Comparative Analysis of Chemical Composition, Anti-Inflammatory Activity and Antitumor Activity in Essential Oils from Siegesbeckia orientalis, S. glabrescens and S. pubescens with an ITS Sequence Analysis

Abstract

Herba Siegesbeckiae (HS), derived from the aerial parts of three plants, Siegesbeckia orientalis (SO), S. glabrescens (SG), and S. pubescens (SP), has been used for the treatment of inflammatory diseases in China for centuries. In the present study, hydrodistillation was applied to extract essential oils from dried SO, SG, and SP aerial parts, and chemical composition analysis by gas chromatography–mass spectrometry (GC-MS) led to the identification of a total of 148 compounds (56 in SO, 62 in SG, and 59 in SP). The main components in the essential oils of SO, SG, and SP differed significantly. In vitro anti-inflammatory activity assays showed that SP essential oils (IC₅₀, 0.97 μg/mL) significantly reduced the ability of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages to release NO, and the SO essential oil (IC₅₀, 14.99 μg/mL) was better than the others at inhibiting the LPS-induced release of cytokine IL-6. Furthermore, the essential oils exhibited antitumor activities (IC₅₀, 37.72–123.16 μg/mL) against Hep3B (liver) and Hela (cervical) cells. Linear regression analysis showed that, caryophyllene oxide peak area percentages showed remarkably high negative correlation coefficients with IC₅₀ values of Hep3B and Hela cytotoxicity, which suggested the contribution of this compound on the cancer cell cytotoxicity of three essential oils. Finally, the ITS1-5.8S-ITS2 region was amplified and sequenced in order to generate genomic reference sequences for each plant. These can be used to identify the origins of the plants, and will assist other research studies related to these three plants.

1. Introduction

Plants produce a wide variety of volatile compounds, including terpenes, alcohols, aldehydes, esters, ethers, ketones, phenols, and oxides, that play important roles in defense, pollinator attraction, signal transduction, and so on. These volatile compounds are also important resources for the pharmaceutical, food, beverages, perfume, and cosmetics industries, and are thus economically significant [1,2,3]. The mixture of plant volatile compounds, which is also known as essential oil, can be extracted using hydrodistillation, steam distillation, supercritical fluid extraction, Soxhlet′s extraction, and ultrasound-assisted extraction [4,5].

Herba Siegesbeckiae (HS), a medicinal material used for the treatment of rheumatic arthritis, malaria, and snake bites in China since centuries ago [6], is derived from the aerial parts of three annual medicinal plants, Siegesbeckia orientalis (SO), S. glabrescens (SG), and S. pubescens (SP), which belong to the genus Siegesbeckia and the family Compositae. The main activities reported for HS extract are anti-inflammatory [7], antiallergic [8], antithrombotic [9], and immunosuppressive [10,11], but other activities have also been identified [12,13]. Recently, it was reported that SP essential oil significantly inhibited the proliferation of hepatocellular carcinoma cells (HepG2, Hep3B, Huh7, SMMC-7721). The 50% proliferation inhibition concentration of the SP essential oils was 42.0–95.2 μg/mL [14]. In the past few decades, through systematic chemical studies, three main categories of compound have been identified in HS: diterpenes, sesquiterpenes, and flavonoids [15,16,17,18]. Pharmacological studies suggested that diterpenoids are the main antirheumatic constituents of HS [19,20]. A series of ent-kaurane and ent-pimarane diterpenoids from HS has been reported [17,18,21,22,23]. However, to the best of our knowledge, a comparative study related to the chemical composition, anti-inflammatory activity, and antitumor activity of essential oils from the three individual plant species has not been reported.

In this study, the hydrodistillation method was used to extract essential oils from dried SO, SG, and SP aerial parts, and chemical composition analysis was carried out using gas chromatography–mass spectrometry (GC-MS). Subsequently, the bioactivities of the oils, including anti-inflammatory and antitumor activities, were evaluated in vitro. Furthermore, in order to provide a reference molecular marker for the plant origin, the ITS1-5.8S-ITS2 genomic regions of the three plants were amplified and sequenced. Our results revealed remarkable differences in the essential oil composition and bioactivities, and in the characteristic ITS sequence, which will facilitate the discrimination of the origins of the three plants and their subsequent utilization.

2. Results and Discussion

2.1. Analysis of Essential Oils

The yield of essential oils from the dried aerial tissue of the three plants was 0.06% (SO), 0.08% (SG), and 0.13% (SP) (w/w). Representative GC-MS chromatograms of the three essential oils are shown in Figure 1. A total of 56 (SO), 62 (SG), and 59 (SP) constituents were identified, representing 98.72% (SO), 93.89% (SG), and 91.35% (SP) of the total peak area. Across the three plants, 148 compounds were identified in total. The retention time, compound name, molecular formula, literature retention indices (RL), experimental retention indices relative to C₈-C₂₀ n-alkanes (RI), and peak area percentages are listed in

Table 1. Chemical composition (peak area percentage) of the essential oils of SO, SG, and SP.

No.	Rt (min)	Compound Name	Molecular Formula	RL	RI	SO	SG	SP
1	6.14	(3E)-Hexenol	C6H12O	855	868		0.05
2	6.59	Hexanol	C6H14O	868	860		0.04
3	6.95	Dibutyl oxide	C8H18O	878	882			0.09
4	7.49	Butyl acrylate	C7H12O2	893	884			0.11
5	7.96	Butyl propionate	C7H14O2	906	894			0.02
6	11.01	Nopinene	C10H16	992	978			0.03
7	11.28	Butyl butyrate	C8H16O2	1000	984			0.07
8	12.24	α-Cymene	C10H14	1025	1027	0.21
9	12.41	Clearene	C10H16	1029	1018			0.07
10	13.57	6-Ethyl-2-methyloctane	C11H24	1059	1046			0.03
11	13.61	2,4-Dimethyldecane	C12H26	1060	1086		0.03
12	15.35	Linalool	C10H18O	1105	1082		0.33
13	15.58	Dehydrocineole	C10H16O	1111	1041		0.03
14	16.45	4-Acetyl-1-methylcyclohexene	C9H14O	1133	1118	0.12
15	16.96	Camphor	C10H16O	1147	1121		0.07
16	17.20	Mentha-1,5-diene-8-ol	C10H16O	1153	1125		0.04
17	17.39	Neryl oxide	C10H16O	1158	1135	0.11
18	17.78	2-Camphanol	C10H18O	1168	1165			0.04
19	17.83	Borneol	C10H18O	1169	1165		0.16
20	17.90	β-Phellandren-8-ol	C10H16O	1171	1178		0.13
21	18.20	4-terpineol	C10H18O	1179	1174	0.01	0.07
22	18.55	p-Cymen-8-ol	C10H14O	1188	1197	0.07
23	18.73	(−)-α-Terpineol	C10H18O	1192	1196	0.08
24	18.78	α-terpineol	C10H18O	1194	1194		0.71	0.04
25	18.83	2-Decanone	C10H20O	1195	1181	0.09
26	19.16	2-Decanol	C10H22O	1204	1218	0.11
27	20.18	Nerol	C10H18O	1233	1228	0.56	2.85	0.48
28	20.42	O-Methylthymol	C11H16O	1239	1231	0.42
29	20.97	2-Hexanoylfuran	C10H14O2	1255	1265			0.04
30	21.16	Geraniol	C10H18O	1260	1256	0.27		0.07
31	21.72	2,4-Dimethylphenethyl alcohol	C10H14O	1276	1292	0.01
32	21.76	Dihydro-4-(3-methyl-2-methylenebutyl)-2(3H)-furanone	C10H16O	1277	1286		0.02
33	21.98	4-Methyldodecane	C13H28	1284	1269			0.03
34	22.02	4,6-Dimethyldodecane	C13H30	1285	1285		0.04
35	22.49	2-Undecanone	C11H22O	1298	1295	0.58
36	22.65	Thymol	C10H14O	1302	1293		0.33
37	22.77	2-Undecanol	C11H24O	1306	1297	0.23
38	23.33	(E,E)-2,4-Decadienal	C10H16O	1322	1321		0.03
39	24.71	Hexahydropseudoionone	C13H26O	1361	1341	0.05
40	24.85	Eugenol	C10H12O2	1365	1359		0.03	0.11
41	25.05	Nerol acetate	C12H20O2	1370	1352		0.13
42	25.24	α-Cubebene	C15H24	1376	1355		0.42
43	25.98	2-Dodecanone	C12H24O	1397	1396	0.45
44	26.48	4,8,8-Trimethyl-2-methylene-4-vinylbicyclo[5.2.0]nonane	C15H24	1409	1407			0.10
45	26.93	cis-Caryophyllene	C15H24	1419	1425	1.30
46	27.00	Caryophyllene	C15H24	1420	1418	14.13	0.21
47	27.19	1,4-Dimethoxy-2-tert-butylbenzene	C12H18O2	1425	1406	0.21	0.02
48	27.67	trans-α-Bergamotene	C15H24	1436	1430		0.89
49	27.72	Aromandendrene	C15H24	1437	1416	0.23
50	27.88	γ-Elemene	C15H24	1440	1431	0.62
51	28.34	cis-α-Bisabolene	C15H24	1451	1478	0.56
52	28.37	α-Farnesene	C15H24	1451	1458			0.12
53	28.56	(E)-β-Famesene	C15H24	1456	1440		0.13
54	29.26	Acoradiene	C15H24	1471	1474	0.61
55	29.39	γ-Muurolene	C15H24	1474	1483	1.42	0.28
56	29.45	Guaia-1(10),11-diene	C15H24	1476	1490		0.21
57	29.57	1,5-Cadinadiene	C15H24	1479	1460			0.11
58	29.64	Curcumene	C15H22	1480	1524	0.51
59	29.8	cis-β-Farnesene	C15H24	1484	1458		3.96
60	30.15	γ-Curcumene	C15H24	1492	1486		0.53
61	30.18	β-Funebrene	C15H24	1492	1479			0.15
62	30.19	Elemol	C15H26O	1492	1512	0.72
63	30.47	β-Himachalene	C15H24	1499	1518		0.19
64	30.54	α-Bisabolene	C15H24	1500	1518	1.83
65	30.73	Cuparene	C15H22	1505	1526		0.04
66	30.79	β-Bisabolene	C15H24	1506	1500	0.31
67	31.12	Valencen	C15H24	1513	1504			0.29
68	31.52	Cadina-1(10),4-diene	C15H24	1523	1526			0.17
69	31.52	Methyl dodecanoate	C13H26O2	1523	1535	0.31
70	31.56	β-Sesquiphellandrene	C15H24	1523	1525		2.74
71	31.78	2-penten-1-ol	C15H24O	1528	1504		3.14
72	32.37	trans-α-Bisabolene	C15H24	1542	1540	24.41
73	32.55	trans-Farnesene epoxide	C15H24O	1546	1540		0.65
74	32.76	(Z)-α-Bisabolene epoxide	C15H24O	1551	1531	1.00
75	32.89	6,10-Dimethyl-3-(1-methylethylidene)cyclodecene	C15H26	1553	1564		0.19
76	32.93	γ-Costol	C15H24O	1554	1542			0.94
77	33.04	Sesquirosefuran	C15H22O	1557	1577		0.15
78	33.09	Ledol	C15H26O	1558	1566	0.16
79	33.26	6-epi-shyobunol	C15H26O	1562	1555		0.09
80	33.46	Globulol	C15H26O	1566	1569	0.22
81	33.52	1,5-Epoxysalvial-4-ene	C15H24O	1568	1560		1.07	0.99
82	33.98	Spathulenol	C15H24O	1578	1577	12.46	0.87	3.44
83	34.21	Caryophyllene oxide	C15H24O	1583	1582	16.88	7.18	21.89
84	34.50	1,2-Dihydronerolidol	C15H28O	1590	1574		0.27
85	34.63	6-(1-methylethyl)-1-naphthalenol	C15H24O	1593	1584			1.51
86	34.64	(−)-Globulol	C15H26O	1593	1590	0.51
87	34.81	Mintketone	C15H24O	1597	1579		1.00	1.53
88	35.13	Rosifoliol	C15H26O	1603	1598	0.33
89	35.15	Cedrol	C15H26O	1603	1583		8.35
90	35.27	Copaborneol	C15H26O	1606	-			0.46
91	35.45	Humulene epoxide	C15H24O	1609	1592	0.25	4.75	2.62
92	35.68	Octahydro-2,2,7a-trimethyl-4-methylene-1,3a-ethano-3aH-inden-5-ol	C15H24O	1613	1599			3.33
93	35.90	Allohimachalol	C15H26O	1617	-			0.48
94	36.12	Z-3-Hexadecen-7-yne	C16H28	1621	1637			0.38
95	36.30	Aromadendrane-4,10-diol	C15H26O2	1624	1619			0.26
96	36.37	Agarospirol	C15H26O	1626	1608		0.89
97	36.45	Bisabolol	C15H26O	1627	1625			0.20
98	36.63	10,10-Dimethyl-2,6-dimethylenebicyclo[7.2.0]undecan-5-ol	C15H24O	1630	1647	0.23		0.67
99	36.78	α-acorenol	C15H26O	1633	1635		1.47
100	36.81	11,11-Dimethyl-4,8-dimethylenebicyclo[7.2.0]undecan-3-ol	C15H24O	1633	1651	0.41		1.20
101	36.92	Isospathulenol	C15H24O	1636	-	0.34
102	37.18	α-Cadinol	C15H26O	1640	1637		0.28	1.35
103	37.40	Cedrenol	C15H24O	1644	1636			1.99
104	37.44	γ-Gurjunenepoxide-(2)	C15H24O	1645	1628		0.83
105	37.68	Sesquibenihiol	C15H24O	1649	1653	0.14	0.30
106	37.70	(−)-Spathulenol	C15H24O	1650	1636			5.14
107	37.88	Himbaccol	C15H26O	1653	1631	1.60
108	37.89	(1S,4R,5S)-1-Methyl-4-(prop-1-en-2-yl)spiro[4.5]dec-7-ene-8-carbaldehyde	C15H22O	1653	1642			0.80
109	37.90	Dehydronerolidol	C15H24O	1653	1632		6.28
110	38.57	trans-Longipinocarveol	C15H24O	1665	1659	2.84		5.87
111	38.79	Isovalencenol	C15H24O	1669	-	0.28
112	38.87	3-Isopropyl-6,7-dimethyltricyclo[4.4.0.0(2,8)]decane-9,10-diol	C15H26O2	1671	1687			0.23
113	38.96	3,7,11-Trimethyl-dodeca-2,4,6,10-tetraenal	C15H22O	1672	1664		3.26
114	39.07	6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalen-2-ol	C15H24O	1674	1690			2.02
115	39.36	1-Isopropyl-4,8-dimethylspiro[4.5]dec-8-en-7-ol	C15H26O	1680	1660		0.28
116	39.41	Germacra-4(15),5E,10(14)-trien-1β-ol	C15H24O	1680	-			14.10
117	39.64	4-(1,5-Dimethylhex-4-enyl)cyclohex-2-enone	C14H22O	1685	1661		12.90
118	40.53	(−)-Isolongifolol, acetate	C17H28O2	1701	1719			1.19
119	40.55	m-Camphorene	C20H32	1701	-		1.79
120	41.03	Pentadecanal	C15H30O	1710	1701	1.65		0.19
121	41.05	Cuparenal	C15H20O	1710	-		3.46
122	41.21	α-Costol	C15H24O	1713	-			0.19
123	42.23	Platambin-1,6-dione	C15H22O2	1731	1740			0.38
124	42.62	Epicedrol	C15H26O	1738	1743			0.81
125	43.62	Ylangenol	C15H24O	1757	-			2.33
126	43.98	Dehydrosaussurea lactone	C15H20O2	1763	1755		5.49	4.85
127	44.32	Valerenic acid	C15H22O2	1769	1759			1.53
128	45.09	β-Acoradienol	C15H24O	1783	-		2.42
129	45.18	4a,5-Dimethyl-3-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalen-1-ol	C15H24O	1785	-	0.58
130	45.92	Aristol-1(10)-en-9-ol	C15H24O	1798	1788	0.93
131	46.27	Naphthalenemethanol	C15H24O	1805	-			1.16
132	46.64	(1-Methyldecyl)cyclohexane	C17H34	1811	1790		0.35
133	47.45	β-Copaen-4α-ol	C15H24O	1826	1820	1.96
134	48.04	Sativene epoxide	C15H24O3	1837	1812		0.13
135	48.10	(+)-Cycloisolongifol-5-ol	C15H24O	1838	-	1.80
136	48.62	2,15-Hexadecanedione	C16H30O	1848	1864	0.66
137	48.73	Phytone	C18H36O	1850	1844		7.83	2.74
138	48.94	6-Methyl-2-(4-methylcyclohex-3-en-1-yl)hepta-1,5-dien-4-ol	C15H24O	1854	1868	1.00
139	49.24	15-Hydroxy-α-muurolene	C15H24O	1859	-	0.35
140	50.06	Diisobutyl phthalate	C16H22O4	1875	1888		1.09	0.29
141	50.62	1-Hexadecanol	C16H34O	1885	1870			0.49
142	50.87	11-Hexadecyn-1-ol	C16H30O	1890	1872	0.44
143	51.21	Z,Z,Z-4,6,9-Nonadecatriene	C19H34	1896	1934	0.32
144	51.87	Geranyl-α-terpinene	C20H32	1908	1962	0.84
145	52.75	Farnesyl acetone	C18H30O	1924	1909		1.17	0.59
146	53.16	Methyl isoheptadecanoate	C18H36O2	1932	1909			0.19
147	54.25	Isophytol	C20H40O	1952	1939		0.26	0.36
148	55.04	Butyl isobutyl phthalate	C16H22O4	1967	1973		0.99	0.49
Sum						98.72	93.89	91.35

RI: Experimental retention indices relative to C₈-C₂₀n-alkanes; RL: Literature retention indices. Compounds shared in common in two or three essential oils are in bold.

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As shown in Figure 1 and Table 1, the composition of the three essential oils differed significantly. The essential oil of SO was dominated by hydrocarbon sesquiterpenes (45.93%), oxygenated sesquiterpenes (44.99%), and oxygenated monoterpenes (1.53%), among which trans-α-bisabolene (24.41%), caryophyllene oxide (16.88%), caryophyllene (14.13%), and spathulenolspathulenol (12.46%) were found to be the main components. The SG essential oil mainly contained oxygenated sesquiterpenes (65.51%), hydrocarbon sesquiterpenes (9.79%), oxygenated diterpenes (9.26%), and oxygenated monoterpenes (4.90%), among which 4-(1,5-dimethylhex-4-enyl)cyclohex-2-enone (12.90%), cedrol (8.35%), phytone (7.83%), caryophyllene oxide (7.18%), dehydronerolidol (6.28%), and dehydrosaussurea lactone (5.49%) were the main components. The essential oil of SP was dominated by oxygenated sesquiterpenes (83.46%), oxygenated diterpenes (3.69%), and hydrocarbon sesquiterpenes (0.94%), with caryophyllene oxide (21.89%), germacra-4(15),5E,10(14)-trien-1β-ol (14.10%), trans-longipinocarveol (5.87%), (−)-spathulenol (5.14%), and dehydrosaussurea lactone (4.85%) as the main components.

Only four chemical components were in common among all three species, namely nerol, spathulenol, caryophyllene oxide, and humulene epoxide. These accounted for 30.15%, 15.65%, and 28.43% of the total peak area in SO, SG, and SP essential oils, respectively (Figure 2). The essential oils of SO and SG shared 5 chemical components, as did the essential oils of SO and SP (Figure 2). SG and SP shared eleven identical components, as shown in Figure 2.

2.2. Anti-Inflammatory Activity

According to traditional use and recently published results, anti-inflammatory [7], antiallergic [8], antithrombotic [9], and immunosuppressive [10,11] effects are reported as the main activities of HS. Diterpenoids were the main antirheumatic constituents of HS [19], of which kirenol was the main chemical constituent. Pharmacological studies suggested that kirenol could inhibit paw swelling, boost up the proliferation of splenic lymphocyte of adjuvant arthritis rats, regulate T lymphocyte subpopulation ratio, and inhibit specific immune response of normal mice [20]. As we know, NO plays an important role in the inflammatory, immune responses, and in thrombotic pathogenesis processes. Inhibition of excess NO production has been used as an assay in the screening of anti-inflammatory, antithrombotic, and immunosuppressive drugs. Therefore, in this study, we treated the mouse macrophage cell line RAW264.7 with lipopolysaccharide (LPS) to induce an inflammatory response and we investigated the inhibitory effects of the three essential oils on NO production by the activated cells. Before the anti-inflammatory activity assay, the cytotoxicity of the three essential oils was evaluated by MTT assay. Our results suggested that the three essential oils had no obvious cytotoxic effects on the RAW264.7 cell line when the concentration was lower than 200 μg/mL (shown in Figure S1).

As shown in Figure 3, the positive control minocycline showed the strongest NO inhibition activity with IC₅₀ = 0.37 μg/mL, which is significantly lower than SG oil (p < 0.05), SO oil (p < 0.01), and SP oil (p < 0.01). Among the three essential oils, SG oil showed the lowest IC₅₀ of 0.97 μg/mL, which is significantly lower (p < 0.01) than SO oil (IC₅₀ = 2.83 μg/mL) and SP oil (IC₅₀ = 13.48 μg/mL). Even though the SG oil showed weaker NO inhibition activity than minocycline, it still has the strongest NO inhibition effect of any crude essential oil as far as we know.

Inflammatory cells can release a variety of inflammatory cytokines which participate in the regulation of the body’s innate immune response and directly kill target cells or mediate apoptosis, thus promoting the repair of damaged tissues. One of these cytokines is IL-6, a critical component of the inflammatory mediator network which plays an important role in the inflammatory response. The ability of the essential oils to inhibit the release of IL-6 was tested in LPS-treated RAW264.7 cells. As indicated in Figure 3, the ability of SO essential oil (IC₅₀, 14.99 μg/mL) to inhibit the release of cytokine IL-6 was significantly weaker (p < 0.01) than that of the positive control (minocycline) (IC₅₀, 7.21 μg/mL), but stronger (p < 0.01) than that of SP (IC₅₀, 25.76 μg/mL) and SG (IC₅₀, 36.41 μg/mL) essential oils.

NO and cytokine IL-6 are important indicators of the cellular inflammatory response. Our results demonstrated that the three essential oils showed significant anti-inflammatory activity. In summary, the three essential oils significantly inhibited the LPS-induced secretion of NO and cytokine IL-6 by the macrophage cell line RAW264.7. These results provide important clues for the identification of possible candidate compounds from the three essential oils, especially SG and SO essential oils.

2.3. Antitumor Activity

The in vitro antitumor activities of the essential oils were evaluated using two cancer cell lines, Hep3B (liver) and Hela (cervical). The cytotoxicity was expressed as IC₅₀ values, as shown in Figure 4. The IC₅₀ of the positive control 5-Fu (4.19 μg/mL in Hep3B, 2.08 μg/mL in Hela) was significantly lower than the three essential oils tested (p < 0.01). Among the three essential oils, SP exhibited the strongest cytotoxicity (38.10 μg/mL for Hep3B, 37.72 μg/mL for Hela). These results suggest that essential oils from the three plants might be used as a natural source of compounds for anticancer compound screening.

A recent study [24] showed that SO ethanol extract significantly inhibited the proliferation of RL 95-2 human endometrial cancer cells. Furthermore, SO ethanol extract was effective against A549 (lung cancer), HepG2 (hepatpma), MDA-MB-231 (breast cancer), and especially on LNCaP (prostate cancer) cell lines. At the same time, it was reported that caryophyllene oxide (IC₅₀ value = 14.90 μg/mL) and caryophyllene (IC₅₀ value = 33.20 μg/mL) were mainly responsible for most cytotoxic activity of SO ethanol extract against RL 95-2 cells. As mentioned previously in Section 2.1, there are only four common compounds in the three essential oils, and caryophyllene oxide is one of the four common components in the three essential oils.

In order to find out the possible relationships between common compounds and two kinds of activities, a linear regression analysis was carried out between percentages and IC₅₀ values. Our results showed that (Figure S2) only caryophyllene oxide peak area percentages showed remarkably strong negative correlation coefficients with IC₅₀ of NO inhibition (R² = 0.959), Hep3B (R² = 0.918), and Hela (R² = 0.980) cytotoxicity. These results suggested the possible important contribution of caryophyllene oxide on the anticancer activity and anti-inflammatory effects of Herba Siegesbeckiae essential oil.

2.4. ITS1-5.8S-ITS2 Sequence

Due to the similarity in morphological characteristics of dried and chopped commercial materials, it is difficult to distinguish the three plants from each other. Therefore, it is important to provide a reliable molecular marker for the identification of the plant origin and for comparison with other research studies. It has been accepted that genomic ribosomal DNA sequences, especially ITS1-5.8S-ITS2, can be used for the interspecific identification of plant origin. Therefore, the full ITS1-5.8S-ITS2 sequence was amplified from genomic DNA of the three plants and sequenced. The accession numbers in Genbank are MH701787 (SO), MH701847 (SG), and MH701848 (SP). Multiple alignment of the ITS1-5.8S-ITS2 sequence from the three plants was carried out using DNAMAN 9.0 software. As shown in Figure S3, across the total sequence of 730–732 bp, the three plants shared 99.27% similarity with each other. There are 16 single nucleotide polyphorism (SNP), which can be used for the development of specific Polymerase chain reaction (PCR) assays to distinguish the origins of samples of these three plants.

3. Conclusions

In this study, hydrodistillation was used to extract essential oils from the dried aerial parts of SO, SG, and SP, and their chemical compositions and bioactivities were analyzed and compared. A total of 56, 62, and 59 constituents were identified in SO, SG, and SP essential oils, respectively, by GC-MS. There was a marked difference between the components of the three essential oils. The essential oils of SO, SG, and SP exhibited various levels of anti-inflammatory and antitumor activity in vitro. The essential oil of SP (IC₅₀, 0.97 μg/mL) significantly reduced NO release by LPS-induced RAW264.7 macrophages, and the essential oil of SO was the most effective at reducing the release of cytokine IL-6. The antitumor activities of the essential oil from SP were much better than SG and SO. Therefore, the essential oils of SO, SG, and SP were significantly different from each other in terms of phytochemical composition, but with similar bioactivities with various activity levels. Among four common compounds in three essential oils, caryophyllene oxide peak area percentages showed remarkably high negative correlation coefficients with IC₅₀ values of Hep3B and Hela cytotoxicity, which suggested the contribution of this compound on the cancer cell cytotoxicity of three essential oils. Finally, in order to provide detailed information about the origin of the plants used in the present work, the complete ITS1-5.8S-ITS2 region was amplified from plant genomic DNA and sequenced. This sequence can be used by other researchers to confirm whether their plant specimens have the same origin as ours or not.

4. Materials and Methods

4.1. Materials

The flowering SP and SG plant materials were collected in October 2017 from Jinzhai, Anhui province, China. The flowering SO plant material was collected in April 2018 from Zhanzhou, Hainan province, China. Voucher specimens were deposited at the Herbarium of Shenyang Pharmaceutical University (Shenyang, China). The voucher numbers were SYPU-P-201710-007 (SP), SYPU-P-201710-008 (SG), and SYPU-P-201804-025 (SO). They were identified by Associate Professor Jia Lingyun in Shenyang Pharmaceutical University (Shenyang, China). The fresh plants were air-dried and ground into a fine powder which was passed through a 20 mesh and stored in sealed plastic bags at −20 °C for future use. RAW264.7, a mouse macrophage cell line, was purchased from the Cell Bank of the Shanghai Institute of Cell Biology and Biochemistry, Chinese Academy of Sciences (Shanghai, China). The cancer cells lines Hep3B (liver) and Hela (cervical) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

4.2. Reagents

He (purity > 99.999%) and N₂ (purity > 99.999%) were supplied by Shenyang Qianzhen Chemical Gas (Shenyang, China). Hexane (HPLC grade) and anhydrous sodium sulfate (analytical grade) were purchased from Shandong Yuwang Chemical Group (Shandong, China). Plant Genomic DNA Extraction Kits, 2X Taq master premix, and GoldView dye were purchased from Kangweishiji Bio. (Beijing, China). Agarose G-10 powder was purchased from Biowest (Hongkong, China). Synthesis of primers ITS1 and ITS4 as well as Sanger sequencing were carried out at Genewiz Bio. Ltd. (Suzhou, China). DMSO and 1% penicillin-streptomycin antibiotics were purchased from Sigma (biology grade, Burlington, VT, USA). RPMI 1640, Eagle’s Minimum Essential Medium, and 10% fetal bovine serum were purchased from Gibco (Waltham, MA, USA). Nitric Oxide Assay Kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). ELISA kits were purchased from Shanghai Joyee Biotechnics Co., Ltd. (Shanghai, China). Minocycline was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). 5-Fluorouracil was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

4.3. DNA Extraction, ITS Amplification, Electrophoresis and Sequencing

Dried leaf tissues were ground into a fine powder. Then, DNA was extracted using a Plant Genomic DNA Extraction Kit following the manufacturer′s instructions. Extracted DNA (1 μL) was used as the template for PCR amplification, with 25 μL 2X Taq master premix, 3 μL 10 μM ITS1 primer (5′-TCCGTAGGTGAACCTGCGG-3′), 3 μL 10 μM ITS4 primer (5′-TCCTCCGCTT ATTGATATGC-3′), and 18 μL double-distilled H₂O. The amplification conditions were as follows: 94 °C for 5 min (1 cycle); 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s (30 cycles); 72 °C for 10 min (1 cycle). PCR product (5 μL) was applied to a 1% agarose gel containing GoldView dye and electrophoresis was carried out for 15 min at 150 V followed by observation under UV light in a Tenon gel imaging system. After confirmation of a strong, single amplified band of about 700 bp, the PCR product was sent for Sanger sequencing using ITS1 and ITS4 as sequencing primers. Sequences were confirmed by comparing them with the original sequencing chromatogram and were connected using Seqman software (Madison, MI, USA).

4.4. Essential Oil Extraction

100 g powder of dried aerial parts were added together with 1 L distilled water into a 2 L round-bottomed bottle, which was connected to a Clevenger-type apparatus with tap water for cooling. The hydrodistillation was continued for 4 h to extract the essential oils. The obtained essential oil was collected from the side arm and dried with anhydrous sodium sulphate, then sealed in a headspace bottle and stored at 4 °C until it was tested.

4.5. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The GC-MS analysis was carried out on a Shimadzu (TQ-8040) series GC-MS system (Tokyo, Japan) equipped with an AOC-20i autosampler. The columns used an HP-5 capillary column (30 m × 0.32 mm, i.d. 0.25 μm) with a stationary phase of 5% diphenyl/95% dimethylpolysiloxane. Helium was used as the carrier gas. The column temperature was maintained at 40 °C for 3 min and then programmed to increase as follows: rising from 40 °C to 130 °C at a rate of 4 °C/min and holding for 2 min; rising from 130 °C to 150 °C at a rate of 2 °C/min and holding for 3 min; rising from 150 °C to 180 °C at a rate of 2 °C/min and holding for 3 min; and finally rising from 180 °C to 210 °C at a rate of 5 °C/min and holding for 5 min. The ionization energy was 70 eV with a scan time of 0.3 s and a mass range of 45–500 AMU. The identification of the compounds was based on the comparison of their retention times and mass spectra with those from the NIST14 and NIST14s (National Institute of Standards and Technologies, Mass Spectra Libraries, Gaithersburg, MD, USA) [25].

4.6. Anti-Inflammatory Activities

4.6.1. Cell Line and Cell Culture

RAW264.7, a mouse macrophage cell line, was grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in a 37 °C humidified incubator containing 5% CO₂.

4.6.2. Cell Viability Assay

The MTT assay was used to evaluate the effect of essential oils on cell viability [26]. RAW264.7 cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well. After overnight growth, cells were treated with different concentrations of essential oils for 1 h, then incubated in the presence or absence of LPS (100 ng/mL) for the next 24 h. Then, 20 μL of MTT solution (5 mg/mL) was added and the cells were cultured for a further 4 h. After that, the supernatant was carefully removed and then the resulting formed azan crystals were dissolved in 100 µL DMSO with horizontal shaking. The absorbance at 490 nm (ref. 570 nm) [24] was measured with a microplate reader.

4.6.3. Measurement of NO and IL-6 Release

RAW264.7 cells were seeded into 96-well plates at a density of 8 × 10⁴ cells/mL and cultured overnight. After pretreatment with different concentrations of essential oils (0.2–200 µg/mL) or the positive control (minocycline) for 1 h, the cells were stimulated with LPS (100 ng/mL) for 24 h. The concentration of NO in the conditioned culture medium was examined with the Nitric Oxide Assay Kit and the release of IL-6 in the supernatants was assayed using ELISA kits according to the manufacturer’s instructions [27,28]. The concentrations were calculated from standard curves.

4.7. Antitumor Assay

4.7.1. Cell Lines and Cell Culture

The cancer cells lines Hep3B (liver) and Hela (cervical) were grown in media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and maintained in a CO₂ incubator at 37 °C in 5% CO₂. The medium was changed every 2 days until the cells reached confluence, at which point they were subcultured.

4.7.2. Cytotoxicity Assay

The essential oils from HS were evaluated for their cytotoxic activities against cells using the MTT cell viability assay with 5-fluorouracil as the positive control. First, cellular suspensions (1 × 10⁵ cells/mL) were cultured in 96-well plates and exposed to different concentrations of the essential oils (1–200 μg/mL) using 3 wells for each concentration. The plates were incubated at 37 °C in 5% CO₂ for 48 h [29]. Then, 200 μL MTT solution was added to each well and the plates were cultured for 4 h. The absorbance of each well was measured at 490 nm using a microplate reader. The data were expressed as the mean percentage of viable cells as compared to the respective control cultures treated with the solvent. The half maximal growth inhibitory concentration (IC₅₀ values) was calculated from the standard curves of the dose-dependent cytotoxicity of the essential oils.

4.8. Statistical Analysis

All activity assay experiments were repeated three times and the data were represented as mean ± SD. Significance analysis was carried out using the t-test in Microsoft Excel 2007. Letters above the columns in Figure 2 and Figure 3 represent significance (A–D: p < 0.01; a,b,d: p < 0.05)