Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2020 / Article
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Research Article | Open Access

Volume 2020 |Article ID 2648759 | https://doi.org/10.1155/2020/2648759

Xiaoyu Xu, Si Shan, Wenlei Wang, Hongning Liu, "Analysis of the Components in Moxa Smoke by GC-MS and Preliminary Discussion on Its Toxicity and Side Effects", Evidence-Based Complementary and Alternative Medicine, vol. 2020, Article ID 2648759, 13 pages, 2020. https://doi.org/10.1155/2020/2648759

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

Academic Editor: Luísa Zuravski
Received20 May 2020
Revised21 Sep 2020
Accepted01 Oct 2020
Published31 Oct 2020

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 [37]. 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 [1416]. 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.

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.

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 15, 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.


No.Rt (min)CompoundRetention indexRelative content (%)

14.038Bicyclo[4.1.0]hept-2-ene7060.033
24.6024-Methyl-1,4-hexadiene7370.175
34.9781-Methylcyclohexene7570.078
45.0931,3,5-Heptatriene7630.115
55.2901,7-Octadiene7740.048
65.4002-Methyl-1-heptene7800.105
75.5141-Octene7860.693
85.947(E)-2-Octene8060.086
96.4581,3-Dimethyl cyclohexene8220.222
106.647(E,E,E)-2,4,6-Octatriene8280.027
117.1115,6-Dimethyl-1,3-cyclohexadiene8430.040
127.7493-Methylenecycloheptene8630.146
138.060(1Z,2Z)-1,2-Di(ethylidene)cyclobutane8730.094
148.334Cyclohexanol8820.703
159.0252-Ethylpyridine9040.095
1610.0642-Methyl-1-octen-3-yne9350.105
1710.1661-Methylcycloheptene9380.141
1811.164Mesitylene9680.307
1911.629Alpha-methyl styrene9810.176
2012.372Gamma-terpinene10040.227
2112.9601,2,4-Trimethylbenzene10220.866
2213.395Trans-beta-methyl styrene10350.371
2314.9731-Phenyl-2-butene10830.228
2415.1811-Methyl-4-(prop-1-en-2-yl)benzene10901.054
2515.901Cosmene11120.163
2616.2102,4-Dimethylstyrene11220.156
2716.3051-Phenyl-1-butene11250.336
2816.4481-Allyl-2-methylbenzene11300.223
2916.749Phenyl acetonitrile11400.610
3017.0512,3-Dimethylphenol11490.687
3117.1551,2,3,4-Tetramethylbenzene11530.203
3217.5101,1a,6,6a-Tetrahydrocycloprop[a]indene11640.258
3318.2411,2-Dimethylindan11880.200
3419.9704-Methylindole12470.200
3520.7927H-Benzocycloheptene12750.200
3621.0452-(2-Hydroxyphenyl)buta-1,3-diene12840.168
3722.3961H-Indene,2,3-dihydro-1,1,3-trimethyl13320.226
3823.4015-Methylindole13690.099
3923.5271,8-Cyclotetradecadiyne13740.108
4024.8161,4-Dimethylnaphthalene14220.227
4125.0731,4,5-Trimethylnaphthalene14320.142
4226.4342,4,6-Trimethylbenzonitrile14850.137
4327.1991-Phenylpyridin-2-one15160.186
4428.4732,4-Dimethoxyacetophenone15680.234
4528.910Spathulenol15860.163
4629.3552-Methylbiphenyl16050.114
4730.735(+)-γ-Gurjunene16650.217
4833.4349-Methylene-9H-fluorene17860.103
4934.4603,7,11,15-Tetramethyl-2-hexadecene18350.062
5034.7102,6,10,14-Tetramethyl-2-hexadecene18470.135
5135.6711-Nonadecene18930.163
5237.669E-15-Heptadecenal19930.123


No.Rt (min)CompoundRetention indexRelative content (%)

14.1063-Methyl-butanenitrile7100.159
24.535Dimethyl aminoacetonitrile7330.191
34.6032,4-Dimethyl-1,3-pentadiene7370.152
45.2363-Methylenecyclohexene7710.095
55.300Cyclooctene7750.030
65.5222-Octene7870.557
75.8372,3-Dimethyl-1,4-hexadiene8020.120
85.957(Z)-2-Octene8060.044
96.462Pyrazine, methyl8220.208
106.8692,5-Dimethylpyrrole8350.088
118.0731,4-Dimethylenecyclohexane8740.051
128.4112,3-Dimethylpyridine8850.166
139.8203-Ethyl-1H-pyrrole9280.295
149.8933,4-Dimethylpyridine9300.509
1510.0782-Ethyl-5,5-dimethyl-1,3-cyclopentadien9350.082
1610.4802-Methylborazine9470.250
1711.2912,5-Cyclooctadien-1-one9710.115
1811.406Benzene9750.060
1911.503Aniline9780.183
2012.1663-Methylstyrene9970.337
2112.385Alpha-phellandrene10040.151
2212.5912-Ethyl-4-methyl-1H-pyrrole10100.295
2313.065o-Cymene10251.118
2413.402Allylbenzene10350.309
2515.0393-Ethyl-o-xylene10850.593
2615.6207-Methylbenzofuran11030.383
2715.921Azulene11130.406
2816.4544-Allyltoluene11300.171
2916.7623-Ethynylaniline11400.367
3017.0653-Methyl-1H-indene11500.557
3117.1631,2,3,4-Tetramethylfulven11530.138
3217.5231,4-Dihydronaphthalene11650.214
3318.2541-Methyl-3-(1-methyl-2-propenyl)benzene11880.188
3418.501Dihydrocarveol11960.140
3518.696Catechol12030.223
3619.0342,6-Dimethylundecane12150.195
3719.250(E)-Cinnamaldehyde12220.391
3819.355Cyclododecene12260.166
3919.758Isoquinoline12390.210
4019.9833-Methylindolizine12470.172
4121.0571,11-Dodecadiene12840.162
4222.7332-Methylhydroquinone13450.174
4322.816Naphthalene, 1,2,3,4-tetrahydro-1, 1-dimethyl13480.125
4423.8252-Methyl-5-(1-methylethenyl)-cyclohexanone13850.131
4523.9553-Methylindole13890.257
4624.8271,3-Dimethylnaphthalene14230.205
4724.9301,6-Dimethylnaphthalene14270.460
4825.7612-Phenyl-1,3-cyclohexadien14590.104
4928.833Phenylephrine15830.020
5031.836Thiazolo[5,4-f]quinolin17130.158
5132.2391,1,2-Trimethylcycloundecane17320.250
5233.442Phenanthrene17870.116
5334.7193,7,11,15-Tetramethyl-2-hexadecene18470.146
5435.480(S)-6,6-Dimethyl-2-azaspiro[4.4]non-1-ene18840.158
5537.6813-Icosene19940.179
5639.61010-Heneicosene (c,t)20930.140
5739.729Heneicosane21000.110


No.Rt (min)CompoundRetention indexRelative content (%)

16.8433-Furaldehyde8341.167
29.8503-Methylheptan-4-one9280.377
39.9982,4-Dimethylpyridine9330.457
410.819Limonene9570.442
512.169Butyl butyrate9976.894
616.330(E)-1-Phenyl-1-butene11260.273
716.771Benzyl(methylidyne)azanium11400.559
818.371cis-2-dodecene11921.064
919.262Tricyclo[3.3.1.0(2,8)]nona-3,6-dien-9-one12220.600
1023.9581-Methylindolizine13900.361


No.Rt (min)CompoundRetention indexRelative content (%)

15.142Thiophene7660.064
26.4882-Methylpyrazine8230.137
39.8112,3-Dimethyl-1H-pyrrole9270.155
411.1706-Methyl-6-ethylfulvene9680.191
512.724Acrylamide10140.098
614.877(−)-Camphor10800.477
714.9742-Methyl-1-phenylpropene10830.137
815.6914-Pyridinol11050.157
917.0593-Phenyl-1,2-butadiene11500.649
1017.518Benzo[2,3]bicyclo[3.1.0]hexane11650.188
1120.713Citral12720.283
1221.0534-Methyl-2H-benzopyrane12840.160
1322.3311,7-Dimethylnaphthalene13300.224
1425.0532,3,6-Trimethylnaphthalene14320.123
1529.065(Z)-8-Hexadecene15930.393
1635.803Nonadecane19000.118
1737.154Dibutyl phthalate19680.425


No.Rt (min)CompoundRetention indexRelative content (%)

14.688Methallyl cyanide7410.065
25.083Cyclopentanone7630.730
35.558Tetrachloroethylene7890.076
46.0104-Aminopyridine8080.470
56.4792-Methylcyclopentanone8230.375
66.5494-Methylpentanenitrile8250.314
76.679(R)-(+)-3-Methylcyclopentanone8290.150
87.8702,6-Dimethylpyridine8670.596
98.091Cyclohexanone8740.378
108.4205,5-Dimethyl-1,3-hexadiene8850.079
118.8162-Ethylpyrazin8980.426
128.9752,3-Dimethylpyrazine9030.126
1310.0002,5-Dimethylpyridine9330.497
1410.3785-Methylfurfural9441.092
1511.178Phenetole9680.185
1611.3461-Isopropylcyclopentene9730.312
1712.1212-Ethyl-6-methylpyridine9960.438
1812.4002-Ethyl-5-methylpyridine10040.282
1912.6875-Ethyl-2-methylpyridine10130.207
2012.7432-Acetyl-5-methylfuran10150.095
2113.5701-Acetyl-2-methyl-1-cyclopentene10400.378
2214.2292-Methyl-6-methylene-2,7-octadien-4-ol10600.540
2314.663Sabinene hydrate10740.750
2414.803p-Tolunitrile10780.511
2515.1682-Methylbenzoxazole10890.568
2615.4192,6-Dimethylphenol10970.820
2715.601Phenylacetone11030.116
2815.6981-Isopropyl-1-cyclohexene11060.226
2916.0454-Ethylphenol11170.681
3016.490Decamethylcyclopentasiloxane11310.372
3116.721Endo-borneol11392.029
3216.9182-Acetyltoluene11450.314
3317.065(-)-Terpinen-4-ol11502.220
3417.2621-(3-Methylphenyl)ethanone11560.818
3517.460(−)-Alpha-terpineol11631.438
3617.581(+)-Dihydrocarvone11670.338
3717.960(+/−)-cis-piperitol11790.350
3818.027Verbenone11810.190
3918.279(−)-cis-carveol11890.357
4018.6102,4-Dimethylanisole12010.248
4119.293Piperitone12230.187
4220.9941-Methylindan-2-one12820.261
4321.356Dodecamethylcyclohexasiloxane12940.193
4422.4853,3-Dimethyl-1-indanone13360.233
4523.314Methyl eugenol13660.229
4623.6692,3-Dimethylnaphthalene13790.146
4726.0012,4-Di-tert-butylphenol14680.644

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.


No.CompoundCAS RNChemical IDPubChem CID

1Toluene108-88-3D0140501140
2Pyrimidine289-95-2C0309869260
31-Methylpyrrole96-54-8C0966547304
4Pyridine110-86-1C0236661049
5Cyclopentanone120-92-3C0072018452
61-Octene111-66-0C0376908125
7Tetrachloroethylene127-18-4D01375031373
8Octane111-65-9C026728356
94-Aminopyridine504-24-5D0157611727
10Ethylbenzene100-41-4C0049127500
11Styrene100-42-5D0200587501
12p-Xylene106-42-3C0312867809
13Furfural98-01-1D0056627362
142,5-Dimethylpyrrole625-84-3C06728612265
152-Furanmethanol98-00-0C0129867361
162-Acetylfuran1192-62-7C03966914505
173-Methylpyridine108-99-6C0536037970
182,6-Dimethylpyridine108-48-5C0130937937
19o-Xylene95-47-6C0261147237
20Butyrolactone96-48-0D0151077302
21Cyclohexanone108-94-1C0364687967
22Phenyl ethyne536-74-3C04473610821
23m-Xylene108-38-3C0312857929
24Propyl benzene103-65-1C0242687668
25Nonane111-84-2C0175738141
262-Ethylpyridine100-71-0C0516727523
27Benzaldehyde100-52-7C032175240
28Cumene98-82-8C0157637406
292,4-Dimethylpyridine108-47-4C0784487936
30Benzofuran271-89-6C1054309223
315-Methylfurfural620-02-0C04806512097
32Phenol108-95-2D019800996
33Limonene138-86-3D00007722222311
343-Ethyltoluene620-14-4C02971912100
351,2,3-Trimethylbenzene526-73-8C01017910686
36Mesitylene108-67-8C0102197947
37Phenetole103-73-1C0794137674
38Eucalyptol470-82-6D0000775912758
39Benzene71-43-2D001554241
40Aniline62-53-3C0236506115
41Alpha-methyl styrene98-83-9C0179157407
42Butyl butyrate109-21-7C0227937983
43Decane124-18-5C01286715600
44Gamma-terpinene99-85-4C0186697461
45Alpha-phellandrene99-83-2C0054037460
465-Ethyl-2-methylpyridine104-90-5C0191967728
47o-Cresol95-48-7C034047335
48Acrylamide79-06-1D0201066579
492-Acetyl-5-methylfuran1193-79-9C05752814514
50p-Cresol106-44-5C0325382879
511,2,4-trimethylbenzene95-63-6C0103137247
52o-Cymene527-84-4C04625710703
53p-Cymene99-87-6C0072107463
54Guaiacol90-05-1D006139460
55m-Cresol108-39-4C042041342
56Allylbenzene300-57-2C1023479309
57Indene95-13-6C0935817219
58Acetophenone98-86-2C0386997410
59Methyl benzoate93-58-3C0446057150
602,6-Dimethylphenol576-26-1C03653111335
61Undecane1120-21-4C02288414257
62Phenylacetone103-79-7C0088637678
634-Pyridinol626-64-2C53414312290
64Naphthalene91-20-3C031721931
65Azulene275-51-4C0055259231
664-Ethylphenol123-07-9C04229131242
674-Allyltoluene3333-13-9C09290376851
68Indolizine274-40-8C0350949230
69Phenyl acetonitrile140-29-4C0067258794
702,3-Dimethylphenol526-75-0C05406710687
711,2,3,4-Tetramethylbenzene488-23-3C02124610263
72(−)-Alpha-terpineol10482-56-1C016775443162
733,5-Dimethylphenol108-68-9C0168347948
74Terpinen-4-ol562-74-3C03401911230
75Dodecane112-40-3C0075488182
76Catechol120-80-9C034221289
775,6-Dimethylbenzimidazole582-60-5C015158675
78Tridecane629-50-5C09407412388
79(E)-Cinnamaldehyde104-55-2C012843637511
80Indole120-72-9C030374798
81Isoquinoline119-65-3C0391098405
82Citral5392-40-5C007076638011
83Hydroquinone123-31-9C031927785
84Biphenyl92-52-4C0105747095
851-Tridecene2437-56-1C02869117095
86Tetradecane629-59-4C02471312389
872-Methylnaphthalene91-57-6C0273847055
881-Methylnaphthalene90-12-0C0259687002
892-Methoxy-4-vinylphenol7786-61-0C014245332
902,6-Dimethylnaphthalene581-42-0C02851911387
912-Methylhydroquinone95-71-6C0623977253
92Methyl eugenol93-15-2C0052237127
935-Methylindole614-96-0C09372611978
942,3-Dimethylnaphthalene581-40-8C09175311386
953-Methylindole83-34-1D0128626736
961,4-Dimethylnaphthalene571-58-4C03196911304
972,4-Di-tert-butylphenol96-76-4C0565597311
98Dibenzofuran132-64-9C023614568
99Phenylephrine59-42-7D0106566041
100Heptadecane629-78-7C01648612398
101Spathulenol6750-60-3C01325892231
1021-Octadecene112-88-9C1097608217
103Chamazulene529-05-5C01387210719
104Phenanthrene85-01-8C031181995
105Octadecane593-45-3C02288311635
106Pinane473-55-2C03021610129
107Nonadecane629-92-5C06158012401
108Hentriacontane630-04-6C04920312410
109Methyl palmitate112-39-0C0190128181
110Ambrettolide123-69-3C0085635365703
111Dibutyl phthalate84-74-2D0039933026
112Icosane112-95-8C0508218222

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.

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.

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 [3033]. 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 [3537]. 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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