Evidence-Based Complementary and Alternative Medicine
Volume 2020 (2020), Article ID 2648759, 13 pages
https://doi.org/10.1155/2020/2648759

Research Article

Analysis of the Components in Moxa Smoke by GC-MS and Preliminary Discussion on Its Toxicity and Side Effects

Xiaoyu Xu,1,2 Si Shan,1,2 Wenlei Wang,1,2 and Hongning Liu1,2

¹Jiangxi Province Key Laboratory of TCM Etiopathogenisis, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330004, China
²Research Center for Differentiation and Development of TCM Basic Theory, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330004, China

Correspondence should be addressed to Si Shan; shansi1987@163.com

Received 20 May 2020; Revised 21 September 2020; Accepted 1 October 2020; Published 31 October 2020

Academic Editor: Luísa Zuravski

Copyright © 2020 Xiaoyu Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Moxibustion plays an important role in the prevention and treatment of diseases and the promotion of human health. In this study, the components in moxa smoke from Jiangxi Poai Biotechnology Co., Ltd., namely, Qing moxa sticks, were absorbed by five solvents (cyclohexane, ethyl acetate, n-butanol, anhydrous ethanol, and water) and identified by gas chromatography-mass spectrometry. The identification results of the smoke from the Qing moxa sticks that was absorbed in liquid are as follows: a total of 294 compounds were identified, including 139 in cyclohexane, 145 in ethyl acetate, 60 in n-butanol, 89 in anhydrous ethanol, and 77 in water, and of those, 112 toxic compounds were identified. Furthermore, Ingenuity Pathway Analysis software and the PubChem database were successfully applied to analyze the toxic compounds. There were 812 target proteins related to the toxic components, 25 molecular networks, and 54 biological pathways. The results showed that the toxic compounds of moxa smoke may have some side effects on the heart, liver, and kidney of humans. This study revealed that the components of moxa smoke are complex and diverse. Due to the findings of toxic compounds in moxa smoke, we recommend that moxibustion rooms should be equipped with ventilation equipment or enough artificial ventilation to ensure the health of patients and practitioners.

1. Introduction

Moxibustion is an important part of clinical treatment in traditional Chinese medicine. In moxibustion, wormwood or other drugs are used to place acupoints or pain points on the body surface for warming meridians and stimulating acupuncture points [1]. As people pay more attention to health, the use of moxibustion to treat diseases in China and other Asian countries is growing [2]. Heat and moxa smoke are produced during moxibustion. The heat of moxibustion has the function of assisting Yang Qi, lifting subsidence, and solidifying. Recent studies have shown that moxa smoke also has antibacterial, antitumor, antiviral, anti-inflammatory, and air purification functions [3–7]. Ancient books on Chinese Medicine contain records of the use of moxa smoke in the treatment of irritable bowel syndrome [8], inflammatory bowel disease [9], and neurological symptoms [10]. Additionally, the antioxidants in moxa smoke play an antiaging role through the penetration of heat [11].

However, some patients feel uncomfortable during moxibustion and can even have noticeable adverse reactions, such as watery eyes and coughing, which has caused people to question the safety of moxa smoke [12, 13]. Some studies have shown that there were harmful components such as monoaromatic hydrocarbons and formaldehyde in moxa smoke [14–16]. The inhalation of these substances induced eustachian tube irritation, throat itching, eye pain, tonsil swelling, and other toxic effects [12]. Therefore, it is very important to determine the toxic compounds in moxa smoke.

The aim of the present study was to analyze the components in Qing moxa smoke based on enrichment with five solvents. A set of smoke absorption devices were designed with cyclohexane, ethyl acetate, n-butanol, anhydrous ethanol, and water as absorbents with the help of an extraction pump to concentrate the moxa smoke in the solvents. The benefits of this device for enrichment of moxa smoke include: (1) moxa sticks can burn completely in the air to avoid incomplete combustion; (2) the devices can detect as many compounds as possible by increasing the concentration of moxa smoke; and (3) the use of different polar solvents can provide reference for the absorption and treatment of moxa smoke. Then, the toxic compounds were queried by the Comparative Toxicogenomics Database (CTD) [17, 18]. In addition, we aimed to estimate the toxic compounds in moxa smoke that would have an impact on the human body by applying Ingenuity Pathway Analysis (IPA) software and the PubChem database to provide an experimental basis for the safety evaluation of moxa smoke [19, 20].

2. Materials and Methods

2.1. Materials

We followed the steps outlined in our patent, “A method of using Terahertz Wave to detect the quality of moxa column,” patent number: ZL 2020 1 0000161.6, which are as follows: (1) sample placement; (2) determination of the background value; (3) measurement of the terahertz wave energy at different bands of the combustion column; (4) data processing; and (5) column quality judgment. If the terahertz wave intensity of each band is stronger than others and the waveform slightly changes, the quality is better. The results revealed which Qing moxa stick had the best quality, and that one was selected for smoke enrichment analysis [21]. Qing moxa sticks (18 × 27 ± 1 mm, Jiangxi Poai Biotechnology Co., Ltd., Poyang, China), which are widely used by the Chinese population, were used in this study. Moxa sticks were encased in Artemisia argyi (Chinese mugwort) floss, which was made of dried A. argyi leaves. The Qing moxa sticks were produced with a 10 : 1 ratio, which means that 10 kg of dried A. argyi leaves were processed into 1 kg of moxa floss. Analytical grade cyclohexane, ethyl acetate, n-butanol, and ethanol were all purchased from Guangdong Xilong Science Co., Ltd., and used as received.

2.2. Sample Preparation

A set of smoke absorption devices was designed as shown in Figure 1. With 1000 mL cyclohexane, ethyl acetate, n-butanol, anhydrous ethanol, or water as the solvent, 50 moxa sticks were burned in the air until combustion was complete. During the combustion process, the air pump control combustion speed was adjusted such that the blank flask did not fill with white smoke, so that the solvent fully absorbed the moxa smoke. The glass ball in the absorption flask had holes in it to reduce the production of bubbles and prevent the solvent from escaping. The absorption solution was emptied from the absorption flask, filtered with a 0.22 μm microporous membrane, and 2 mL of each solution was added into the sample bottle for gas chromatography-mass spectroscopy (GC-MS) analysis.

Figure 1: A smoke absorption device. (1) Smoke hood. (2) Absorption flask. (3) Blank flask. (4) Buffer flask.

2.3. GC-MS Analysis

An Agilent Technologies 7890 GC system (Agilent Technologies Inc., Palo Alto, CA, USA) coupled with an Agilent Technologies 5975 mass spectrometer (Agilent Technologies Inc.) was used for moxa smoke analysis. A HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used to separate compounds. High-purity helium was applied as the carrier gas. The following conditions were used: column flow rate: 1.0 mL/min; split injection, split ratio: 100 : 1; injection volume: 1 μL; and injection port temperature: 250°C. The temperature procedure was as follows: 0–3 min, 40–40°C; 3–39 min, 40–220°C; 39–43 min, 220–220°C; 43–49 min, 220–280°C; and 49–50 min, 280–280°C.

The MS working conditions were as follows: the electron ionization energy was 70 eV, the full-scan acquisition was used in the range of 50–650 m/z, the ion source temperature was 230°C, the transmission ion temperature was 280°C, and the four-stage pole temperature was 150°C. The identification of each peak in the total ion flow chromatogram was automatically retrieved from the National Institute of Standards and Technology (NIST) 11.L as the standard mass spectrometry database and verified with standard mass spectrometry. Some components were confirmed with the retention value of a standard sample. The identified components were semiquantified by comparing the peak area of each component with the total peak area, and the relative percentage of components was calculated by the peak area normalization method.

2.4. Network Toxicological Analysis

The compounds identified by the NIST 11.L were then queried for related toxicity through the CTD database (https://ctdbase.org/about/). Then, the molecular information corresponding to the toxic compounds of moxa smoke was obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/) [22]. In addition, the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) was used to predict toxic compounds relevant targets, and exporting Uniprot ID. Next, the molecular networks of toxic compound target proteins and its biological pathways were constructed by IPA software (Qiagen, Redwood City, CA, USA).

3. Results

3.1. Total Ion Chromatogram

The total ion chromatograms (TIC) of moxa smoke from solvents by GC-MS are shown in Figure 2 [23, 24]. As shown in Figure 2, the compounds in moxa smoke were detected within 40 min.

(a)

(b)

(c)

(d)

(e)

Figure 2: Total ion chromatograms of five solvents by GC-MS. (a) Cyclohexane. (b) Ethyl acetate. (c) n-Butanol. (d) Anhydrous ethanol. (e) Water. Only compounds unique to each solvent with a relative content greater than 0.5% are tagged in the figure.

3.2. GC-MS Analysis Results

A total of 294 compounds, including 139 in cyclohexane, 145 in ethyl acetate, 60 in n-butanol, 89 in anhydrous ethanol, and 77 in water were found and identified in Qing moxa smoke. As shown in Tables 1–5, only 52 unique compounds were detected in cyclohexane smoke absorption liquid, 57 in ethyl acetate, 10 in n-butanol, 17 in anhydrous ethanol, and 47 in water, and other components were identified in more than one solvent. Toluene, pyridine, 2-methylpyridine, 2-methyl-2-cyclopenten-1-one, 2-furanmethanol, 2-acetylfuran, phenol, eucalyptol, o-cresol, indole, and biphenyl were detected in all five solvents and are shown in Figure 3, but the same components had different concentrations in different solvents. This shows that the components of moxa smoke were absorbed differently by different polar solvents.

Table 1: Relative content (%) of unique compounds in cyclohexane.

No.
Rt (min)
Compound
Retention index
Relative content (%)

1
4.038
Bicyclo[4.1.0]hept-2-ene
706
0.033

2
4.602
4-Methyl-1,4-hexadiene
737
0.175

3
4.978
1-Methylcyclohexene
757
0.078

4
5.093
1,3,5-Heptatriene
763
0.115

5
5.290
1,7-Octadiene
774
0.048

6
5.400
2-Methyl-1-heptene
780
0.105

7
5.514
1-Octene
786
0.693

8
5.947
(E)-2-Octene
806
0.086

9
6.458
1,3-Dimethyl cyclohexene
822
0.222

10
6.647
(E,E,E)-2,4,6-Octatriene
828
0.027

11
7.111
5,6-Dimethyl-1,3-cyclohexadiene
843
0.040

12
7.749
3-Methylenecycloheptene
863
0.146

13
8.060
(1Z,2Z)-1,2-Di(ethylidene)cyclobutane
873
0.094

14
8.334
Cyclohexanol
882
0.703

15
9.025
2-Ethylpyridine
904
0.095

16
10.064
2-Methyl-1-octen-3-yne
935
0.105

17
10.166
1-Methylcycloheptene
938
0.141

18
11.164
Mesitylene
968
0.307

19
11.629
Alpha-methyl styrene
981
0.176

20
12.372
Gamma-terpinene
1004
0.227

21
12.960
1,2,4-Trimethylbenzene
1022
0.866

22
13.395
Trans-beta-methyl styrene
1035
0.371

23
14.973
1-Phenyl-2-butene
1083
0.228

24
15.181
1-Methyl-4-(prop-1-en-2-yl)benzene
1090
1.054

25
15.901
Cosmene
1112
0.163

26
16.210
2,4-Dimethylstyrene
1122
0.156

27
16.305
1-Phenyl-1-butene
1125
0.336

28
16.448
1-Allyl-2-methylbenzene
1130
0.223

29
16.749
Phenyl acetonitrile
1140
0.610

30
17.051
2,3-Dimethylphenol
1149
0.687

31
17.155
1,2,3,4-Tetramethylbenzene
1153
0.203

32
17.510
1,1a,6,6a-Tetrahydrocycloprop[a]indene
1164
0.258

33
18.241
1,2-Dimethylindan
1188
0.200

34
19.970
4-Methylindole
1247
0.200

35
20.792
7H-Benzocycloheptene
1275
0.200

36
21.045
2-(2-Hydroxyphenyl)buta-1,3-diene
1284
0.168

37
22.396
1H-Indene,2,3-dihydro-1,1,3-trimethyl
1332
0.226

38
23.401
5-Methylindole
1369
0.099

39
23.527
1,8-Cyclotetradecadiyne
1374
0.108

40
24.816
1,4-Dimethylnaphthalene
1422
0.227

41
25.073
1,4,5-Trimethylnaphthalene
1432
0.142

42
26.434
2,4,6-Trimethylbenzonitrile
1485
0.137

43
27.199
1-Phenylpyridin-2-one
1516
0.186

44
28.473
2,4-Dimethoxyacetophenone
1568
0.234

45
28.910
Spathulenol
1586
0.163

46
29.355
2-Methylbiphenyl
1605
0.114

47
30.735
(+)-γ-Gurjunene
1665
0.217

48
33.434
9-Methylene-9H-fluorene
1786
0.103

49
34.460
3,7,11,15-Tetramethyl-2-hexadecene
1835
0.062

50
34.710
2,6,10,14-Tetramethyl-2-hexadecene
1847
0.135

51
35.671
1-Nonadecene
1893
0.163

52
37.669
E-15-Heptadecenal
1993
0.123

Table 2: Relative content (%) of unique compounds in ethyl acetate.

No.
Rt (min)
Compound
Retention index
Relative content (%)

1
4.106
3-Methyl-butanenitrile
710
0.159

2
4.535
Dimethyl aminoacetonitrile
733
0.191

3
4.603
2,4-Dimethyl-1,3-pentadiene
737
0.152

4
5.236
3-Methylenecyclohexene
771
0.095

5
5.300
Cyclooctene
775
0.030

6
5.522
2-Octene
787
0.557

7
5.837
2,3-Dimethyl-1,4-hexadiene
802
0.120

8
5.957
(Z)-2-Octene
806
0.044

9
6.462
Pyrazine, methyl
822
0.208

10
6.869
2,5-Dimethylpyrrole
835
0.088

11
8.073
1,4-Dimethylenecyclohexane
874
0.051

12
8.411
2,3-Dimethylpyridine
885
0.166

13
9.820
3-Ethyl-1H-pyrrole
928
0.295

14
9.893
3,4-Dimethylpyridine
930
0.509

15
10.078
2-Ethyl-5,5-dimethyl-1,3-cyclopentadien
935
0.082

16
10.480
2-Methylborazine
947
0.250

17
11.291
2,5-Cyclooctadien-1-one
971
0.115

18
11.406
Benzene
975
0.060

19
11.503
Aniline
978
0.183

20
12.166
3-Methylstyrene
997
0.337

21
12.385
Alpha-phellandrene
1004
0.151

22
12.591
2-Ethyl-4-methyl-1H-pyrrole
1010
0.295

23
13.065
o-Cymene
1025
1.118

24
13.402
Allylbenzene
1035
0.309

25
15.039
3-Ethyl-o-xylene
1085
0.593

26
15.620
7-Methylbenzofuran
1103
0.383

27
15.921
Azulene
1113
0.406

28
16.454
4-Allyltoluene
1130
0.171

29
16.762
3-Ethynylaniline
1140
0.367

30
17.065
3-Methyl-1H-indene
1150
0.557

31
17.163
1,2,3,4-Tetramethylfulven
1153
0.138

32
17.523
1,4-Dihydronaphthalene
1165
0.214

33
18.254
1-Methyl-3-(1-methyl-2-propenyl)benzene
1188
0.188

34
18.501
Dihydrocarveol
1196
0.140

35
18.696
Catechol
1203
0.223

36
19.034
2,6-Dimethylundecane
1215
0.195

37
19.250
(E)-Cinnamaldehyde
1222
0.391

38
19.355
Cyclododecene
1226
0.166

39
19.758
Isoquinoline
1239
0.210

40
19.983
3-Methylindolizine
1247
0.172

41
21.057
1,11-Dodecadiene
1284
0.162

42
22.733
2-Methylhydroquinone
1345
0.174

43
22.816
Naphthalene, 1,2,3,4-tetrahydro-1, 1-dimethyl
1348
0.125

44
23.825
2-Methyl-5-(1-methylethenyl)-cyclohexanone
1385
0.131

45
23.955
3-Methylindole
1389
0.257

46
24.827
1,3-Dimethylnaphthalene
1423
0.205

47
24.930
1,6-Dimethylnaphthalene
1427
0.460

48
25.761
2-Phenyl-1,3-cyclohexadien
1459
0.104

49
28.833
Phenylephrine
1583
0.020

50
31.836
Thiazolo[5,4-f]quinolin
1713
0.158

51
32.239
1,1,2-Trimethylcycloundecane
1732
0.250

52
33.442
Phenanthrene
1787
0.116

53
34.719
3,7,11,15-Tetramethyl-2-hexadecene
1847
0.146

54
35.480
(S)-6,6-Dimethyl-2-azaspiro[4.4]non-1-ene
1884
0.158

55
37.681
3-Icosene
1994
0.179

56
39.610
10-Heneicosene (c,t)
2093
0.140

57
39.729
Heneicosane
2100
0.110

Table 3: Relative content (%) of unique compounds in n-butanol.

No.
Rt (min)
Compound
Retention index
Relative content (%)

1
6.843
3-Furaldehyde
834
1.167

2
9.850
3-Methylheptan-4-one
928
0.377

3
9.998
2,4-Dimethylpyridine
933
0.457

4
10.819
Limonene
957
0.442

5
12.169
Butyl butyrate
997
6.894

6
16.330
(E)-1-Phenyl-1-butene
1126
0.273

7
16.771
Benzyl(methylidyne)azanium
1140
0.559

8
18.371
cis-2-dodecene
1192
1.064

9
19.262
Tricyclo[3.3.1.0(2,8)]nona-3,6-dien-9-one
1222
0.600

10
23.958
1-Methylindolizine
1390
0.361

Table 4: Relative content (%) of unique compounds in anhydrous ethanol.

No.
Rt (min)
Compound
Retention index
Relative content (%)

1
5.142
Thiophene
766
0.064

2
6.488
2-Methylpyrazine
823
0.137

3
9.811
2,3-Dimethyl-1H-pyrrole
927
0.155

4
11.170
6-Methyl-6-ethylfulvene
968
0.191

5
12.724
Acrylamide
1014
0.098

6
14.877
(−)-Camphor
1080
0.477

7
14.974
2-Methyl-1-phenylpropene
1083
0.137

8
15.691
4-Pyridinol
1105
0.157

9
17.059
3-Phenyl-1,2-butadiene
1150
0.649

10
17.518
Benzo[2,3]bicyclo[3.1.0]hexane
1165
0.188

11
20.713
Citral
1272
0.283

12
21.053
4-Methyl-2H-benzopyrane
1284
0.160

13
22.331
1,7-Dimethylnaphthalene
1330
0.224

14
25.053
2,3,6-Trimethylnaphthalene
1432
0.123

15
29.065
(Z)-8-Hexadecene
1593
0.393

16
35.803
Nonadecane
1900
0.118

17
37.154
Dibutyl phthalate
1968
0.425

Table 5: Relative content (%) of unique compounds in water.

No.
Rt (min)
Compound
Retention index
Relative content (%)

1
4.688
Methallyl cyanide
741
0.065

2
5.083
Cyclopentanone
763
0.730

3
5.558
Tetrachloroethylene
789
0.076

4
6.010
4-Aminopyridine
808
0.470

5
6.479
2-Methylcyclopentanone
823
0.375

6
6.549
4-Methylpentanenitrile
825
0.314

7
6.679
(R)-(+)-3-Methylcyclopentanone
829
0.150

8
7.870
2,6-Dimethylpyridine
867
0.596

9
8.091
Cyclohexanone
874
0.378

10
8.420
5,5-Dimethyl-1,3-hexadiene
885
0.079

11
8.816
2-Ethylpyrazin
898
0.426

12
8.975
2,3-Dimethylpyrazine
903
0.126

13
10.000
2,5-Dimethylpyridine
933
0.497

14
10.378
5-Methylfurfural
944
1.092

15
11.178
Phenetole
968
0.185

16
11.346
1-Isopropylcyclopentene
973
0.312

17
12.121
2-Ethyl-6-methylpyridine
996
0.438

18
12.400
2-Ethyl-5-methylpyridine
1004
0.282

19
12.687
5-Ethyl-2-methylpyridine
1013
0.207

20
12.743
2-Acetyl-5-methylfuran
1015
0.095

21
13.570
1-Acetyl-2-methyl-1-cyclopentene
1040
0.378

22
14.229
2-Methyl-6-methylene-2,7-octadien-4-ol
1060
0.540

23
14.663
Sabinene hydrate
1074
0.750

24
14.803
p-Tolunitrile
1078
0.511

25
15.168
2-Methylbenzoxazole
1089
0.568

26
15.419
2,6-Dimethylphenol
1097
0.820

27
15.601
Phenylacetone
1103
0.116

28
15.698
1-Isopropyl-1-cyclohexene
1106
0.226

29
16.045
4-Ethylphenol
1117
0.681

30
16.490
Decamethylcyclopentasiloxane
1131
0.372

31
16.721
Endo-borneol
1139
2.029

32
16.918
2-Acetyltoluene
1145
0.314

33
17.065
(-)-Terpinen-4-ol
1150
2.220

34
17.262
1-(3-Methylphenyl)ethanone
1156
0.818

35
17.460
(−)-Alpha-terpineol
1163
1.438

36
17.581
(+)-Dihydrocarvone
1167
0.338

37
17.960
(+/−)-cis-piperitol
1179
0.350

38
18.027
Verbenone
1181
0.190

39
18.279
(−)-cis-carveol
1189
0.357

40
18.610
2,4-Dimethylanisole
1201
0.248

41
19.293
Piperitone
1223
0.187

42
20.994
1-Methylindan-2-one
1282
0.261

43
21.356
Dodecamethylcyclohexasiloxane
1294
0.193

44
22.485
3,3-Dimethyl-1-indanone
1336
0.233

45
23.314
Methyl eugenol
1366
0.229

46
23.669
2,3-Dimethylnaphthalene
1379
0.146

47
26.001
2,4-Di-tert-butylphenol
1468
0.644

Figure 3: Relative contents (%) of common compounds in the five solvents.

As shown in Figure 3, the common components from moxa smoke in the five solvents included toluene (0.650%–3.872%), pyridine (0.137%–2.847%), 2-methylpyridine (0.267%–1.878%), 2-methyl-2-cyclopenten-1-one (0.412%–1.649%), 2-furanmethanol (0.526%–1.320%), 2-acetylfuran (0.266%–1.092%), phenol (2.686%–5.405%), eucalyptol (1.037%–1.605%), o-cresol (0.661%–1.419%), indole (0.780%–1.257%), and biphenyl (0.179%–0.338%). Among the above common components, the relative contents of phenol were more than 2% in all solvents. Phenol is a corrosive compound that is a strong irritant, which can lead to acute poisoning, skin ulcers, and tissue burns and can even be life-threatening [25, 26]. However, the amount of harmful substances produced by moxibustion will dictate the negative impact on the human body, and the duration of exposure to a moxa fume environment will determine if damage is caused to the body. There is no unified answer to these questions, which requires a large amount of case analysis and clinical trials.

3.3. Toxic Compounds of Moxa Smoke

The toxicity of compounds was determined based on the CTD database (https://ctdbase.org/about/), which provided abundant toxicological information for researchers. Among the 294 compounds detected in the moxa smoke absorption liquid, 112 compounds were confirmed to be toxic. Further study is needed to explore the toxicity of the 112 compounds. Table 6 provides details of the 112 toxic compounds.

Table 6: Toxic compounds in moxa smoke.

No.
Compound
CAS RN
Chemical ID
PubChem CID

1
Toluene
108-88-3
D014050
1140

2
Pyrimidine
289-95-2
C030986
9260

3
1-Methylpyrrole
96-54-8
C096654
7304

4
Pyridine
110-86-1
C023666
1049

5
Cyclopentanone
120-92-3
C007201
8452

6
1-Octene
111-66-0
C037690
8125

7
Tetrachloroethylene
127-18-4
D013750
31373

8
Octane
111-65-9
C026728
356

9
4-Aminopyridine
504-24-5
D015761
1727

10
Ethylbenzene
100-41-4
C004912
7500

11
Styrene
100-42-5
D020058
7501

12
p-Xylene
106-42-3
C031286
7809

13
Furfural
98-01-1
D005662
7362

14
2,5-Dimethylpyrrole
625-84-3
C067286
12265

15
2-Furanmethanol
98-00-0
C012986
7361

16
2-Acetylfuran
1192-62-7
C039669
14505

17
3-Methylpyridine
108-99-6
C053603
7970

18
2,6-Dimethylpyridine
108-48-5
C013093
7937

19
o-Xylene
95-47-6
C026114
7237

20
Butyrolactone
96-48-0
D015107
7302

21
Cyclohexanone
108-94-1
C036468
7967

22
Phenyl ethyne
536-74-3
C044736
10821

23
m-Xylene
108-38-3
C031285
7929

24
Propyl benzene
103-65-1
C024268
7668

25
Nonane
111-84-2
C017573
8141

26
2-Ethylpyridine
100-71-0
C051672
7523

27
Benzaldehyde
100-52-7
C032175
240

28
Cumene
98-82-8
C015763
7406

29
2,4-Dimethylpyridine
108-47-4
C078448
7936

30
Benzofuran
271-89-6
C105430
9223

31
5-Methylfurfural
620-02-0
C048065
12097

32
Phenol
108-95-2
D019800
996

33
Limonene
138-86-3
D000077222
22311

34
3-Ethyltoluene
620-14-4
C029719
12100

35
1,2,3-Trimethylbenzene
526-73-8
C010179
10686

36
Mesitylene
108-67-8
C010219
7947

37
Phenetole
103-73-1
C079413
7674

38
Eucalyptol
470-82-6
D000077591
2758

39
Benzene
71-43-2
D001554
241

40
Aniline
62-53-3
C023650
6115

41
Alpha-methyl styrene
98-83-9
C017915
7407

42
Butyl butyrate
109-21-7
C022793
7983

43
Decane
124-18-5
C012867
15600

44
Gamma-terpinene
99-85-4
C018669
7461

45
Alpha-phellandrene
99-83-2
C005403
7460

46
5-Ethyl-2-methylpyridine
104-90-5
C019196
7728

47
o-Cresol
95-48-7
C034047
335

48
Acrylamide
79-06-1
D020106
6579

49
2-Acetyl-5-methylfuran
1193-79-9
C057528
14514

50
p-Cresol
106-44-5
C032538
2879

51
1,2,4-trimethylbenzene
95-63-6
C010313
7247

52
o-Cymene
527-84-4
C046257
10703

53
p-Cymene
99-87-6
C007210
7463

54
Guaiacol
90-05-1
D006139
460

55
m-Cresol
108-39-4
C042041
342

56
Allylbenzene
300-57-2
C102347
9309

57
Indene
95-13-6
C093581
7219

58
Acetophenone
98-86-2
C038699
7410

59
Methyl benzoate
93-58-3
C044605
7150

60
2,6-Dimethylphenol
576-26-1
C036531
11335

61
Undecane
1120-21-4
C022884
14257

62
Phenylacetone
103-79-7
C008863
7678

63
4-Pyridinol
626-64-2
C534143
12290

64
Naphthalene
91-20-3
C031721
931

65
Azulene
275-51-4
C005525
9231

66
4-Ethylphenol
123-07-9
C042291
31242

67
4-Allyltoluene
3333-13-9
C092903
76851

68
Indolizine
274-40-8
C035094
9230

69
Phenyl acetonitrile
140-29-4
C006725
8794

70
2,3-Dimethylphenol
526-75-0
C054067
10687

71
1,2,3,4-Tetramethylbenzene
488-23-3
C021246
10263

72
(−)-Alpha-terpineol
10482-56-1
C016775
443162

73
3,5-Dimethylphenol
108-68-9
C016834
7948

74
Terpinen-4-ol
562-74-3
C034019
11230

75
Dodecane
112-40-3
C007548
8182

76
Catechol
120-80-9
C034221
289

77
5,6-Dimethylbenzimidazole
582-60-5
C015158
675

78
Tridecane
629-50-5
C094074
12388

79
(E)-Cinnamaldehyde
104-55-2
C012843
637511

80
Indole
120-72-9
C030374
798

81
Isoquinoline
119-65-3
C039109
8405

82
Citral
5392-40-5
C007076
638011

83
Hydroquinone
123-31-9
C031927
785

84
Biphenyl
92-52-4
C010574
7095

85
1-Tridecene
2437-56-1
C028691
17095

86
Tetradecane
629-59-4
C024713
12389

87
2-Methylnaphthalene
91-57-6
C027384
7055

88
1-Methylnaphthalene
90-12-0
C025968
7002

89
2-Methoxy-4-vinylphenol
7786-61-0
C014245
332

90
2,6-Dimethylnaphthalene
581-42-0
C028519
11387

91
2-Methylhydroquinone
95-71-6
C062397
7253

92
Methyl eugenol
93-15-2
C005223
7127

93
5-Methylindole
614-96-0
C093726
11978

94
2,3-Dimethylnaphthalene
581-40-8
C091753
11386

95
3-Methylindole
83-34-1
D012862
6736

96
1,4-Dimethylnaphthalene
571-58-4
C031969
11304

97
2,4-Di-tert-butylphenol
96-76-4
C056559
7311

98
Dibenzofuran
132-64-9
C023614
568

99
Phenylephrine
59-42-7
D010656
6041

100
Heptadecane
629-78-7
C016486
12398

101
Spathulenol
6750-60-3
C013258
92231

102
1-Octadecene
112-88-9
C109760
8217

103
Chamazulene
529-05-5
C013872
10719

104
Phenanthrene
85-01-8
C031181
995

105
Octadecane
593-45-3
C022883
11635

106
Pinane
473-55-2
C030216
10129

107
Nonadecane
629-92-5
C061580
12401

108
Hentriacontane
630-04-6
C049203
12410

109
Methyl palmitate
112-39-0
C019012
8181

110
Ambrettolide
123-69-3
C008563
5365703

111
Dibutyl phthalate
84-74-2
D003993
3026

112
Icosane
112-95-8
C050821
8222

3.4. Targets of Toxic Compounds

Through the PubChem database (http://pubchem.ncbi.nlm.nih.gov/), molecular information for the 112 toxic compounds in moxa smoke was identified, and the corresponding number of “Canonical SMILES” was obtained. Then, using the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) to predict the 112 relevant targets of the toxic compounds, the UniProt ID was exported. In addition, the UniProt ID was analyzed with IPA software to obtain the targets of toxic compounds. There were 812 targets for the toxic compounds in moxa smoke, compared to 810 identified with the IPA database.

3.5. Molecular Networks of Toxic Compounds

The UniProt IDs of the 810 target proteins of the 112 toxic compounds were imported into the IPA bioanalysis software. Under the “tox analysis” module, IPA was used to construct the molecular networks of target proteins. A total of 25 molecular networks were constructed for 112 toxic compounds, with a maximum score of 43, as shown in Figure 4. The results showed that these target proteins were related to cell signal transduction, nucleic acid metabolism, inflammatory response, organ damage, and cell apoptosis. Therefore, this can be used to frame a correlation study on moxa smoke.

Figure 4: Molecular network with a maximum score of 43. Each node in the figure represents 1 molecule, the solid lines represent a direct interaction between two molecules, and the dotted lines represent an indirect interaction between two molecules.

3.6. Biological Pathways of Toxic Compounds

Using the “tox analysis” module in the IPA software, a total of 54 biological pathways were found for the 112 toxic compounds. The main biological pathways of the toxic compounds from moxa smoke included cardiotoxicity, hepatotoxicity, and nephrotoxicity. Consequently, the toxic compounds of moxa smoke may have some side effects on the human heart, liver, and kidneys. A heat map of the biological pathway of toxic compounds is shown in Figure 5. According to it, the pathway with the highest −log ( value) was cardiac arteriopathy, which was classified as cardiotoxicity, with a value of 79.429. Drug target molecules acting on this pathway include ABCB1, ABCC8, ACE, ADORA1, ADORA2A, ADORA2B, ADORA3, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, ALDH5A1, ALOX5AP, AR, ASIC3, CA1, CA12, CA13, CA14, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA9, CACNA2D1, CETP, CNR1, CYP2C19, CYP2C9, DPP4, ESR1, ESR2, F10, F2, F2R, FADS1, FKBR1A, FLT1, FLT4, GAA, GABRA1∗, GABRA2∗, GABRA3∗, GABRA5∗, GABRB2∗, GABRB3∗, GABRG2∗, GLP1R, GLRA1, GRIA4, HCAR2, HMGCR, HRH2, HTT, ICAM1, INSR, ITGAL, ITGB2, KCNA5, KCNJ11, KDM1A, KDR, MTNR1A, MTNR1B, MTOR, NOS3, NPC1L1, NR3C1, NR3C2, OPRD1, OPRK1, OPRM1, PDE10A, PDE11A, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE5A, PDE7A, PDE7B, PGR, PLA2G2A, PLA2G7, PLG, PPARA, PPARG, PRCP, PRKCH, PTGER1, PTGER2, PTGER3, PTGER4, PTGIR, PTGS1, PTGS2, RHOA, S1PR1, SCARB1, SCN10A, SCNSA, SCN9A, SELE, SERPINE1, SLC6A4, SOAT1, TBXA2R, TERT, TLR4, TNF, TNNT2∗, TSPO, TUBB1, TUBB3, VDR, VEGFA, and XDH. This also guides the development of follow-up toxicology experiments and research on the effects of moxa smoke on the organs of Sprague Dawley rats.

Figure 5: A heat map of the biological pathway of toxic compounds. The pathway scores are displayed using a purple color gradient, where darker purple corresponds to higher scores (increased statistical significance).

4. Discussion

GC-MS was applied to study the compounds in moxa smoke absorbed in five different polar solvents from Qing moxa sticks. This study found that a total of 294 compounds were identified, including 139 in cyclohexane, 145 in ethyl acetate, 60 in n-butanol, 89 in anhydrous ethanol, 77 in water, and 11 in all five polar solvents. Among the 294 compounds detected in the moxa smoke absorption liquid, 112 compounds were confirmed to be toxic. With the “tox analysis” module, IPA was used to construct molecular networks of target proteins. The results showed that these target proteins were related to cell signal transduction, nucleic acid metabolism, inflammatory response, organ damage, and cell apoptosis. At the same time, the main biological pathways of the toxic compounds from moxa smoke included cardiotoxicity, hepatotoxicity, and nephrotoxicity. The safety of smoke has become greater concern. The question of whether moxa smoke is harmless or not has become key to restricting the use of moxibustion.

At present, most studies on moxa smoke have shown that it has many pharmacological effects. A study [27] showed that the superoxide anion scavenging activity of moxa smoke was superoxide dismutase 24.4 U/mg, which was slightly higher than that of partially purified moxa extract and alkali-lignin, but lower than that of sodium ascorbate, gallic acid, and catechin, which further confirmed the antioxidant and pro-oxidative effects of moxa smoke. The methanol extract of moxa smoke has the functions of antioxidation and eliminates free radicals [28]. Another study demonstrated that moxa smoke can improve sperm concentration and promote sperm movement in rats [29]. Although it was suggested that the toxic compounds in moxa smoke were harmful to the human heart, liver, and kidneys, low and middle concentrations had no effects. Moxa smoke at higher concentrations might destroy heart, liver, and kidney function. In fact, it has been reported that moxa smoke can cause related symptoms, such as eustachian tube and throat itching, eye pain, tonsil enlargement, and other symptoms [30–33]. Tar contains two-tenths of a million of a kind of thick cyclic aromatic hydrocarbon called benzo(a)pyrene, which is a strong carcinogen [34]. In a few cases, patients undergoing moxibustion treatment or after treatment had erythema, blisters, and other hypersensitive symptoms, and these conditions disappeared after leaving the moxa smoke environment [35–37]. Research results show that moxibustion may have a greater impact on some people with chronic pharyngitis, leading to coughing due to moxa smoke allergy, but these symptoms gradually improved after ventilation [38]. Some scholars have placed rats in a dynamic exposure cabinet and observed the content of Ox-LDL in their serum. The results showed that the content of Ox-LDL decreased gradually with the increase of moxa smoke concentration, suggesting that moxa smoke can reduce the degree of platelet aggregation. Therefore, it may improve microcirculation and promote metabolism of the body. Low concentrations of moxa smoke have no noticeable damage to vascular endothelium, while medium concentrations can cause a certain degree of vascular endothelium damage [39, 40].

The moxa sticks were encased in A. argyi floss, which is made of dried A. argyi leaves. There have been many experimental studies on the toxicity of A. argyi, which were not limited to conventional acute toxicity, subacute toxicity, or chronic toxicity. Domestic scholars have conducted in-depth studies on the hepatorenal toxicity, embryonic toxicity, and genetic toxicity caused by A. argyi. The research objects were not limited to the whole animal, but also extended to the cellular level, and the intrinsic mechanism of some toxicity of A. argyi was also discussed. The relationship between quantity, time and toxicity, and a safe time span for use were also discussed. However, some of the results showed that A. argyi had hepatotoxicity, especially the essential oil of A. argyi [41, 42]. The dosage of A. argyi or moxa sticks used in toxic experiments was more than 10 to 200 times the clinical dosage. According to the results of this paper, we carried out toxicological experiment of moxa smoke in rats. We followed the steps outlined in our patent, “A device for enriching moxa smoke and its analytical method,” patent number: CN202010327163.6 [43]. Rats exposed to 756650 mg/m³ concentration of moxa smoke (concentration of moxa smoke in 50 moxa sticks) were compared with the control group, and the structure of myocardial cell, hepatic cell, and the renal tubules showed changes (Supplementary Figure S1) such as cardiac hypertrophy, degeneration and necrosis, and dilatation of renal tubules, respectively.

In a word, we should not discuss the toxicity in terms of toxicity in isolation but should comprehensively consider the clinical use characteristics of traditional Chinese medicine. However, in clinical application, we should pay attention to its “toxicity” to human body and try to avoid overuse. Therefore, moxibustion rooms should have installed ventilation equipment or the room should have adequate artificial ventilation so that the health of patients and practitioners can be guaranteed. The safety of compounds in moxa smoke needs to be further studied. The results of this study provide a basis for a safety evaluation of moxa smoke in the future.

Data Availability

The data used to support the findings of this study are included within the article and in the supplementary figure. The prior studies (and datasets) are cited at relevant places within the text as references [21, 43].

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

L.H.N. conceptualized the study. S.S. and X.X.Y. contributed to methodology and formal analysis, validated the study, and wrote, reviewed, and edited the manuscript. W.W.L. provided software. X.X.Y. wrote and prepared the original draft. L.H.N was responsible for project administration. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

This work was financially subsidized by the 2018 First Class Discipline Construction Project of Jiangxi Province (no. JXSYLXK-ZHYAO145). The authors thank LetPub (http://www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Supplementary Materials

Supplementary Figure S1: microscopic observations of heart, liver, and kidney pathology. (Supplementary Materials)

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