GC-MS and Chemometric Contrastive Analysis of the Essential Oils from the Leaves of Chimonanthus salicifolius S.Y.Hu and Chimonanthus nitens Oliv. in China

Abstract

Background/Objective: Chimonanthus salicifolius S.Y.Hu (C. salicifolius) is one of the most important herbs in She nationality of China. For the similar appearance of C. salicifolius and Chimonanthus nitens Oliv. (C. nitens), they are often used in confusion in folk. In order to preliminarily estimate whether C. salicifolius is the pharmaceutical base source of C. nitens, in this study, the essential oil components of C. salicifolius and C. nitens from different locations in China were analyzed for the first time. Methods: The essential oil components of C. salicifolius and C. nitens from different locations in China were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Results: The two essential oils contained 67 and 71 compounds, respectively, and with 18 common components. The primary components of volatile oil of C. salicifolius were eucalyptol, linalool, caryophyllene, δ-cadinene and limonene, while those of C. nitens were eucalyptol, linalool, 2-bornanone and α-terpineol. PLS-DA and HCA were performed on 18 common main components. The C. salicifolius species sources (L1 and L2) were in a cluster, the C. salicifolius species sources (L3) and the C. nitens species sources (S1-S3) were in another cluster. The differential substances of the two clusters were eucalyptol, linalool, caryophyllene, 2-bornanone, limonene and α-terpineol. In the cell experiment, the essential oils of C. salicifolius and C. nitens exerted evident anti-inflammatory effects. Conclusions: Our work indicated that there were similarity in types, contents and anti-inflammatory effects of essential oil of C. salicifolius and C. nitens from different germplasm resources. This study determined preliminarily that C. salicifolius could be used as the medicinal base of source of C. nitens.

Keywords

analysis essential oil GC-MS

Introduction

Chimonanthus salicifolius S.Y.Hu (C. salicifolius) is a semi-evergreen shrub and included in “Zhejiang Province Traditional Chinese Medicine Concoctions Regulations” (2015 edition),¹ which is mainly distributed in Jiangxi, Zhejiang and Anhui provinces in China. C. salicifolius is a common medicinal herb and used widely by the She nationality. The tender leaves of C. salicifolius are often made into a kind of mellow herbal tea, commonly known as “Xiangfeng Tea” or “Golden Tea”,² which can relieve wind, detoxificate, digest food and regulate intestines, etc.³ In daily life, this kind of medicinal tea is usually used to treat colds, flu and stomach distension and acidity, which has been honored as the “First Taste of She Nationality Medicine”.⁴ Modern research has shown that C. salicifolius has a variety of pharmacological effects such as antioxidant, antibacterial, lipid-lowering, gastrointestinal protection, vascular protection and so on.^5–8 Essential oils are the key bioactive substances of C. salicifolius.²

Chimonanthus nitens Oliv. (C. nitens) is an evergreen shrub of the genus chimonanthus and collected in the “Jiangxi Province Chinese Medicine Preparation Specifications” (2008 edition),⁹ which also can display the efficacy of detoxifying wind and detoxification. C. nitens is mainly produced in Dexing, Wuyuan and Huizhou and often used to treat colds, chronic bronchitis and heatstrokein clinic.¹⁰ C. nitens had been developed into many patent medicine product such as Chimonanthus nitens leaf granules, Chimonanthus nitens drops, sachets, tea cakes and so on. Essential oils of C. nitens are important secondary metabolites with a wide range of biological activities such as anti-inflammatory, antibacterial and anti-acute lung injury.^11–14

C. salicifolius and C. nitens both belong to the genus of aromatic Chimonanthus and have similar pharmacological activities.¹⁵ Their leaves have good medicinal value and are widely used in folklore as an edible herbal tea. For the similar appearance of C. salicifolius and C. nitens, they are often used in confusion to treat colds and coughs in folk, especially in northeastern Jiangxi Province.¹⁶ C. salicifolius is a traditional medicinal material used by the She Nationality, but it is not included in the Jiangxi Province Chinese Medicine Preparation Specifications. In addition C. nitens are currently mainly from the wild, its clinical application and production is limited. Essential oils are their main medicinal ingredients. Up to now, there was no a systematic and comparative analysis report about the essential oil components of C. salicifolius and C. nitens. Above all, it is very necessary to analyze the essential oil components of C. salicifolius and C. nitens from different locations in China. Hierarchical Cluster Analysis (HCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were adopted to demonstrate variation in the composition of the different species essential oils. Our study can preliminarily determine that C. salicifolius may be used as the medicinal source of C. nitens.

Materials and Methods

Plant Material and Cell

The experimental materials of C. salicifolius and C. nitens were collected from various habitats, and detailed information was showed in Table 1. All samples were planted in Jiangxi Youmei Pharmaceutical Co. Ltd in March 2020, and identified by one of the authors (Xiaoqun He). In this study, from different locations in China of C. salicifolius and C. nitens were planted under the same plot with consistent growing, climatic conditions, and the same management conditions. On November 1, 2023, all samples were harvested and air-dried for preservation.

Table 1.

Content of Essential Oils in C. salicifolius and C. nitens Leaves of Different Locations in China.

Plant sample	Numbers	Location	Collection site	Yields%^a
C. salicifolius	L1	Lishui	28°21′7″ N, 118°59′32″ E, 319 m asl	2.75 ± 0.01
C. salicifolius	L2	Huangshan	29°29′6″ N, 118°13′12″ E, 648 m asl	2.66 ± 0.01
C. salicifolius	L3	Wuyuan	29°13′36″ N, 117°58′35″ E, 188 m asl	2.96 ± 0.01
C. nitens	S1	Lishui	28°21′7″ N, 118°59′32″ E, 319 m asl	2.75 ± 0.01
C. nitens	S2	Wuyuan	29°13′36″ N, 117°58′35″ E, 188 m asl	3.18 ± 0.01
C. nitens	S3	Huizhou	23°37′38″ N, 113°52′24″ E, 522 m asl	2.74 ± 0.02

Extracted yield based on 100 g crude powder. All the data are mean (±SD) of triplicate measurements.

RAW264.7 cell Line were purchased from the ATCC (Manassas, VA) and grown in DMEM supplemented with 1% penicillin-streptomycin and 10% FBS at 37°C and 5% CO₂.

Essential Oil Extraction

The essential oil was extracted by hydrodistillation. The raw material (100.0 g) was accurately weighed and crushed, and the crushed sample was subsequently transferred to a Soxhlet extractor and 600 ml of distilled water was added, then refluxed in a Soxhlet extractor for 4 h. Each sample was extracted three times, and the extracted essentia oils were combined. After layering, we got the volume of essential oils, dried with anhydrous sodium sulfate, and stored in brown sealed vials at 4 °C before analysis.

GC-MS Analyses

Essential oil compounds were determined by Agilent 8890-7250 GC/Q-TOF gas chromatography-mass spectrometry system. The chromatographic column was HP-5MS (30 m × 320 μm × 1.8 μm), The injection volume was 1 μl. Programmed temperature increased as follow: the initial temperature at 60 °C, held for 0.5 min, first increase to 90 °C at 25 °C/min and held for 2 min, then increase to 100 °C at 1 °C/min and held for 1 min, then increase to 160 °C at 100 °C/min and held for 0 min, finally, increase to 220 °C at 2 °C/min held for 7 min. High-purity He gas was used as the carrier gas at a flow rate of 1 ml/min, with an inlet temperature of 250 °C and a split ratio of 100:1.

The MS ionization potential was 70 eV and scan range 50-550 amu; the ionization current was 350 μA, and the ion source and transmission line temperatures were 230 °C and 280 °C, respectively. All samples were analyzed in triplicate, and the relative standard deviation was less than 2%.

The essential oil components were identified by the van den Dool and Kratz linear equations with reference to the homologous series of n-alkanes to determine their retention index (RI). The authenticity of the components was further confirmed by comparing the MS data of the components with those of standard compounds in the NIST 20 chemical workstation, Adams and Satyal libraries.^17,18 The relative content of each compound was calculated by peak area normalization method.

Cell Experiments

CCK-8 Assay

Logarithmically growing RAW264.7 cells (1 × 10⁵/well) were added into a 96-well plate with gradient-diluted compounds and incubated at 37°C under 5% CO₂ for 24 h, 20 µL of CCK-8 solution (Beyotime, Shanghai, China) was added and incubated for 30 min. Optical density (OD) was measured using a K6600-A reader (KAIAO, Beijing, China) at 450 nm and 630 nm, and the viability was calculated.

Measurement of Cytokines Levels in Cells

RAW264.7 cells (1 × 10⁵/well) were seeded into a 96-well plate. The groups of Dexamethasone (DEX) (National Institute for Food and Drug Control, Beijing, China), and the essential oil of C. salicifolius and C. nitens (1 μg/mL) were pretreated for 2 h, then treated with 1 μg/mL LPS for 24 h.

After 24 h, the cell supernatants were gathered, and the contents of cytokines TNF-α, IL-6 in the supernatant were measured following the procedures outlined in the ELISA kit manuals (Proteintech, Wuhan, China).

Statistical Analysis

Partial Least Squares Discriminant Analysis (PLS-DA) was adopted to characterize the correlation between the essential oil components and different locations in China of C. salicifolius and C. nitens. Hierarchical Cluster Analysis (HCA) was carried out to examine the similarity of oil samples based on the percentages of 18 common major components and to verify the previous PLS-DA analysis. The six essential oil samples were treated as operational taxonomic units. Pearson correlation was used to measure similarity, and the unweighted pair-group method with arithmetic average was used to define the clusters. The data were analyzed by PLS-DA using SIMCA 14.1, and cluster analysis was performed using the heat-mapper program.

The cell experimental data were analyzed by SPSS version 20 and GraphPad Prism 9.0 software. One-way ANOVA or Kruskal–Wallis tests were selected for data analysis according to the data distribution and homogeneity of variance. Asterisks indicatestatistical significance (*, P < .05; **, P < .01; ***, P < .001; ****, P < .0001).

Results

Contrastive Analysis of Essential Oils of C. salicifolius and C. nitens Leaves

The essential oils were prepared from C. salicifolius and C. nitens from four locations in China resources. Essential oils contents of them ranged from 2.66% to 2.96% and 2.74% to 3.18%, respectively, shown in Table 1. The results suggested that essential oils contents of C. salicifolius and C. nitens from different germplasm resources were no significant difference.

Contrastive Analysis of Chemical Compositions of the Essential Oils

There were 43, 42, 31 compounds identified from the essential oils of samples L1 to L3 of C. salicifolius, respectively. A total of 67 compounds were identified in the three essential oils from different germplasm samples of C. salicifolius, randing from 84.05 to 98.77% (Table 2, L1-L3). According to chemical structrue, the essential oil is mainly found by terpenes (49.06-58.16%) and alcohols (19.41-31.49%). There were 21 common components presented in C. salicifolius L1-L3, and eucalyptol, linalool, caryophyllene, δ-cadinene and limonene were identified as the major constituents of the essential oil. Terpenes were the highest (49.0-58.1%), followed by alcohols (19.4-31.4%) and ketones/aldehyde (0.3-10.6%) (Figure 1). Alkanes and amines are unique to sample L1, pyrans spoted in sample L2. The differences of content and composition could be affected by the growing environment such as climate, soil, light, and altitude and so on.

Figure 1.

Percentage Share of Groups of Components of Essential Oils of C. salicifolius and C. nitens.

Table 2.

Essential Components of C. salicifolius and C. nitens Leaves from Different Locations Resources.

No.	Compounds	RT/min	RI_db	RI_calc	Relative Content/%						Method of identification
No.	Compounds	RT/min	RI_db	RI_calc	L1	L2	L3	S1	S2	S3	Method of identification
1	Tetrahydropyran	3.342	914	914	0.00	0.52	0.00	0.00	0.06	0.00	a,b,c
2	Butyrolactone	3.347	915	915	0.00	0.00	0.00	0.03	0.00	0.00	a,b,c
4	Allyl isobutyrate	3.398	920	918	0.45	0.47	0.46	0.00	0.00	0.00	a,b,c
5	1-Hexanol	3.439	921	921	0.00	0.24	0.10	0.01	0.06	0.00	a,b,c
6	Tricyclene	3.471	924	924	0.04	0.00	0.00	0.00	0.00	0.00	a,b,c
7	Cyclofenchene	3.477	924	924	0.00	0.00	0.00	0.05	0.00	0.00	a,b,c
8	α-Thujene	3.495	926	926	0.00	0.00	0.05	0.00	0.00	0.00	a,b,c
9	α-Pinene	3.609	936	934	0.00	0.00	0.00	2.92	0.00	3.14	a,b,c
10	3,6,6-Trimethyl-2-norpinene	3.607	936	934	2.11	1.61	2.81	0.00	2.14	0.00	a,b,c
11	3-Hydroperoxyhexane	3.646	940	937	0.00	0.00	0.00	0.04	0.00	0.00	a,b,c
12	1-Methylpentyl hydroperoxide	3.760	946	947	0.04	0.00	0.00	0.02	0.00	0.00	a,b,c
13	Camphene	3.822	950	950	1.76	0.11	0.24	2.05	0.54	1.09	a,b,c
14	β-Phellandrene	4.146	977	974	0.49	0.00	0.23	0.24	0.18	0.24	a,b,c
15	β-Pinene	4.231	981	981	2.71	0.66	0.77	0.73	0.60	0.79	a,b,c
16	6-Methylhept-5-en 7-2-one	4.292	985	985	0.00	0.00	0.00	0.02	0.00	0.00	a,b,c
17	Myrcene	4.354	993	990	1.72	0.51	0.86	0.57	0.39	0.36	a,b,c
18	Dehydro-1,8-cineole	4.420	996	995	0.03	0.00	0.00	0.02	0.00	0.02	a,b,c
19	α-Phellandrene	4.652	1007	1007	0.58	0.08	1.34	0.37	0.29	0.36	a,b,c
20	p-Cymene	5.032	1022	1023	0.29	0.00	0.34	0.13	0.08	0.10	a,b,c
21	Limonene	5.120	1027	1027	1.92	2.38	2.94	0.87	0.87	0.71	a,b,c
22	Eucalyptol	5.201	1031	1031	15.03	12.70	39.16	29.26	27.70	41.49	a,b,c
23	β-Ocimene	5.486	1043	1043	1.09	0.67	0.90	0.18	0.39	0.19	a,b,c
24	γ-Terpinene	5.783	1056	1056	0.16	0.41	0.31	0.05	0.10	0.07	a,b,c
25	Linalool oxide (furanoid)	6.160	1070	1071	0.00	0.00	0.00	0.00	0.03	0.00	a,b,c
26	4-Carene	6.585	1092	1090	0.08	1.02	0.89	0.04	0.23	0.04	a,b,c
27	Linalool	6.858	1102	1100	4.82	5.38	24.61	25.65	24.52	5.04	a,b,c
28	3-Hexenyl Butyrate	7.003	1103	1102	0.00	0.00	0.00	0.05	0.00	0.00	a,b,c
29	2-Bornanone	8.455	1119	1118	10.62	1.74	0.31	11.24	3.16	8.49	a,b,c
30	Borneol	9.265	1126	1126	5.88	0.00	0.00	4.09	0.12	2.49	a,b,c
31	Tetrahydro-2-(methoxymethyl)-furan	9.324	1127	1127	0.00	0.00	0.00	0.00	1.43	0.00	a,b,c
32	Terpinen-4-ol	9.751	1130	1131	0.49	0.70	0.22	0.16	0.10	0.28	a,b,c
33	α-Terpineol	10.336	1138	1138	1.36	2.00	1.55	2.00	3.95	4.40	a,b,c
34	Isobutyric acid anhydride	10.156	1139	1136	0.00	0.00	0.00	0.00	0.19	0.00	a,b,c
35	Methyl tetrahydrofurfuryl ether	11.070	1146	1145	0.00	1.63	2.09	0.53	2.53	0.49	a,b,c
36	Isopropyl pyruvate	12.248	1158	1158	0.00	0.41	0.00	0.00	0.01	0.02	a,b,c
37	Neral	12.883	1168	1165	0.00	0.00	0.00	0.06	0.00	0.14	a,b,c
38	Nerol	13.635	1173	1173	3.10	0.00	0.00	0.31	0.11	0.63	a,b,c
39	Geranial	14.648	1184	1184	0.00	0.00	0.00	0.04	0.00	0.00	a,b,c
40	3-(1-Methyl-2-propen-1-yl)-1,5-cyclooctadiene	14.760	1185	1185	0.00	0.00	0.00	0.00	0.18	0.00	a,b,c
41	Bornyl acetate	15.461	1196	1193	1.08	0.60	0.00	1.13	0.10	0.72	a,b,c
42	2,6-Dimethylocta-5,7-dien-2-ol	16.827	1121	1120	0.00	0.00	0.00	0.00	0.05	0.00	a,b,c
43	δ-Terpinyl acetate	16.833	1122	1121	0.02	0.00	0.00	0.16	0.00	0.44	a,b,c
44	α-Terpinyl acetate	17.890	1156	1156	2.02	0.69	1.85	0.36	0.12	1.17	a,b,c
45	α-Cubebene	18.591	1180	1179	0.00	0.33	0.00	0.03	0.38	0.06	a,b,c
46	Neryl acetate	18.735	1188	1188	0.99	0.00	0.00	0.19	0.06	0.48	a,b,c
47	β-Elemene	19.012	1193	1193	0.46	4.22	0.06	0.34	0.20	0.38	a,b,c
48	Caryophyllene	19.627	1423	1421	11.72	5.98	3.82	0.45	1.50	3.57	a,b,c
49	Clovene	19.724	1426	1426	0.00	0.00	0.03	0.00	0.00	0.00	a,b,c
50	γ-Elemene	19.932	1437	1437	0.00	2.04	0.00	0.00	0.00	0.00	a,b,c
51	α-Guaiene	20.037	1442	1442	0.00	0.00	0.00	0.01	0.11	0.04	a,b,c
52	Humulene	20.334	1457	1457	0.00	1.41	0.00	0.00	0.00	0.00	a,b,c
53	Aristolene	20.388	1460	1460	0.00	1.30	0.00	0.00	0.00	0.00	a,b,c
54	α-Gurjunene	20.451	1462	1463	0.00	2.71	0.00	0.00	0.00	0.00	a,b,c
55	Naphthalene	20.772	1480	1480	0.39	0.00	0.00	0.00	0.00	0.00	a,b,c
56	β-Copaene	20.858	1484	1484	0.44	0.00	0.97	0.20	0.57	3.53	a,b,c
57	Germacrene D	20.871	1485	1484	0.00	1.82	0.00	0.00	0.00	0.00	a,b,c
58	β-Selinene	20.960	1489	1489	0.00	2.74	0.00	0.12	0.12	0.00	a,b,c
59	α-Farnesene	21.099	1496	1496	0.73	0.00	0.00	0.00	0.00	0.00	a,b,c
60	1-Isopropenyl-4α,8-dimethyl-1,2,3,4,4α,5,6,7-octahydronaphthalene	21.130	1498	1498	0.00	3.18	0.00	0.21	0.00	0.00	a,b,c
61	Bicyclogermacrene	21.153	1499	1499	2.48	0.00	0.00	0.00	0.00	0.80	a,b,c
62	α-Muurolene	21.218	1502	1502	1.15	2.24	0.37	0.00	0.63	0.35	a,b,c
63	α-Bulnesene	21.335	1507	1508	0.00	0.00	0.00	0.00	0.23	0.00	a,b,c
64	α-Farnesene	21.345	1509	1509	0.88	0.00	0.00	0.00	0.00	0.00	a,b,c
65	4,7-Dimethyl-1-propan-2-yl-1,2,3,5,6,8α-hexahydronaphthalene	21.356	1511	1509	0.00	0.00	0.00	0.00	0.00	0.44	a,b,c
66	γ-Cadinene	21.481	1516	1516	0.74	0.85	0.21	0.02	0.23	0.22	a,b,c
67	γ-Gurjunene	21.565	1520	1520	0.00	0.31	0.00	0.00	0.00	0.00	a,b,c
68	δ-Cadinene	21.665	1525	1525	1.90	7.00	1.32	0.82	8.42	2.17	a,b,c
69	1,2,3,4,4α,7-Hexahydro-1,6-dimethyl-4-(1-methylethyl)-Naphthalene -4,7-dimethylnaphthalene	21.829	1533	1534	0.00	0.00	0.00	0.00	0.11	0.00	a,b,c
70	Valencene	21.902	1538	1537	0.00	1.13	0.00	0.00	0.00	0.00	a,b,c
71	α-Cadinene	21.941	1539	1539	0.20	0.00	0.17	0.00	0.24	0.07	a,b,c
72	β-Maaliene	21.950	1540	1540	0.00	2.37	0.00	0.00	0.00	0.00	a,b,c
73	Selina-3,7(11)-diene	22.031	1544	1544	0.00	1.44	0.00	0.00	0.00	0.00	a,b,c
74	α-Calacorene	21.132	1545	1545	0.00	0.00	0.00	0.08	0.08	0.07	a,b,c
75	Elemol	22.179	1551	1551	0.21	8.72	0.00	2.11	0.12	0.85	a,b,c
76	Nerolidol	22.437	1563	1564	0.00	0.00	0.00	0.00	0.00	0.05	a,b,c
77	β-Calacorene	22.466	1566	1566	0.00	0.00	0.00	0.01	1.20	0.05	a,b,c
78	Cadinol	22.724	1579	1579	0.62	0.00	0.00	0.00	0.00	0.00	a,b,c
79	Spathulenol	22.779	1581	1582	0.00	0.00	0.00	0.00	0.14	0.09	a,b,c
80	2-Methylene-4,8,8-trimethyl-4-vinylbicyclo[5,2,0]nonane	22.908	1588	1588	0.00	0.00	0.08	0.01	0.04	0.18	a,b,c
81	Guaiol acetate	23.090	1598	1598	0.00	0.00	0.00	0.00	0.00	0.05	a,b,c
82	3,7-Dimethyl-2,6-octadienyl butyrate	23.342	1610	1609	0.00	0.00	0.00	0.00	0.00	0.27	a,b,c
83	α-Corocalene	23.726	1626	1626	0.00	0.00	0.00	0.00	0.34	0.00	a,b,c
84	γ-Eudesmol	23.948	1635	1635	0.00	6.21	0.00	0.80	0.00	0.71	a,b,c
85	α-Muurolol	24.256	1648	1649	0.59	0.00	0.00	0.00	0.08	0.00	a,b,c
86	β-Eudesmol	24.382	1655	1654	0.00	3.53	0.00	0.40	0.15	0.00	a,b,c
87	α-Eudesmol	24.447	1657	1657	0.00	4.71	0.00	0.95	0.00	2.37	a,b,c
88	α-Cadinol	24.457	1658	1658	2.34	0.00	0.00	0.00	1.22	0.00	a,b,c
89	Cadalene	24.941	1679	1679	0.00	0.00	0.00	0.00	0.50	0.00	a,b,c
90	1,3-Dioxolane	24.538	1662	1661	0.27	0.00	0.00	0.00	0.00	0.00	a,b,c
91	2-Propanamine	24.700	1668	1668	0.05	0.00	0.00	0.00	0.00	0.00	a,b,c
92	15-Hydroxy-α-muurolene	25.337	1697	1696	0.00	0.00	0.37	0.00	0.00	0.00	a,b,c
Terpenes					49.06	58.04	58.16	39.51	48.26	59.91
Alcohols					19.41	31.49	26.48	36.48	30.62	16.91
Ketones/Aldehyde					10.62	1.74	0.31	11.39	3.16	8.63
Ester					4.56	3.80	4.40	2.42	3.01	3.64
Hydrocarbons					0.04	3.18	0.08	0.27	0.33	0.62
Others					0.36	0.52	0.00	0.06	1.52	0.00
Total					84.05	98.77	89.43	90.13	86.90	89.71

Relative content are the mean (±SD) of different samples of C. salicifolius and C. nitens of three measurements.

RI_calc: The Kováts retention index relative to C7-C40 n-alkanes on HP-5MS column.

RI_db: The reference retention index obtained from the databases.^17,18

a: Identification by retention index (RI).

b: identification by comparing with authentic specific compounds.

c: identification by comparing of mass spectra (MS).

RT: Retention time(min).

Percentage share of groups of essential components of essential oils of C. salicifolius and C. nitens from different locations in China had similarities. 49, 51, 47 compounds were identified from essential oil of C. nitens S1-S3, respectively (Table 2). A total of 71 compounds were found in the three essential oils from different locations in China samples of C. nitens, randing from 84.37 to 90.13% (Table 2, S1-S3). The C. nitens essential oil was rich in terpenes (39.51-59.91%), alcohols (16.91-36.48%) and ketones/aldehyde (3.16-11.39%) (Figure 1). As it can be seen in Table 2, 31 common constituents were identified from essential oils of S1-S3. Eucalyptol, linalool, 2-bornanone and α-terpineol were identified as the major constituents of the essential oil. Terpenes, alcohols and ketones were the common components of 3 samples of C. nitens. Pyrans and furans were restricted to sample S2. All samples contained aldehydes except sample S2, all contained alkanes except sample S3. The difference of composition and content of C. salicifolius and C. nitens may be due to species source and growth environment.

92 compounds were identified from the essential oils of C. salicifolius and C. nitens, of which 18 were common (Table 3). Both were rich in terpenes and alcohols. In addition, both contained ketones and esters. C. salicifolius possessed 22 unique constituents, of which the contents of γ-elemene, α-gurjunene, bicyclogermacrene and β-maaliene were relatively high. C. nitens possessed 25 unique components, of which α-pinene, tetrahydro-2-(methoxymethyl)-furan and β-calacorene were relatively high. Compared with previous reports,^19,20 there were no obvious different in main components of the essential oils of C. salicifolius and C. nitens. Eucalyptol, linalool, δ-cadinene, caryophyllene and 2-bornanone were the main common components in C. salicifolius and C. nitens, but with some differents in contents. The contents of eucalyptol, linalool and 2-bornanone were significantly higher in C. nitens than C. salicifolius. However, C. salicifolius had higher contents of limonene and caryophyllene compared to C. nitens. The relative contents of β-elemene, β-selinene, elemol, γ-eudesmol and β-eudesmol in sample L2 were significantly higher than those in other samples. The above results indicated that there were similarities in the types of components and relative contents of essential oils of C. salicifolius and C. nitens from different locations in China, but we should not ignore the differences between them.

Table 3.

Common Compounds of Essential Oils of C. salicifolius and C. nitens Leaves.

NO.	Compounds	Relative Content/%
NO.	Compounds	L1	L2	L3	S1	S2	S3
1	Camphene	1.76	0.11	0.24	2.05	0.54	1.09
2	β-Pinene	2.71	0.66	0.77	0.73	0.60	0.79
3	Myrcene	1.72	0.51	0.86	0.57	0.39	0.36
4	α-Phellandrene	0.58	0.08	1.34	0.37	0.29	0.36
5	Limonene	1.92	2.38	2.94	0.87	0.87	0.71
6	Eucalyptol	15.03	12.70	39.16	29.26	27.70	41.49
7	β-Ocimene	1.09	0.67	0.90	0.18	0.39	0.19
8	γ-Terpinene	0.16	0.41	0.31	0.05	0.10	0.07
9	4-Carene	0.08	1.02	0.89	0.04	0.23	0.04
10	Linalool	4.82	5.38	24.61	25.65	24.52	5.04
11	2-Bornanone	10.62	1.74	0.31	11.24	3.16	8.49
12	Terpinen-4-ol	0.49	0.70	0.22	0.16	0.10	0.28
13	α-Terpineol	1.36	2.00	1.55	2.00	3.95	4.40
14	α-Terpinyl acetate	2.02	0.69	1.85	0.36	0.12	1.17
15	β-Elemene	0.46	4.22	0.06	0.34	0.20	0.38
16	Caryophyllene	11.72	5.98	3.82	0.45	1.50	3.57
17	γ-Cadinene	0.74	0.85	0.21	0.02	0.23	0.22
18	δ-Cadinene	1.90	7.00	1.32	0.82	8.42	2.17

Relative content are the mean (±SD) of different samples of C. salicifolius and C. nitens of three measurements.

PLS-DA and HCA Analysis of the Essential Oils Composition of C. salicifolius and C. nitens

PLS-DA and HCA were performed on 18 common main components of our study (Figures 2 and 3). In PLS-DA, the fitting parameter R²X (0.61), R²Y(0.986) and Q²(0.77) indicated that the model was well fitted with high stability and prediction rate. The closer R²Y value was to 1, the more stable the model was Q² value greater than 0.5 indicated good predictive ability. The PLS-DA scores of C. salicifolius and C. nitens samples from different locations in China were shown in Figure 2. As we could see, all samples could be effectively categorized into 2 groups with clear boundaries. Sample L1 and L2 were clustered into a group, and sample L3, S1-S3 were clustered into another group. Eucalyptol, linalool, caryophyllene, 2-bornanone, limonene and α-terpineol were the main compounds causing the clustering difference. PC1 (44.2%) was shown mainly by the eucalyptol, linalool, 2-bornanone and α-terpineol in the positive score, and in a minor contribution by caryophyllene and limonene in negative scores. PC2 (16.8%) was represented mainly by a negative score of eucalyptol, linalool, caryophyllene and limonene and positive scores of the 2-bornanone and α-terpineol. The results indicated that there were no obvious differences in essential compositions and contents of essential oils of C. salicifolius and C. nitens from different locations in China.

Figure 2.

PLS-DA Score Plot of Essential Components of C. salicifolius and C. nitens Leaves.

Figure 3.

Cluster Analysis Diagram of Common Components of C. salicifolius and C. nitens Leaves.

The HCA (Figure 3) was an unsupervised pattern recognition method, which was used to divide the studied objects into multiple groups and each group having the highest similarity and difference. The dendrogram could directly show classification of test samples. Figure 3 suggested that the 6 samples were divided into two clusters. Clusters I included sample L1 and sample L2, and cluster II included sample L3, S1-S3. Sample L3 was classified in cluster II because of the high content of eucalyptol and linalool. The differential substances between sample L3 and cluster I were mainly 2-bornanone, β-elemene and γ-cadinene. The HCA result was basically consistent with the result of PLS-DA. To some extent, there were differences between the essential oils of C. salicifolius and C. nitens, but there was some correlation between the two species.

Effect of the Essential Oils of C. salicifolius and C. nitens on the Contents of Cytokines in Cell Supernatants

The results indicated that essential oils showed minimal cytotoxicity to RAW264.7 at 1 µg/mL, while DEX exhibited mild cytotoxicity at 20 µg/mL. Drug concentrations that ensured a cell survival rate of over 90% were used for subsequent experiments. Thus, the maximum non-toxic concentration of essential oil was identified as 1 μg/mL, and for DEX, it was 10 μg/mL (Figure 4A-B).

Figure 4.

The Cytotoxicity and Anti-inflammatory Activity of Essential Oils. (A, B) RAW264.7 Cells were Treated with Gradient-Diluted Essential Oils or DEX for 24 h and Cell Viability was Measured by CCK8. Data Represent the mean ± SD of Three Independent Experiments. (C, D) RAW264.7 Cells were Pre-Treated with Essential Oils (1 µg/mL) or DEX (10 µg/mL) for 2 h Followed by LPS (1 µg/mL) Stimulation for 24 h, Cell Culture Supernatant was Collected to Measure Quantity of TNF-α and IL-6 by ELISA. Data were Analyzed Using One-Way ANOVA, Asterisks Indicate Statistical Significance (****P < .0001).

Essential oil regulated a series of production induced by LPS, such as TNF-α and IL-6. After treatment with essential oil, the release of cytokines induced by LPS in the supernatant was significantly reduced, suggesting that essential oils exerted evident anti-inflammatory activity. (Figure 4C-D).

Discussion

Similar to previous findings on the essential oil yields of C. salicifolius and C. nitens, both them contain abundant essential oils and have an aromatic aroma.^21,22 Our results showed that the essential oil yields of them were higher than in the literature respectively, It may be due to differences in harvesting season and growth environment.

The main constituents of the essential oils from the leaves of C. salicifolius and C. nitens. are terpenoids. The main volatile components in C. salicifolius, C. nitens and Chimonanthus zhejiangensis include terpenoids such as α-pinene, laurene, eucalyptol, a-cubebene β-elemene, caryophyllene and 3- (4,8-dimethyl-3,7-nonadienyl) furan.²³ 1,8- Eucalyptin and geranylgeranyl were the main terpenoids in the leaves of Chimonanthus grammatus, while β-ocimene and acetic acid linalool ester were unique to the leaves of Chimonanthus praecox.²³ However, the main volatile components identified in this study are different from those identified in that previous study. For instance, the major volatile components laurene and 3- (4,8-dimethyl-3,7-nonadienyl) furan could not be found. This may be due to differences in extraction method, different locations, cultivation environments, and harvesting season of the samples. Eucalyptol and caryophyllene has been identified in essential oil of Chimonanthus in many reports. In the present investigation, eucalyptol and caryophyllene were the major compounds of the essential oils from C. salicifolius and C. nitens, similar to the findings in the above report. This result also indicated a close correlation between C. salicifolius and C. nitens.

Eucalyptol is the mian effective ingredient in the essential oils of many aromatic plants, which is widely used in medicine, cosmetics and fragrance industry. Eucalyptol has multiple biological activities, such as anti-bacteria, anti-inflammatory, anti-oxidative, anti-tumor, analgesic, and neuroprotective effects.^24–29 Due to excellent antibacterial and anti-inflammatory activities, eucalyptol has achieved good results in the treatment of respiratory diseases.³⁰ Eucalyptol was the highest in the essential oils of C. salicifolius and C. nitens, and was the main active substance in the treatment of colds, bronchitis, and so on.²⁵ Linalool, as another major essential compound, also has multiple pharmacological effects, such as analgesic, anxiolytic, anti-inflammatory, anti-tumor and anti-bacterial.^31–34 Caryophyllene has anti-inflammatory and antioxidant effects and is abundant in C. salicifolius.³⁵ All of them play an important role in bioactivities of the essential oils of C. salicifolius.

The essential oil of leaves of C. nitens is used to treat variant rhinitis.¹⁰ In China, C. nitens has been developed into a traditional Chinese medicine-Shanlameiye Keli, which are used for wind-heat colds, fever, chills, sore throat. It has played an important role in the treatment of the novel coronavirus pneumonia. C. salicifolius and C. nitens both belong to the genus of aromatic Chimonanthus, and their leaves have good medicinal value and are widely used in folklore as an edible herbal tea. As congeners, C. salicifolius and C. nitens are both rich in essential oils with similar composition and type of essential oils. In the cell experiment, both the essential oils of C. salicifolius and C. nitens had anti-inflammatory effects, and there was no obvious difference between them. The above results indicate that they have the same resource utilization between C. salicifolius and C. nitens.

In this study, we preliminarily explored the resource substitutability between C. salicifolius and C. nitens, focusing mainly on their essential oil components and anti-inflammatory activity. However, the research has certain limitations and did not fully cover all relevant factors. Future research plans include expanding the samples and further investigating other potential active components and efficacy evaluations to more comprehensively assess the substitutability of these two plants. Nevertheless, the present study for the first time provided a scientific basis for the substitution of two Chimonanthus species from the perspective of chemical composition and biological activity, which had important reference value for the sustainable use of traditional Chinese medicine resources.

Conclusions

Samples of C. salicifolius and C. nitens were analyzed by GS-MS and chemometrics. A total of 92 compounds were identified from the 6 samples, of which 18 were common components. The main ingredients were eucalyptol, linalool, caryophyllene, 2-bornanone, limonene and α-terpineol, δ-cadinene. Among them, the contents of linalool, caryophyllene, 2-bornanone and δ-cadinene varied significantly among the different locations in China. The results of statistical studies showed that all samples were clearly divided into two clusters, sample L3 clustered with S2, S1, S3, suggesting some correlation between the two species.

There was no obvious difference in the yield and anti-inflammatory effects and composition of essential oil of C. salicifolius and C. nitens. From the perspective of volatile components and anti-inflammatory effects, this study provides scientific support for expanding the sources of C. nitens. Therefore, this study provides a substance basis, which C. salicifolius may be included as one of the sources of C. nitens medicinal materials.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X251333914 - Supplemental material for GC-MS and Chemometric Contrastive Analysis of the Essential Oils from the Leaves of Chimonanthus salicifolius S.Y.Hu and Chimonanthus nitens Oliv. in China

Supplemental material, sj-doc-1-npx-10.1177_1934578X251333914 for GC-MS and Chemometric Contrastive Analysis of the Essential Oils from the Leaves of Chimonanthus salicifolius S.Y.Hu and Chimonanthus nitens Oliv. in China by Yamin Wang, Yanzhen Hu, Xiaoqun He, Xusheng Huang, Di Zhang, Cui Hu, Junshen Xu and Xue Li in Natural Product Communications

Footnotes

Acknowledgements

This research was supported by the Science and technology project of Jiangxi Provincial Administration of Traditional Chinese Medicine (2021B634), Health Commission science and technology plan project of Jiangxi Provincial (202510622), Natural Science Foundation of Jiangxi Province (20212BAB206073), and the Earmarked Fund for China Agriculture Research System-21(CARS-21). In addition, we thank sincerely Dr Xusheng Huang to carry out the anti-inflammatory activity experiment, so we added him as one of the authors in our article.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

ORCID iDs

Yamin Wang

Xue Li

Ethical Considerations

Ethical Approval is not applicable for this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Earmarked Fund for China Agriculture Research System-21(CARS-21).

Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

All the data for this study are available in the manuscript.

Supplemental Material

Supplemental material for this article is available online.

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