, 4aβ, 8aS∗)]-1,1a,5,6,7,8-Hexahydro-4a,8,8-trimethyl-cyclopropa[d]naphthalen-2(4aH)-one 82.710 — — — — 0.124 (S)
386 Murolan-3,9(11)-diene-10-peroxy 83.248 — — — — 0.267 (S)
387 Chromone derivative 85.396 0.109 (C) — 0.08 (C) — —
388 Chromone derivative 85.818 0.132 (C) 0.217 (C) 0.151 (C) — —
389 Chromone derivative 86.783 2.245 (C) 1.812 (C) 1.697 (C) 3.633 (C) —
390 Chromone derivative 87.187 0.104 — — 0.112 —
391 Chromone derivative 87.539 — — — — 0.331 (C)
392 Chromone derivative 88.724 0.159 — — — —
393 Chromone derivative 88.880 0.118 — 0.123 — —
394 Chromone derivative 88.891 — — — — —
395 Chromone derivative 88.897 — 0.152 (C) — 0.175 —
396 Chromone derivative 89.209 0.14 (C) 0.662 (C) 0.345 (C) — 0.334 (C)
397 Chromone derivative 89.515 — — 0.064 (C) 0.14 (C) —
398 Chromone derivative 89.769 — — 0.221 (C) — —
399 Chromone derivative 89.787 — 0.125 (B) — — —
400 Chromone derivative 89.844 0.286 — — — —
401 Chromone derivative 89.867 — 0.108 (B) — — —
402 Chromone derivative 90.595 — — 3.447 (C) — —
403 Chromone derivative 90.601 1.127 (C) — — — —
404 Chromone derivative 90.630 — — — 0.179 (C) —
405 Chromone derivative 90.641 — 3.239 (C) — — —
406 Chromone derivative 90.676 — — — — 7.398 (C)
407 Chromone derivative 91.000 — — 0.197 (C) — —
408 Chromone derivative 91.000 0.121 (C) — — — —
409 Chromone derivative 91.017 — 0.148 (C) — — —
410 Chromone derivative 91.485 — — 3.502 (C) — —
411 Chromone derivative 91.485 1.32 (C) — — — —
412 Chromone derivative 91.571 — — — — 0.542 (C)
413 Chromone derivative 91.571 — 4.593 (C) — — —
414 Chromone derivative 91.629 — — — 0.105 (C) —
415 Chromone derivative 91.710 — — 0.649 (C) — —
416 Chromone derivative 91.716 0.132 (C) — — — —
417 Chromone derivative 91.727 — — — — 1.405 (C)
418 Chromone derivative 91.745 — 0.561 (C) — — —
419 Chromone derivative 91.803 — — 0.592 (C) — —
420 Chromone derivative 91.808 0.481 (C) — — — —
421 Chromone derivative 91.837 — — — 0.411 —
423 Chromone derivative 91.837 — 0.539 (C) — — —
422 Chromone derivative 91.953 — — 0.88 (C) — —
424 Chromone derivative 91.953 0.434 (C) — — — —
425 Chromone derivative 91.987 — 0.796 (C) — — —
426 Chromone derivative 92.571 — — — — 0.143 (C)
427 Chromone derivative 92.894 — 0.254 (B) — — —
428 Chromone derivative 92.935 — — — — 1.293 (C)
429 Chromone derivative 92.958 — — 0.221 (C) — —
430 Chromone derivative 92.969 — 0.485 (B) — — —
431 Chromone derivative 93.339 — — — — 0.388 (C)
432 Chromone derivative 93.784 — — — — 0.6 (C)
433 Chromone derivative 94.136 — — — — 0.14 (C)
434 Chromone derivative 94.356 — — 0.731 — —
435 Chromone derivative 94.361 — — — — 0.125 (C)
436 Chromone derivative 94.361 0.596 (C) — — — —
437 Chromone derivative 94.390 — 0.832 (C) — — —
438 Chromone derivative 94.795 — — 0.323 (C) — —
439 Chromone derivative 94.800 0.187 (C) — — — —
440 Chromone derivative 94.812 — 0.371 (C) — — —
441 Chromone derivative 95.228 — — — — 1.254 (C)
442 Chromone derivative 95.228 — — 0.596 (C) — —
443 Chromone derivative 95.257 — 0.614 (C) — — —
444 Chromone derivative 95.268 0.165 (C) — — — —
445 Chromone derivative 95.609 — — — — 0.217 (C)
446 Chromone derivative 95.748 0.243 (C) — — — —
447 Chromone derivative 95.754 — — 0.237 (C) — —
448 Chromone derivative 95.765 — — — 0.478 (B) —
449 Chromone derivative 95.771 — 0.206 (C) — — —
450 Chromone derivative 95.834 — — — — 0.414 (C)
451 Chromone derivative 95.904 0.291 (C) — — — —
452 Chromone derivative 95.921 — — — 0.678 (B) —
453 Chromone derivative 95.967 0.713 (C) — — — —
454 Chromone derivative 95.973 — — 3.243 (C) — —
455 Chromone derivative 96.019 — 2.703 (C) — — —
456 Chromone derivative 96.088 — — — — 7.2 (C)
457 Chromone derivative 96.308 — — 0.178 (C) — —
458 Chromone derivative 96.308 0.192 (C) — — — —
459 Chromone derivative 96.319 — — — 0.177 (B) —
460 Chromone derivative 96.343 — 0.281 (C) — — —
461 Chromone derivative 96.372 — — — — 0.498 (C)
462 Chromone derivative 96.395 — — 0.267 (C) — —
463 Chromone derivative 96.400 0.313 (C) — — — —
464 Chromone derivative 96.412 — — — 0.381 —
465 Chromone derivative 96.418 — 0.43 (B) — — —
466 Chromone derivative 96.556 — 0.138 (B) — — —
467 Chromone derivative 96.632 — — 0.058 (C) — —
468 Chromone derivative 96.637 0.312 (C) — — — —
469 Chromone derivative 96.649 — — — 0.236 (B) —
470 Chromone derivative 96.655 — 0.16 (B) — — —
471 Chromone derivative 96.718 — — — — 0.332 (C)
472 Chromone derivative 96.753 — 0.206 — — —
473 Chromone derivative 97.088 — — — — 0.464 (C)
474 Chromone derivative 97.319 — — — — 0.176 (C)
475 Chromone derivative 97.469 — — — — 0.171 (C)
476 Chromone derivative 97.700 — — — — 0.469 (C)
477 Chromone derivative 98.341 — — — — 0.863 (C)
478 Chromone derivative 98.399 — 0.103 — — —
479 Chromone derivative 98.982 — — — — 0.104 (C)
480 Chromone derivative 99.242 — — — — 0.374 (C)
481 Chromone derivative 99.566 — — — — 0.264 (C)
482 Chromone derivative 99.751 — — — — 0.192 (C)
483 Chromone derivative 100.034 — — — — 0.281 (C)
484 Chromone derivative 100.346 — — — — 0.577 (C)
Total 97.620 97.164 99.859 97.040 96.079
  • B: aromatic compound; C: chromone derivative; S: sesquiterpenes; —: not detected.

2.4. LPS-Stimulated TNF-α and IL-1α Release in RAW264.7 Cells

2.4.1. Isolation and Culture of RAW264.7 Cells

RAW264.7 cells in logarithmic growth phase were washed twice with phosphate-buffered saline (PBS) and inoculated in 96-well plates at a density of 1 × 104 cells per well, and 100 μL of cell suspension was added to each well. Three compound wells were set in each group and cultured at 37°C in 5% CO2 for 24 h.

2.4.2. Measurement of TNF-α and IL-1α Production

The cells were incubated with 1 ng/mL LPS in the presence of indomethacin, AAW, BCDA, and AWIT (20, 40 and 80 μg/mL) and cultured at 37°C and 5% CO2 for 24 h. Then, the levels of TNF-α and IL-1α in the cell-free culture supernatant were determined by ELISA kits. Briefly, 10 μL of supernatant was mixed with an equal volume of reagents A and B [1 : 1 (v/v)] in a 96-well flat-bottom plate. The absorbance at 540 nm was measured after 10 min using an ELISA reader. The amounts of TNF-α and IL-1α were calculated from a standard curve created using known concentrations of standards.

3. Results and Discussion

3.1. GC-MS Analysis

n-Hexane, methanol, DMSO, and CH2Cl2 were used to collect the chemical constituents of incense smoke from agarwood. The GC-MS peaks of incense smoke samples collected using CH2Cl2 were the most intense among the peaks obtained using the above solvents. Therefore, CH2Cl2 was selected to dissolve the chemical constituents of smoke samples (agarwood and AS). Finally, 484 compounds in total (Table 1 and Figure 1) were identified from the incense smoke samples (AAW, BCDA, AWIT, and AS) and the samples obtained by CH2Cl2 extraction of sticks from AWIT. The numbers of compounds identified in incense smoke from AAW, BCDA, AWIT, AS, and EAWIT were 167, 158, 141, 127, and 131, respectively. Aromatics and chromone derivatives were the main chemical constituents in AAW, BCDA, and AWIT; among all chemical constituents, aromatics represented 69.617, 55.038, and 60.483%, and chromone derivatives represented 9.252, 17.725, and 16.946%, respectively.

The chemical constituents of incense smoke may be quantifiable. Therefore, the chemical constituents of incense smoke from agarwood produced by AWIT were compared with the corresponding constituents from agarwood produced by AAW and BCDA. A total of 61 compounds in the AWIT sample, representing 54.837%, were also found in the AAW and BCDA samples. The major compounds (relative content >1%) were phenylethylene (7.973%); ethylbenzene (6.384%); 2,3,5,6-tetrafluoroanisole (4.130%); 5-(2-thienyl)-4-pyrimidinamine (2.402%); 2-methoxy-4-(1-propen-1-yl)-phenol (2.379%); 2,6-dimethoxyphenol (2.178%); syn-3,3,5,6,8,8-hexamethyl-tricyclo[5.1.0.0(2,4)]oct-5-ene (1.762%); 1-(2,4,6-trimethylphenyl)-3-(2-propynyl)-thiourea (1.680%); phenylacetylene (1.612%); 1-hydroxy-6-methylphenazine (1.555%); 4-hydroxy-3-methoxystyrene(1.512%) 3,5-dimethoxy-4-hydroxyphenylacetic acid (1.391%); 2,2,4,6,6-pentamethylheptane (1.152%); 4-(4-methoxyphenyl)-2-butanone (1.124%); 4-phenyl-2-butanone (1.060%); and benzaldehyde (1.008%). Moreover, chromone derivatives and sesquiterpenes are the main components responsible for pharmacodynamic effects [23–25]. In this experiment, 21 compounds, representing 16.946%, were identified as chromone derivatives according to the peaks at m/z 91, 121, 137, 107, 160, 176, 190, 220, 250, 266, 280, 282, 296, 310, 312, 326, 328, and 342 [22], and 16 compounds were identified as sesquiterpenes, representing 6.768%. In short, aromatic compounds were the main chemical constituents of incense smoke from agarwood, including AWIT, AAW, and BCDA samples.

To identify whether agarwood (AAW, AWIT, and BCDA) contained chemical constituents of AS, incense smoke produced from AS was tested by the same method. No sesquiterpenes were detected among the chemical constituents of the smoke, and chromones only represented 4.569%, which was less than the contents in AWIT (16.946%), AAW (9.252%), and BCDA (17.725%). Finally, 29 compounds, representing 32.627%, were also found in AWIT, AAW, and BCDA. The main compounds (relative amount >1%) were phenylethylene (5.132%); 2-methoxy-4-(1-propen-1-yl)-phenol (3.254%); 3,5-dimethoxy-4-hydroxybenzaldehyde (2.920%); 4-hydroxy-3-methoxystyrene (2.254%); 3,5-dimethoxy-4-hydroxyphenylacetic acid (2.150%); vanillin (1.842%); acetosyringone (1.499%); guaiacol (1.362%); ethylbenzene (1.313%); benzaldehyde (1.146%); homovanillyl alcohol (1.119%); 2,6-dimethoxy-4-(2-propen-1-yl)-phenol (1.029%); and phenylacetylene (1.013%). These components may be from the residue of A. sinensis, a sticky powder, making agarwood powder bind, used in the preparation of sticks of AS (making sticks from pure AS alone is difficult, so the addition of a sticky powder is necessary) or agarwood in A. sinensis (AS can form agarwood in the process of storage).

The data for incense smoke from agarwood (AAW, AWIT, and BCDA) showed that low-molecular-weight aromatic compounds (LACs) represented more than 55% of the total constituents. Michiho Ito et al. reported that chromone derivatives could be converted and produce the pleasant smell of agarwood through the generation of LACs in the process of heating [26,27] (Scheme 1). Chromone derivatives are among the main chemical constituents of agarwood. They can generate unique and different LACs at high temperature (when burned). As a result, many LACs were detected in the agarwood smoke. To verify the results, an extraction experiment of sticks from AWIT was carried out at room temperature (to avoid high temperature). The results showed that chromone derivatives, sesquiterpenes, and aromatics were the main chemical constituents, representing 26.547, 26.767, and 26.941% of the total constituents, respectively. Few chemical constituents of EAWIT were observed before 40 min (tR), as shown in Figure 1, while there was far higher number of peaks after 58 min (tR), which is indicative of chromone derivatives and sesquiterpenes. Interestingly, the chemical constituents of incense smoke showed the opposite trend in Figure 1. The results indicated that high-molecular-weight compounds might be cracked into low-molecular-weight compounds at high temperature. In other words, some chromone derivatives and sesquiterpenes might be converted into low-molecular-weight compounds, which is consistent with the reported literature [26, 27]. Therefore, low-molecular-weight compounds accounted for a high percentage of the incense smoke obtained from agarwood during burning. Moreover, some studies suggested that the inhalation of some LACs had a sedative or hypnotic effect on mice and that benzylacetone in particular reduces mouse locomotor activity [28–30]. Hence, inhalation of the pleasant aroma generated by agarwood during heating could lead to pharmacological effects.

Details are in the caption following the image
Scheme 1
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LACs generated from agaroterol on heating.

3.2. Effect of Chemical Constituents on TNF-α and IL-1α Release in LPS-Stimulated RAW264.7 Cells

As shown in Tables 2 and 3, normal inactivated RAW264.7 cells produced low amounts of TNF-α and IL-1α after 24 h of incubation at 37°C, and exposure to LPS induced higher amounts of TNF-α and IL-1α. In contrast, under indomethacin treatment, AAW, BCDA, and AWIT produced a concentration-dependent decrease at concentrations of 20, 40, and 80 μg/mL. The TNF-α and IL-1α levels of model group were significantly higher than those of the normal group (P < 0.05 or P < 0.01). The incense components of AAW, BCDA, AWIT, and indomethacin significantly reduced TNF-α and IL-1α levels (P < 0.05, P < 0.01, or P < 0.001), showing better anti-inflammatory effects. These results showed that the anti-inflammatory activities of AAW, AWIT, and indomethacin were comparable and superior to that of BCDA.

Table 2. LPS-induced release of TNF-α in RAW264.7 cells.
Drug/dose   80 μg/mL 40 μg/mL 20 μg/mL
Normal 189.09 ± 15.25      
Model 236.09 ± 18.79 ∗∗      
Indomethacin   152.39 ± 16.67### 169.14 ± 18.23### 200.19 ± 19.42#
AAW   157.69 ± 15.98### 181.79 ± 19.45## 209.19 ± 21.03#
BCDA   165.09 ± 16.12### 194.84 ± 17.67## 213.04 ± 22.43#
AWIT   154.89 ± 17.13### 187.84 ± 18.37## 212.84 ± 19.35#
  • Note. This result is the average of three parallel experiments.  ∗∗P < 0.01 vs normal;  ###P < 0.001,  ##P < 0.01,  #P < 0.05 vs model.
Table 3. LPS-induced release of IL-1α in RAW264.7 cells.
Durg/dose   80 μg/mL 40 μg/mL 20 μg/mL
Normal 15.00 ± 1.78      
Model 20.43 ± 2.32 ∗      
Indomethacin   10.38 ± 2.12## 12.52 ± 2.65## 17.87 ± 1.85#
AAW   10.78 ± 2.56## 15.72 ± 2.57# 19.10 ± 2.58
BCDA   15.05 ± 1.74# 18.99 ± 2.97 22.19 ± 2.94
AWIT   10.88 ± 1.97## 12.15 ± 2.72## 22.52 ± 2.93
  • Note. This result is the average of three parallel experiments.  ∗P < 0.05 vs normal;  ##P < 0.01,  #P < 0.05 vs model.

4. Conclusions

The chemical constituents of incense smoke from AAW, BCDA, AWIT, AS, and EAWIT were analyzed by GC-MS, and 484 compounds were identified. Aromatic compounds were the main chemical constituents of incense smoke from AAW, BCDA, and AWIT. A total of 61 aromatic compounds from AWIT, representing 54.837%, were also found in AAW and BCDA. All experimental data suggested that aromatic compounds were the main chemical constituents in agarwood smoke and that some chromone derivatives could be cracked into LACs at high temperature. Furthermore, agarwood incense smoke showed anti-inflammatory activities by inhibiting lipopolysaccharide- (LPS-) induced TNF-α and IL-1α release in RAW264.7 cells.

Conflicts of Interest

The authors declare that they have no conflicts of interest in any form.

Authors’ Contributions

De-Qian Peng, Zhang-Xin Yu contributed equally to this work.

Acknowledgments

This research work was financially supported by the Natural Science  Foundation of Hainan Province (no. 217291), the Science and Technology  Programs from Hainan Province of China  (no. ZDKJ2016004), and the  Major  Science  and Technology  Innovation  Project  of  the  Chinese  Academy of  Medical  Sciences  (no. 2016-I2M-2-003). The authors are grateful to Hai-Yan Wang (an English teacher) of Qiqihar University and Ass. Prof. Lu-Jia Mao of Hainan Medical University for revising the manuscript.

    Data Availability

    The data used to support the findings of this study are available from the corresponding author upon request.

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