Identification of in vitro-in vivo components of Caoguo using accelerated solvent extraction combined with gas chromatography-mass spectrometry integrated with network pharmacology on indigestion.

BACKGROUND: Caoguo (Tsaoko Fructus), a traditional Chinese medicine, is widely used as medicine and dietary spices. Volatile components are among its important bioactive constituents used to treatment of abdominal distension and pain, but the mechanism is not clear up to now. The purpose of this study was to develop a simple, sensitive, and accurate method to analyze and identify components of Caoguo in vitro and in vivo, and further investigate the therapeutic mechanism of Caoguo on indigestion using network pharmacology.
METHODS: Caoguo were extracted by accelerated solvent extraction (ASE) and n-hexane:ethyl acetate (1:1, v/v) was selected as the extraction solvent. Gas chromatography-mass spectrometry (GC-MS) was adopted to analyze and identify the volatile components in vitro and in vivo. Network pharmacology including protein-protein network construction, Gene Ontology (GO) enrichment and pathway enrichment analysis and component-target-pathway network construction was applied.
RESULTS: By comparing the retention times and mass spectrometry data, as well as retrieving the reference literature, a total of 169 components were tentatively identified in Caoguo extract and 43 components were identified in rats plasma samples for the first time. The results of network pharmacology analysis indicated that the potential mechanism was mainly associated with regulation of lipolysis in adipocytes and serotonergic synapse signaling pathway, which might be responsible for the effect of indigestion.
CONCLUSIONS: Caoguo was first extracted by ASE and the volatile chemical components in vivo were first identified by GC-MS. Coupled with network pharmacology analysis, a network of component-target-pathway was constructed to reveal the possible mechanism of Caoguo in treatment of indigestion. This study provided a new reference method for the extraction and analysis of Caoguo, laid a chemical basis for in-depth studies on pharmacodynamics and pharmacology, and revealed an updated understanding of the therapeutic effects of Caoguo on indigestion. 2021 Annals of Translational Medicine. All rights reserved.

Introduction

Caoguo (Tsaoko Fructus), is the dried and mature fruit of Amomum tsao-ko Crevost et Lemaire, exhibits a broad biological activities such as anti-infectious, anti-oxidant, anti-proliferation and α-glucosidase inhibitory activity (1-5). As a herb, Caoguo can used as medicine and dietary spices, which has pharmacological effects including reduce plasma and liver lipids, reduce plasma glucose levels, adjust gastrointestinal metabolic function, anti-inflammatory, analgesic and neuroprotective effects (6-8). Caoguo Zhimu Ddecoction with CaoGuo as the monarch was used in clinical for treatment of epilepsy and chronic renal failure (9,10). Mongolian medicine containing twenty-one Chinese medicines was used in chronic aplastic anemia therapy (11).

Volatile oil is the main components in Caoguo, many methods such as reflux extraction, steam distillation, supercritical fluid CO2 extraction, ultrasonic extraction and microwave extraction have been adopt for volatile oil extraction from Caoguo in recent years (12-16). Different extraction methods and solvents both impact on the components and contents of volatile oil, causing distinct extraction efficiency and yields. Traditional extraction methods such as soaking extraction and ultrasonic extraction are easy to operate, but they are both time-consuming process and a large amount of medicinal materials and solvents are always required, causing difficult to extract the chemical components that are unstable and easily oxidized. While supercritical fluid CO2 extraction method don’t need organic solvents in the whole extraction process and has a higher extraction efficiency for oil, but it is generally suitable for extraction of lipophilicity and small molecular substances and cost much than other extraction methods due to expensive equipment. Accelerated solvent extraction (ASE) technique, as a fast and efficient extraction method with low solvent consumption, has been adopt for active components extraction in many traditional Chinese medicine herbs such as Xanthii Fructus, Salvia miltiorrhiza, Aucklandia lappa Decne (17,18).

The network pharmacology can construct a network model to explain the relationship among the compounds-targets-pathways-diseases by combining the system pharmacology and systems biology. As a paradigm, network pharmacology has been used more and more in traditional Chinese medicine research for the purpose to predict the multi-targets of the mechanism of diseases treatment (19-21).

This study is aimed to apply ASE combined gas chromatography-mass spectrometry (GC-MS) method to extract and identify the in vivo and in vitro volatile constituents of Caoguo. Based on the identified components, network pharmacology analysis was proceeded to predict the protein/gene targets and signaling pathways of Caoguo in the treatment of indigestion. We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi.org/10.21037/atm-21-3245).

Methods

Reagents and materials

Dried Caoguo, derived from the Yunnan province (China), was purchased from Beijing Tongrentang of Chinese medicinal material in Shenyang, Liaoning, China. Reference standards (purity >98%) of α-pinene, β-pinene, eucalyptol, nerolidol, and α-terpineo were purchased from SinoStandards (Chengdu, Sichuan, China) for GC-MS analysis. Distilled water was provided by Wahaha (Hangzhou, Zhejiang, China). Methanol (chromatographic grade) was purchased from Sigma-Aldrich Company (Shanghai, China). Ethyl acetate and hexane of chromatographic grade were obtained separately from Tianjin Fuyu Chemical Limited Company (Tianjin, China) and Shandong Yuwang Industrial (Yucheng, Shandong, China).

Preparation of Caoguo extract

The Caoguo samples were extracted by ASE, which was carried out with a Dionex ASE 350 (Thermo Fisher Scientific, Waltham, MA, USA). All extractions were equipped with 34 mL capacity stainless steel cells. A cellulose filter was placed at the bottom of the extraction cell, and then 5 g Caoguo powder mixed with a small amount of diatomaceous earth was poured into the extraction cell. Then, the remaining void was filled with diatomaceous earth, with a cellulose filter placed on the top. Mixture solvent of n-hexane:ethyl acetate (1:1, v/v) was used as extraction solvent. Three static cycles of each 15 min under pressure of 1,500 psi (10.3 MPa) and temperature of 110 °C, then the extraction cell was flushed with solvent (100% of the cell volume) and purged with nitrogen for 90 s. All the extracts were collected in 250 mL bottles and filtered.

Animals dosing and sampling

A total of 12 male pathogen-free Sprague-Dawley (SD) rats weighing 180–220 g were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. Rats were housed and bred under a 12 h dark-light cycle at suitable room temperature (20–25 °C) and humidity (40–70%). Rats were placed in this conditions for one week with free access to water and standard rodent food before experiment. Experiments were performed under a project license (No.: SYPU-IACUC-C2018-12-26-103) granted by institutional ethics board of Shenyang Pharmaceutical University, in compliance with Shenyang Pharmaceutical University institutional guidelines for the care and use of animals. All the rats were randomly divided into two groups, with six in each group, and fasted overnight prior to the experiments. Caoguo extract was redissolved with 0.5% carboxymethyl cellulose sodium (CMC-Na) solution and diluted to an appropriate concentration (0.1 g/mL). A volume of 10 mL/kg (equivalent to raw medicine 100 g/kg) was intragastrically administrated to rats in the treatment group and the same volume of 0.5% CMC-Na was intragastrically administrated to rats in the vehicle group, doses in both groups were once every two hours for a total of three times. After the last dosing, plasma samples (~0.5 mL) were collected into heparinized tubes from the post-orbital venous plexus at 0.25, 0.5, 075, 1, 1.5, 2, and 3 h. Then, the samples were centrifuged with speed of 4,000 rpm for 10 min. The samples were stored at −80 °C before analysis.

Sample preparation of Caoguo extract and rats plasma for GC-MS analysis

The Caoguo extract processed by ASE was dissolved in an appropriate amount of n-hexane/ethyl acetate (1:1, v/v) and vortex-mixing for 5 min, after stand still for 3 min and then was centrifuged at 4,000 rpm for 10 min. The supernatant was transferred to sampler vials and an aliquot of 1 µL was injected into the GC-MS system for analysis.

Plasma samples of 200 µL were mixed with 200 µL n-hexane, vortexed for 5 min and then rested for 5 min. Then, the samples were centrifuged at 4,000 rpm for 10 min. The supernatant was transferred to sampler vials and an aliquot of 1 µL of the organic extracts supernatant was injected into the GC-MS system for analysis.

GC-MS conditions and compound identification

The volatile compounds were analyzed using a GC-MS system, comprising a Trace 1300 GC system combined with a Triplus RSH autosampler, an ISQ MS system, and X calibur software for data analysis (all components manufactured by Thermo Fisher). Separation was accomplished on a TS-5MS column (30 m × 0.25 mm, 0.25 µm; Thermo Fisher). Helium was used as carrier gas at a flow rate of 1.0 mL/min, and split injection was used with a ratio of 10:1. Temperature programming conditions for qualitative analysis were as follows: the initial oven temperature was set at 40 °C for 3 min, increased to 130 °C by increments of 10 °C/min keeping for 5 min, finally increased to 280 °C at a rate of 6 °C/min and retained for 15 min. The inlet temperature was set at 280 °C. Electron ionisation mode (EI) with ionization energy at 70 eV was used for mass analysis operating in full scan mode (m/z 60-500).

The chemical components were identified by comparing to the reference spectra from the National Institute of Standards and Technology (NIST) library combined with literature, reference standards and manual analysis. Information of volatile chemical components in Caoguo was also searched from the literature and collected to conduct an Excel database for compound identification and comparison.

Active components screening and target genes prediction

The chemical compounds were obtained from the traditional Chinese medicine systems pharmacology database (TCMSP) database (http://tcmspw.com/) coupled with identification in this study. Then bioactive compounds with oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18 were selected for further study. The targets of the bioactive components above were collected from the TCMSP database.

Target genes for indigestion

Indigestion-related target proteins were collected from the biological targets related to indigestion which were selected from the GeneCards (https://www.genecards.org/) database and then put them into UniProt databases (http://www.uniprot.org/) to search the reviewed target genes of human species

Protein-protein interaction (PPI) network

The acquired intersection target genes were submitted to the Search Tool for the Retrieval of Interacting Genes/Proteins (http://string-db.org/) with the organism set to “homo sapiens”, the PPIs with the confidence score of >0.4 were reserved and then ranked in network by Analyze Network tool in Cytoscape. Furthermore, a PPI network of closely related proteins was made by using the network visualization software Cytoscape (http://cytoscape.org/).

Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and the compound-target-pathway network construction

The GO enrichment analysis and KEGG pathway enrichment analysis were proceeded. The species were set to “homo sapiens” to predict the potential biological mechanisms in biological process, cell component, molecular function and illustrate the target genes expressed in the pathways.

The network composed of the bioactive compounds, target genes and enriched pathways were constructed by importing data into Cytoscape software.

Results

Volatile components identification in vitro and in vivo

The typical total ion chromatogram of the Caoguo extract is shown in . All the main components were completely separated within 57 min. The identification results of the GC-MS analysis of Caoguo extract including compound names, molecular weight, molecular formula, retention time and content are presented in . A total of 169 components were identified in Caoguo extract processed by ASE. Their relative contents were calculated by peak area normalization method. The compounds in Caoguo extract were classified into 18 categories. The prevailing compounds found in Caoguo extract were 39 alcohols (37.1%), 21 aldehydes (11.71%), 5 sterols (10.37%), 21 esters (10.27%), 19 ketones (6.21%), 11 alkanes (4.56%), 16 organic acids (4.27%), 3 terpenes (3.23%), 6 alkylene oxides (3.20%) and 11 olefins (3.01%).

Figure 1

The GC-MS chromatogram of Caoguo extract processed by ASE. GC-MS, gas chromatography-mass spectroscopy; ASE, accelerated solvent extraction.

Table 1

The identified compounds of Caoguo processed by ASE

No.	Retentiontime (min)	Compound name	Molecular formula	Molecular weight	Content (%)
1	4.53	2-Methyl-2-propan-2-yloxirane	C6H12O	100.09	0.03
2	4.65	Hexan-3-ol	C6H14O	102.10	0.01
3	4.71	Hexanal	C6H12O	100.09	0.15
4	4.83	Hex-3-enal	C6H10O	98.07	0.02
5	6.48	13-Heptadecyn-1-ol	C17H32O	252.25	0.08
6	6.59	2-Butylfuran	C8H12O	124.09	0.02
7	6.77	Heptanal	C7H14O	114.10	0.77
8	8.26	3-Methylpentanoic acid	C6H12O2	116.08	0.02
9	8.39	6-Methylhept-5-en-2-one	C8H14O	126.10	0.08
10	8.48	α-Pinene	C10H16	136.13	1.23
11	8.58	3-Ethenylhex-2-en-1-ol	C8H14O	126.10	0.01
12	8.67	Octanal	C8H16O	128.12	0.55
13	8.94	β-Pinene	C10H16	136.13	1.44
14	9.08	1-Methyl-2-propan-2-ylbenzene	C10H14	134.11	0.20
15	9.20	Eucalyptol	C10H18O	154.14	3.81
16	9.30	(3Z)-3,7-Dimethylocta-1,3,6-triene	C10H16	136.13	0.01
17	9.48	Z,Z,Z-1,4,6,9-Nonadecatetraene	C19H32	260.25	0.04
18	9.57	1,1-Dimethyl-2-(2-methylbut-3-en-2-yl)cyclopropane	C10H18	138.14	0.02
19	9.63	(E)-Oct-2-enal	C8H14O	126.10	0.05
20	9.67	3,7,7-Trimethylbicyclo[4.1.0]hept-3-ene	C10H16	136.13	0.56
21	9.91	Heptanoic acid	C7H14O2	130.10	0.14
22	10.21	2-Hexyl-furan	C10H16O	152.12	0.19
23	10.29	3-Methyl-2-(2-methyl-2-butenyl)-furan	C10H14O	150.10	0.06
24	10.34	1-Methyl-4-prop-1-en-2-ylcyclohexan-1-ol	C10H18O	154.14	0.12
25	10.40	Nonanal	C9H18O	142.14	0.61
26	10.48	(Z)-Icos-11-enoic acid	C20H38O2	310.29	0.08
27	11.17	2,7,7-Trimethyl-3-oxatricyclo[4.1.1.02,4]octane	C10H16O	152.12	0.15
28	11.36	(8Z,11Z,14Z)-icosa-8,11,14-trienoic acid	C20H34O2	306.26	0.07
29	11.44	Oleic Acid	C18H34O2	282.26	0.37
30	11.52	1,3-Dimethylcyclohexene	C8H14	110.11	0.17
31	11.62	4,7,7-Trimethylbicyclo[4.1.0]hept-4-en-3-ol	C10H16O	152.12	0.22
32	11.69	2-Pentadec-12-ynoxyoxane	C20H36O2	308.27	0.10
33	11.81	α-Terpineol	C10H18O	154.14	3.27
34	11.91	Benzene-1,2-diol	C6H6O2	110.04	0.11
35	11.98	[(1S,3S,5S)-4-Methylidene-1-propan-2-yl-3-bicyclo[3.1.0]hexanyl] acetate	C10H16O	152.12	0.63
36	12.12	2-Methylheptan-2-ol	C8H18O	130.14	0.06
37	12.18	1-Ethenyl-4-ethoxybenzene	C10H12O	148.09	0.03
38	12.32	(R)-3,7-Dimethyl-6-octen-1-ol	C10H20O	156.15	0.15
39	12.42	3-Methyl-3-(4-methylpent-3-enyl)oxirane-2-carbaldehyde	C10H16O2	168.12	0.04
40	12.75	Geraniol	C10H18O	154.14	4.88
41	12.88	(Z)-Dec-2-enal	C10H18O	154.14	0.80
42	12.99	Dec-2-en-1-ol	C10H20O	156.15	0.77
43	13.05	(2E)-3,7-Dimethylocta-2,6-dienal	C10H16O	152.12	0.97
44	13.16	Hexadec-9-enoic acid	C16H30O2	254.22	0.03
45	13.34	Ethyl iso-allocholate	C26H44O5	436.32	0.03
46	13.48	α-Ethylbenzeneacetaldehyde	C10H12O	148.09	0.82
47	13.63	2,6-Dimethyl-octa-2,6-diene-1,8-diol	C10H18O2	170.13	0.49
48	13.73	2-[(Z)-Octadec-9-enoxy]ethanol	C20H40O2	312.30	0.02
49	13.83	Tetracyclo[5.2.1.01,5.05,9]decan-8-one	C10H12O	148.09	0.34
50	14.02	(2S,3R)-2,6,6-Trimethylbicyclo[3.1.1]heptane-2,3-diol	C10H18O2	170.13	2.34
51	14.31	11,12-Dioxatetracyclo[4.3.1.1(3,10).1(2,5)]dodecane	C10H14O2	166.10	0.79
52	14.39	1a,2,3,3a,4,5,6,7-Octahydroindeno[1,7a-b]oxirene	C9H14O	138.10	0.96
53	14.55	1,2-Dimethylcyclooctane	C12H24	168.19	0.21
54	14.63	2,3-Dihydro-1H-indene-4-carbaldehyde	C10H10O	146.07	0.28
55	14.75	3-Ethyl-4-methyl-3-hepten-2-on	C10H18O	154.14	0.56
56	14.91	2-Propenoic acid octyl ester	C11H20O2	184.15	0.45
57	15.06	n-Decanoic acid	C10H20O2	172.15	0.46
58	15.17	1,4-Cyclododecanedione	C12H20O2	196.15	0.15
59	15.29	5-Methyl-2-prop-1-en-2-ylhex-4-enal	C10H16O	152.12	0.06
60	15.42	2-Methyl-3-phenylprop-2-enal	C10H10O	146.07	0.25
61	15.55	Geranyl acetate	C12H20O2	196.15	0.60
62	15.66	2-Cyclopentylcyclopentan-1-one	C10H16O	152.12	0.10
63	15.77	(2Z)-3,7-Dimethyl-2-octen-1-yl 2-methylpropanoate	C14H26O2	226.19	0.40
64	15.93	Bicyclo(3.1.1)heptane-2,3-diol,2,6,6-trimethyl	C10H18O2	170.13	0.53
65	16.07	Vanillin	C8H8O3	152.05	0.49
66	16.15	(E)-2-Dodecenoic acid	C10H18O2	170.13	0.21
67	16.42	Cis-bicyclo[4.4.0]decan-1-ol-3-one	C10H16O2	168.12	0.13
68	16.63	6-Methyl-7-oxabicyclo[4.1.0]heptan-2-one	C10H16O2	168.12	0.73
69	16.95	p-Menth-1-en-3-one	C10H16O2	168.12	0.25
70	17.05	1-(2-Nitro-2-propen-1-yl)cyclohexene	C9H13NO2	167.09	0.31
71	17.20	Tricyclo[7.1.0.01,3]decane-2-carbaldehyde	C11H16O	164.12	0.60
72	17.46	2-Methylene-5α-cholestan-3α-ol	C28H48O	400.37	0.12
73	17.54	(E)-Docos-13-enoic acid	C22H42O2	338.32	0.11
74	17.64	2-Hydroxy-6-methyl-3-(1-methylethyl)-2-Cyclohexen-1-one	C10H16O2	168.12	0.28
75	17.78	(E)-1-Ethoxy-4,4-dimethylpent-2-ene	C9H18O	142.14	0.50
76	18.01	(3-Cyclopropyl-7-bicyclo[4.1.0]heptanyl)methanol	C11H18O	166.14	0.59
77	18.16	Ethyl 8-[3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl]octanoate	C20H36O4	340.26	0.65
78	18.25	2-Dodecenal	C12H22O	182.17	0.74
79	18.41	Trans-2-dodecen-1-ol	C12H24O	184.18	1.42
80	18.59	3-(4-Hydroxyphenyl) propanal	C9H10O2	150.07	0.19
81	19.59	3-Cyclohexene-1-methanol,2-hydroxy-α, α,4-trimethyl	C10H18O2	170.13	0.38
82	19.70	9,12,15-Octadecatrienoic acid	C25H40O6	436.28	0.03
83	19.86	(3S,3aR,3bR,4S,7R,7aR)-3,7-Dimethyl-4-(propan-2-yl)octahydro-1H-cyclopenta[1,3]cyclopropa[1,2]benzen-3-ol	C15H26O	222.20	0.68
84	20.02	1-[(Z)-3-Ethoxy-1-Propenyl]-1-Cyclohexene	C11H18O	166.14	0.24
85	20.10	5-Tert-butylpyrogallol	C10H14O3	182.09	0.25
86	20.44	Cyclopentaneacetaldehyde,2-formyl-3-methyl-α-methylene	C10H14O2	166.10	3.71
87	20.72	(4Z)-11-Oxabicyclo[8.1.0]undec-4-ene	C10H16O	152.12	2.06
88	20.78	[s- (Z, Z)]-α, α,4,8-Tetramethyl-3,7-Cyclodecadiene-1-methanol	C15H26O	222.20	1.48
89	20.99	(3-Cyclopropyl-7-bicyclo[4.1.0]heptanyl)methanol	C11H18O	166.14	0.57
90	21.13	Nerolidol	C15H26O	222.20	4.46
91	21.61	Bicyclo[3.3.1]nonane-2,9-diol	C9H16O2	156.12	0.46
92	21.88	Methyl octadeca-2,5-diynoate	C19H30O2	290.22	0.08
93	22.09	2-Dodecenoic acid	C12H22O2	198.16	0.36
94	22.22	[(E)-Dec-2-enyl] acetate	C12H22O2	198.16	0.61
95	22.31	(2E,4Z,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide	C16H25NO	247.19	0.14
96	22.47	Methyl (8E,11E,14E)-docosa-8,11,14-trienoate	C23H40O2	348.30	0.21
97	22.76	2-Methyl-5-propan-2-yl-7-azabicyclo[4.1.0]heptane	C10H19N	153.15	0.18
98	22.89	Guaia-1(10),11-diene	C15H24	204.19	0.46
99	23.12	3,7-Dioxatetracyclo[6.4.0.02,6.04,9]dodecane	C10H14O2	166.10	0.14
100	23.33	2-[(2R,4aR,8aS)-4a-Methyl-8-methylidene-1,2,3,4,5,6,7,8a-octahydronaphthalen-2-yl]propan-2-ol	C15H26O	222.20	0.39
101	23.40	α-Acorenol	C15H26O	222.20	2.06
102	23.47	4-Hydroxy-3,5-dimethoxybenzaldehyde	C9H10O4	182.06	0.31
103	23.72	Pentadec-2-yn-1-ol	C15H28O	224.21	0.15
104	23.79	Caryophyllene oxide	C15H24O	220.18	0.09
105	23.85	3-Cyclohex-3-en-1-ylpropanal	C9H14O	138.10	0.14
106	23.95	Tricyclo[5.2.2.1(2,6)]dodecan-12-ol	C12H20O	180.15	1.41
107	24.18	1,4-Benzenedicarboxaldehyde,2,5-dimethyl	C10H10O2	162.19	0.16
108	24.39	Methyl (E)-heptadec-10-en-8-ynoate	C18H30O2	278.22	0.06
109	25.08	3-Ethoxy-1,4,4a,5,6,7,8,8a-octahydroisoquinoline	C11H19NO	181.15	0.35
110	25.34	(2E,6E)-9-(3,3-Dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol	C15H26O2	238.19	0.06
111	25.59	3-Oxatetracyclo[5.5.0.01,8.04,8]dodecan-6-one	C11H14O2	178.10	0.28
112	25.95	2(1H)-Naphthalenone,4a,5,6,7,8,8a-hexahydro-7à-isopropyl-4aá,8aá-dimethyl	C15H24O	220.18	0.15
113	26.44	(E)-Undec-2-enoic acid	C11H20O2	184.15	0.19
114	26.64	Tricyclo[5.2.1.04,8]decan-5-ol	C10H16O	152.12	1.55
115	26.74	(1R,4aR,7R,8aR)-7-(2-hydroxypropan-2-yl)-1,4a-Dimethyl-2,3,4,5,6,7,8,8a-octahydronaphthalen-1-ol	C15H28O2	240.21	0.49
116	27.39	1-Ethenoxy-3,7-dimethylocta-2,6-diene	C12H20O	180.15	0.19
117	27.57	Isoaromadendrene epoxide	C15H24O	220.18	0.18
118	27.64	3,5,9-Trimethyl-2-methylidenetricyclo[6.3.0.01,5]undec-3-ene	C15H22	202.17	0.16
119	27.83	4,8a-Dimethyl-6-prop-1-en-2-yl-2,3,5,6,7,8-hexahydro-1H-naphthalen-2-ol	C15H24O	220.18	0.18
120	28.11	(2-Methyl-5-prop-1-en-2-ylcyclohex-2-en-1-yl) 2,2-dimethylpropanoate	C15H24O2	236.18	0.38
121	28.28	5-(2,3-Dimethyl-3-tricyclo[2.2.1.02,6]heptanyl)pentan-2-one	C14H22O	206.17	0.21
122	28.42	2-Heptadec-7-ynoxyoxane	C22H40O2	336.30	0.06
123	29.35	Hexadecanoic acid	C16H32O2	256.24	0.78
124	29.56	Trans-Z-à-Bisabolene epoxide	C15H24O	220.18	0.39
125	29.68	[(2Z,6E)-10,11-Dihydroxy-3,7,11-trimethyldodeca-2,6-dienyl] acetate	C17H30O4	298.21	0.20
126	29.76	Methyl pentacosa-10,12-diynoate	C26H44O2	388.33	0.21
127	29.87	Methyl tricosa-10,12-diynoate	C24H40O2	360.30	0.37
128	30.32	2,6-Dimethyl-3-octyl-1,2,3,4,4a,5,6,7,8,8a-decahydronaphthalene	C20H38	278.30	0.16
129	30.39	2,6,6,8-Tetramethyltricyclo[5.3.1.01,5]undec-8-en-3-one	C15H22O	218.17	0.36
130	30.49	(6E,10E)-3,7,11,15-Tetramethylhexadeca-1,6,10,14-tetraen-3-ol	C20H34O	290.26	0.12
131	30.79	31,32-Dioxapentacyclo[20.8.1.17,16.01,22.07,16]dotriacontane	C30H52O2	444.40	0.35
132	30.85	Methyl 8-(2-hexyl-5,6-dihydro-2H-naphthalen-4a-yl)octanoate	C25H40O2	372.30	0.41
133	31.44	α-Ylangene	C15H24	204.19	0.68
134	31.52	(5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenoic acid	C20H30O2	302.22	0.38
135	31.70	Methyl octadeca-11,14-diynoate	C19H30O2	290.22	0.40
136	31.82	Urs-12-en-28-ol	C30H50O	426.39	0.66
137	32.09	Cholest-22-ene-21-ol,3,5-dehydro-6-methoxy-, pivalate	C33H54O3	498.41	0.23
138	32.15	3,7,11-Trimethyldodeca-1,6,10-trien-3-yl formate	C16H26O2	250.19	0.34
139	32.23	(2E,6E)-1-Methoxy-3,7,11-trimethyldodeca-2,6,10-triene	C16H28O	236.21	1.34
140	32.34	Methyl (6E,9E,12E,15E)-docosa-6,9,12,15-tetraenoate	C23H38O2	346.29	0.13
141	32.63	2,3-Dihydroxypropyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate	C21H36O4	352.26	0.06
142	32.83	5,6,7,8,9,10,11,12,13,14-Decahydrocyclododeca[b]pyridine	C15H23N	217.18	0.72
143	32.91	2-[4-Methyl-6-(2,6,6-trimethylcyclohexen-1-yl)hexa-1,3,5-trienyl]cyclohexene-1-carbaldehyde	C23H32O	324.25	0.14
144	33.04	4,6,6-Trimethyl-2-(3-methylbuta-1,3-dienyl)-3-oxatricyclo[5.1.0.02,4]octane	C15H22O	218.17	1.36
145	33.27	Octadec-6-ynenitrile	C18H31N	261.25	0.44
146	33.51	(3E,6E)-6-Methyl-5-propan-2-ylidenedeca-3,6,9-trien-2-one	C14H20O	204.15	0.35
147	33.90	5-Hydroxy-6-octylquinoline-7,8-dione	C17H21NO3	287.15	0.64
148	34.19	Cyclohexanepropanol,2,2-dimethyl-6-methylene	C12H22O	182.17	0.66
149	34.53	3,7,11,15-Tetramethylhexadeca-1,6,10,14-tetraen-3-ol	C20H34O	290.26	0.45
150	34.61	3-Ethyl-3-hydroxy-5alpha-androstan-17-one	C21H34O2	318.26	0.35
151	34.68	2-Methyl-4-(2,6,6-trimethylcyclohexen-1-yl)but-2-en-1-ol	C14H24O	208.18	0.51
152	34.78	1-Methyl-4-[(2Z)-6-methylhepta-2,5-dien-2-yl]-7-oxabicyclo[4.1.0]heptane	C20H34O	290.26	0.45
153	35.06	(2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-ol	C20H34O	290.26	0.28
154	35.19	(8E)-2,6,6,10-Tetramethylundeca-8,10-diene-3,7-dione	C15H24O2	236.18	0.37
155	35.66	7,9-Octadecadiynoic acid, DMOX derivative	C22H35NO	329.27	0.56
156	35.71	9,11-Octadecadiynoic acid, DMOX derivative	C22H35NO	329.27	0.48
157	35.97	4α-Methylcholesta-8,24-dien-3α-ol	C28H46O	398.35	1.40
158	36.05	5-Hydroxy-4-nitro-3,4,4a,5,6,7,8,8a-octahydro-2H-naphthalen-1-one	C10H15NO4	213.10	0.46
159	37.91	Bis(6-methylheptyl) benzene-1,2-dicarboxylate	C24H38O4	390.28	2.97
160	38.95	2-Cyclohexyl-4a,7-dimethyl-4,5,6,8a-tetrahydro-3H-1,2-benzoxazine-3-carbonitrile	C17H26N2O	274.20	0.82
161	40.36	[(2E)-3,7-Dimethylocta-2,6-dienyl] hexadecanoate	C26H48O2	392.37	0.95
162	40.70	(Z)-Docos-13-enamide	C22H43NO	337.33	0.64
163	42.41	Geranyl oleate	C28H50O2	418.38	0.97
164	42.51	[(2E)-3,7-Dimethylocta-2,6-dienyl] (9Z,12Z,15Z)-octadeca-9,12,15-trienoate	C28H46O2	414.35	0.35
165	45.41	Vitamin E	C29H50O2	430.38	0.86
166	47.16	Ergost-5-en-3α-ol	C28H48O	400.37	0.73
167	47.84	Stigmasterol	C29H48O	412.37	2.13
168	49.17	γ-Sitosterol	C29H50O	414.39	5.99
169	52.53	Stigmast-4-en-3-one	C29H48O	412.37	0.42

ASE, accelerated solvent extraction.

The total ion chromatograms of Caoguo extract in rat plasma is depicted in . The results of GC-MS analysis of the volatile components of Caoguo extract processed by ASE in rat plasma is shown in . Comparing to the plasma sampled from the vehicle group, 43 constituents were identified in rats plasma after dosing Caoguo extract. Among the 43 components, the prevailing compounds were attributed to 15 alkanes (30.37%), 6 esters (20.13%), 4 olefins (15.37%), 5 alcohols (6.57%) and 6 organic acids (3.52%). The following five compounds including bis(6-methylheptyl) benzene-1,2-dicarboxylate (13.72%), cholesta-3,5-diene (8.61%), 11-decyltetracosane (5.12%), 11-pentan-3-ylhenicosane (4.88%) and methyl (E)-octadec-6-enoate (3.85%) were higher in the plasma.

Figure 2

The total ion chromatograms of Caoguo processed by ASE in rats plasma. ASE, accelerated solvent extraction.

Table 2

The identified compounds of Caoguo processed by ASE in rats plasma

No.	Retentiontime (min)	Compound name	Molecular formula	Molecular weight	Content (%)
1	4.04	Cyclohepta-1,3,5-triene	C7H8	92.06	3.25
2	6.13	1,3-Xylene	C8H10	106.08	2.22
3	6.46	(Z)-Octadec-9-enal	C18H34O	266.26	3.08
4	7.42	(5Z,8Z,11Z,14Z,17Z)-Icosa-5,8,11,14,17-pentaenoic acid	C20H30O2	302.22	0.10
5	8.23	β-Pinene	C10H16	136.13	2.78
6	9.21	Eucalyptol	C10H18O	154.14	1.43
7	9.74	2,4,6-Trimethyldecane	C13H28	184.22	0.29
8	10.41	Nonanal	C9H18O	142.14	0.87
9	10.63	3-Methyl-2,3-dihydro-benzofuran	C9H10O	134.07	0.22
10	11.81	(4Z,6Z,9Z)-Nonadeca-4,6,9-triene	C19H34	262.27	0.43
11	13.21	6-Methyloctadecane	C19H40	268.31	0.22
12	20.19	3,7,11-Trimethyldodecan-1-ol	C15H32O	228.25	0.21
13	21.62	[(2E)-3,7-Dimethylocta-2,6-dienyl] 3-methylbutanoate	C15H26O2	238.19	0.73
14	21.93	Hexadec-9-enoic acid	C16H30O2	254.22	0.60
15	22.23	(22E)-Ergosta-5,22-dien-3-yl acetate	C30H48O2	440.37	0.74
16	24.58	2,6,10,15-Tetramethylheptadecane	C21H44	296.34	2.39
17	24.76	2,6,10-Trimethyltetradecane	C17H36	240.28	0.39
18	25.49	2-Methylicosane	C21H44	296.34	1.60
19	25.59	Icosan-2-ylcyclohexane	C26H52	364.41	0.91
20	31.53	(8Z,11Z,14Z)-Icosa-8,11,14-trienoic acid	C20H34O2	306.26	0.25
21	31.62	Methyl (E)-octadec-6-enoate	C19H36O2	296.27	3.85
22	32.35	2-Pentadecyl-1,3-dioxocane	C21H42O2	326.32	0.02
23	32.80	5,8-Diethyldodecane	C16H34	226.27	0.14
24	33.24	Oleic Acid	C18H34O2	282.26	0.09
25	34.31	(2-Phenyl-1,3-dioxolan-4-yl)methyl (Z)-octadec-9-enoate	C28H44O4	444.32	2.07
26	34.53	1,25-Dihydroxyvitamin D3,	C30H52O3Si	488.37	0.06
27	35.93	Hexadecan-2-ol	C16H34O	242.26	1.06
28	37.90	Bis(6-methylheptyl) benzene-1,2-dicarboxylate	C24H38O4	390.28	13.72
29	38.06	(2-Phenyl-1,3-dioxolan-4-yl)methyl (Z)-octadec-9-enoate	C28H44O4	444.32	0.07
30	38.51	2-Methyloctadecane	C19H40	268.31	2.39
31	38.68	16-Hydroxy-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-1(12)-ene-6,2'-oxane]-11-one	C27H40O4	428.29	0.08
32	38.77	Ethyl iso-allocholate	C26H44O5	436.32	1.02
33	39.73	7-Hexylicosane	C26H54	366.42	3.00
34	40.65	1,1,3,3,5,5,7,7,9,9,11,11-Dodecamethylhexasiloxane	C12H38O5Si6	430.13	0.86
35	40.73	(Z)-Icos-11-enamide	C20H39NO	309.30	2.17
36	40.91	11-Pentan-3-ylhenicosane	C26H54	366.42	4.88
37	42.14	Cholesta-3,5-diene	C27H44	368.34	8.61
38	43.27	11-Decyltetracosane	C34H70	478.55	5.12
39	44.69	3-Ethyl-5-(2-ethylbutyl)octadecane	C26H54	366.42	3.70
40	45.23	Cholesterol	C27H46O	386.35	3.81
41	46.40	9-Hexylheptadecane	C23H48	324.38	3.10
42	47.07	2-(3-Acetoxy-4,4,14-trimethylandrost-8-en-17-yl)-Propanoic acid	C27H42O4	430.31	0.41
43	48.45	1-Chloroheptacosane	C27H55CL	414.40	3.03

ASE, accelerated solvent extraction.

Among the chemical components, β-pinene, nonanal, eucalyptol, ethyl iso-allocholate, oleic acid, hexadec-9-enoic acid, (5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenoic acid, (8Z,11Z,14Z)-Icosa-8,11,14-trienoic acid and bis(6-methylheptyl) benzene-1,2-dicarboxylate were identified both in Caoguo extract and rat plasma samples.

Network pharmacology analysis

According to the screen criteria, twelve active components with 52 target genes in Caoguo were filtered out in the subsequent analysis ().

Table 3

Twelve active compounds in Caoguo were retrieved from databases and previous experiments

MOL ID	Molecule name	Target name	Target
MOL000073	ent-Epicatechin	Prostaglandin G/H synthase 1	PTGS1
		Estrogen receptor	ESR1
		Prostaglandin G/H synthase 2	PTGS2
		Heat shock protein HSP 90	HSP90
		Beta-lactamase	#N/A
		mRNA of PKA Catalytic Subunit C-alpha	#N/A
MOL000074	(4E,6E)-1,7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one	Estrogen receptor	ESR1
		Peroxisome proliferator activated receptor gamma	PPARG
		Prostaglandin G/H synthase 2	PTGS2
		Beta-2 adrenergic receptor	ADRB2
		Mitogen-activated protein kinase 14	MAPK14
		Leukotriene A-4 hydrolase	LTA4H
		mRNA of PKA Catalytic Subunit C-alpha	#N/A
MOL000085	beta-daucosterol_qt	Progesterone receptor	PGR
MOL000096	(-)-catechin	Prostaglandin G/H synthase 1	PTGS1
		Estrogen receptor	ESR1
		Prostaglandin G/H synthase 2	PTGS2
		Heat shock protein HSP 90	HSP90
		Beta-lactamase	#N/A
		mRNA of PKA Catalytic Subunit C-alpha	#N/A
		Nuclear receptor coactivator 2	NCOA2
		Calmodulin	CALM
		Fatty acid synthase	FASN
		Peroxisome proliferator-activated receptor gamma	PPARG
		Krueppel-like factor 7	KLF7
MOL000098	quercetin	Prostaglandin G/H synthase 1	PTGS1
		Androgen receptor	AR
		Peroxisome proliferator activated receptor gamma	PPARG
		Prostaglandin G/H synthase 2	PTGS2
		Heat shock protein HSP 90	HSP90
		Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, gamma isoform	PIK3CG
		Nuclear receptor coactivator 2	NCOA2
		Dipeptidyl peptidase IV	DPP4
		Aldose reductase	AR
		Trypsin-1	PRSS1
		DNA topoisomerase II	TOP2
		Thrombin	#N/A
		Potassium voltage-gated channel subfamily H member 2	KCNH2
		Sodium channel protein type 5 subunit alpha	SCN5A
		Coagulation factor Xa	F10
MOL005970	Eucalyptol	Prostaglandin G/H synthase 1	PTGS1
		Dopamine D1 receptor	DRD1/DRD5
		Muscarinic acetylcholine receptor M3	CHRM3
		Thrombin	#N/A
		Muscarinic acetylcholine receptor M1	CHRM1
		Sodium channel protein type 5 subunit alpha	SCN5A
		Muscarinic acetylcholine receptor M5	CHRM5
		Prostaglandin G/H synthase 2	PTGS2
		Carbonic anhydrase II	CA2
		Muscarinic acetylcholine receptor M4	CHRM4
		Retinoic acid receptor RXR-alpha	RXRA
		Delta-type opioid receptor	OPRD1
		Acetylcholinesterase	ACHE
		5-hydroxytryptamine 2A receptor	HTR2A
		Alpha-1A adrenergic receptor	ADRA1A
		Muscarinic acetylcholine receptor M2	CHRM2
		Beta-2 adrenergic receptor	ADRB2
		Alpha-1D adrenergic receptor	ADRA1D
		Sodium-dependent serotonin transporter	SLC6A4
		Mu-type opioid receptor	OPRM1
		Heat shock protein HSP 90	HSP90
		Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, gamma isoform	PIK3CG
		Neuronal acetylcholine receptor protein, alpha-7 chain	CHRNA7
		Ig gamma-1 chain C region	IGHG1
		Nuclear receptor coactivator 2	NCOA2
		Nuclear receptor coactivator 1	NCOA1
MOL005030	gondoic acid	Prostaglandin G/H synthase 1	PTGS1
		Nuclear receptor coactivator 2	NCOA2
MOL006202	LAX	Prostaglandin G/H synthase 1	PTGS1
		Prostaglandin G/H synthase 2	PTGS2
MOL002879	Diop	Sodium channel protein type 5 subunit alpha	SCN5A
		Beta-2 adrenergic receptor	ADRB2
		Muscarinic acetylcholine receptor M3	CHRM3
MOL000449	Stigmasterol	Progesterone receptor	PGR
		Mineralocorticoid receptor	NR3C2
		Nuclear receptor coactivator 2	NCOA2
		Alcohol dehydrogenase 1C	ADH1C
		Ig gamma-1 chain C region	IGHG1
		Retinoic acid receptor RXR-alpha	RXRA
		Nuclear receptor coactivator 1	NCOA1
		Prostaglandin G/H synthase 1	PTGS1
		Prostaglandin G/H synthase 2	PTGS2
		Alpha-2A adrenergic receptor	ADRA2A
		Sodium-dependent noradrenaline transporter	SLC6A2
		Sodium-dependent dopamine transporter	SLC6A3
		Beta-2 adrenergic receptor	ADRB2
		Aldose reductase	AR
		Urokinase-type plasminogen activator	PLAU
		Leukotriene A-4 hydrolase	LTA4H
		Amine oxidase [flavin-containing] B	MAOB
		Amine oxidase [flavin-containing] A	MAOA
		mRNA of PKA Catalytic Subunit C-alpha	#N/A
		Chymotrypsinogen B	CTRB1
		Muscarinic acetylcholine receptor M3	CHRM3
		Muscarinic acetylcholine receptor M1	CHRM1
		Beta-1 adrenergic receptor	ADRB1
		Sodium channel protein type 5 subunit alpha	SCN5A
		5-hydroxytryptamine 2A receptor	HTR2A
		Alpha-1A adrenergic receptor	ADRA1A
		Gamma-aminobutyric-acid receptor alpha-3 subunit	GABRA3
		Muscarinic acetylcholine receptor M2	CHRM2
		Alpha-1B adrenergic receptor	ADRA1B
		Gamma-aminobutyric acid receptor subunit alpha-1	GABRA1
		Neuronal acetylcholine receptor protein, alpha-7 chain	CHRNA7
MOL002320	γ-sitosterol	Progesterone receptor	PGR
		Nuclear receptor coactivator 2	NCOA2
MOL001507	stigmast-4-en-3-one	Progesterone receptor	PGR

There are 19 identified target genes focused on bioactive compounds and indigestion including ESR1, PTGS2, ADRB1, PLAU, PGR, RXRA, PTGS1, SLC6A4, PPARG, MAPK14, DPP4, SCN5A, F10, ADRB2, NR3C2, PIK3CG, HTR2A, LTA4H, OPRD1 by intersection ().

Table 4

Nineteen potential targets of Caoguo related to indigestion

Target	Protein name
ADRB1	Beta-1 adrenergic receptor
ADRB2	Beta-2 adrenergic receptor
DPP4	Dipeptidyl peptidase IV
ESR1	Estrogen receptor
F10	Coagulation factor Xa
HTR2A	5-hydroxytryptamine 2A receptor
LTA4H	Leukotriene A-4 hydrolase
MAPK14	Mitogen-activated protein kinase 14
NR3C2	Mineralocorticoid receptor
OPRD1	Delta-type opioid receptor
PGR	Progesterone receptor
PIK3CG	Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, gamma isoform
PLAU	Urokinase-type plasminogen activator
PPARG	Peroxisome proliferator activated receptor gamma
PTGS1	Prostaglandin G/H synthase 1
PTGS2	Prostaglandin G/H synthase 2
RXRA	Retinoic acid receptor RXR-alpha
SCN5A	Sodium channel protein type 5 subunit alpha
SLC6A4	Sodium-dependent serotonin transporter

The PPI network is showed in contained 17 nodes and twenty-six edges, shows the interaction of PTGS2, PPARG, MAPK14, PGR, SLC6A4, ADRB2, ESR1, PLAU, PTGS1, ADRB1, OPRD1, LTA4H, DPP4, HTR2A, PIK3CG, NR3C2, RXRA ranked according to degree that indicates the importance for treatment of indigestion by Caoguo.

Figure 3

A PPI network (the size and colour indicate the importance of the target genes proportional to their degree and edges represent associations between protein and protein). PPI, protein-protein interaction.

showed the results of GO enrichment analysis of the target genes in biological processes, cell components and molecular functions. The enriched biological processes were dominated by steroid hormone mediated signaling pathway (GO:0043401) and transcription initiation from RNA polymerase II promoter (GO:0006367). Cell components terms mainly contained plasma membrane(GO:0005886). In terms of GO molecular functions, steroid hormone receptor activity (GO:0003707) and enzyme binding (GO:0019899) were mainly enriched.

Figure 4

GO enrichment. (A) Biological processes; (B) cell components; (C) molecular functions. GO, Gene Ontology.

According to the KEGG enrichment analysis, there were 17 signal pathways enriched and analyzed (). Based on the results of KEGG, signal pathway of regulation of lipolysis (hsa04923) and serotonergic synapse (hsa04726) played the important role in treatment of indigestion among the enriched pathways which were displayed in . The component-target-pathway network was constructed with 12 active components, 52 target genes and 17 pathways (), revealed the probable mechanism of Caoguo in treatment of indigestion which were related to multiple active compounds, target genes and pathways.

Figure 5

KEGG enrichment analysis of 17 signal pathways.

Figure 6

Regulation of lipolysis in adipocytes signal pathway.

Figure 7

Serotonergic synapse signal pathway.

Figure 8

The component-target-pathway network (the yellow node represents the herbal medicine; the 12 purple nodes represent the active components in Caoguo; the 52 rhombuses represent the target genes of the active components in Caoguo, in which 19 red nodes represent the intersecting targets; the 17 triangles represent the pathways and two of them marked in red considered as important).

Discussion

Accelerated solvent extraction (ASE) is a new extraction method which uses organic solvents at high pressure and temperature above boiling point. The ASE procedure was introduced by Dionex (Sunnyvale, CA, USA) in recent years. It uses organic solvents to extract solid and semi-solid samples at higher temperatures (50–200 °C) and pressure (10.3–20.6 MPa) than those used in traditional solvent extraction procedures (22). High temperature can increase the solubility of the target components in the solvent and reduce solvent viscosity, which is beneficial to the diffusion of solvent molecules into the matrix and increases the dissolution rate of them (23). The high pressure can increase the boiling point of the solvent to above normal boiling point and keep the solvent in a liquid state at this elevated temperature (24). Compared with the traditional extraction method, ASE can significantly improve the extraction rate, shorten analysis time, and reduce environmental pollution. Additionally, as one of the best methods for sample preparation, ASE can reduce or even eliminate errors caused by individual differences in manual operation and increase the sensitivity, accuracy, and reproducibility of analytical tests. In this study, Caoguo was first extracted by ASE which was able to extract a plenitude of potential chemical constituents than other methods reported before (16,25).

Gas chromatography-mass spectroscopy (GC-MS) is one of the most versatile and widely applied technology in modern volatile constituents identification (26) due to its inherent advantages of high resolution (27), rapid separation, low cost, and easy linkage with sensitive and selective detectors (28). Mass spectrometry is a better technique which affords rather complete structural information (29), and it is suitable for the analysis of complex mixtures (30). Some methods based on GC or GC-MS have been reported to detect volatile components in Caoguo (31), but there is still no published data on the in vitro-in vivo components analysis. This is the first time using the GC-MS to identify the components of Caoguo in vivo.

Recent years, with the popularization of network pharmacology, it has been used in traditional Chinese medicine research, which could reveal the relationships between disease and active components of traditional Chinese medicine by linking the chemical constituents, targets and pathways to establish a compound-target-pathway network to explore the possible mechanisms in disease treatment. Niu et al. used network pharmacology to predict the active components and targets of Pterocypsela elata for treatment of cerebral ischemia (32). The anti-Alzheimer’s effects of Bushen Tiansui formula was studied by Zhang et al. using network pharmacology approach (33). In a review, Luo et al. summarized the methodology, application and prospective of network pharmacology technology providing us a new research strategy in the area of Chinese medicine (34).

In this study, 12 identified active components of Caoguo were considered to have potential key roles in the treatment of indigestion by acting on 19 targets. Based on KEGG enrichment analysis, 17 signal pathways were enriched and analyzed, 2 of which had strong significance and relation. A total of 7 key targets were involved in the top two enriched pathways including regulation of lipolysis in adipocytes (hsa04923) (related to the key target ADRB1, ADRB2, PTGS2, PIK3CG, PTGS1) and serotonergic synapse (hsa04726) (related to the key target HTR2A, PTGS2, SLC6A4, PTGS1).

In the pathway of regulation of lipolysis in adipocytes, Caoguo plays a pharmacological role by promoting the effect of epinephrine and norepinephrine on lipid metabolism and inhibiting the inhibitory effect of insulin and prostaglandin on lipid metabolism. Adrenoceptor beta 1 (ADRB1) and adrenoceptor beta 2 (ADRB2) are responsible to activate the regulation of lipolysis in adipocytes. The assembly of G proteins produces a large number of second messengers cAMP which are dispersed into the cells and then activate PKA which enable lipase activation to participate in lipid metabolism and transfer triglycerides into glycerol and fatty acids to the liver. The protein PIK3CG is responsible for the PI3K-Akt insulin signal pathway. Activation of PI3K/Akt/mTOR pathways may be involved in pathogenesis of the gastrointestinal tract (35). The PI3K/Akt signaling pathways is involved in soluble uric acid and the gut excretion in human intestinal cells (36). The proteins PTGS1 and PTGS2 are responsible for arachidonic acid metabolism, which then affects the release of PGE2, causing the direct activation of longitudinal smooth muscle contraction of the colon in rats (37).

In the regulation of serotonergic synapse pathway, Caoguo plays a pharmacological role by inhibiting 5-HT reuptake and promoting the combination of 5-HT to 5-hydroxytryptamine receptor. Solute carrier family 6 member 4 (SLC6A4) is responsible for the reuptake process by SERT. Up-regulation of SERT levels in the midbrain and thalamus may relate to pathogenesis of the gut (38). SERT reuptakes excessive 5-HT and participates in the regulation of gastrointestinal motility, thus intestinal dysregulation may be due to up-regulation of SERT expression, leading to the development of gastrointestinal diseases (39). Additionally, HTR2A is responsible for the activation of the 5-HT receptor 2A which leads to release of calcium ion and play an important role in controlling gastric emptying (40).

Conclusions

In this article, a systematic study of the essential volatile chemical components in Caoguo extract and in rats plasma were conducted by application of ASE technique and GC-MS method, which played an important role in the development, modernization, and quality control of Caoguo formulations. Network pharmacology was also introduced to reveal the possible mechanism of Caoguo in treatment of indigestion. This study provided a new strategy using ASE combined with GC-MS for Caoguo extraction and analysis, while enriched our knowledge about its volatile components and the therapeutic material basis. In next work, the prediction signaling pathways need be confirmed and verified by further study.

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