Essential Oils of Zingiber Species from Vietnam: Chemical Compositions and Biological Activities

Abstract

Mosquito-borne diseases are a large problem in Vietnam as elsewhere. Due to environmental concerns regarding the use of synthetic insecticides as well as developing insecticidal resistance, there is a need for environmentally-benign alternative mosquito control agents. In addition, resistance of pathogenic microorganisms to antibiotics is an increasing problem. As part of a program to identify essential oils as alternative larvicidal and antimicrobial agents, the leaf, stem, and rhizome essential oils of several Zingiber species, obtained from wild-growing specimens in northern Vietnam, were acquired by hydrodistillation and investigated using gas chromatography. The mosquito larvicidal activities of the essential oils were assessed against Culex quinquefasciatus, Aedes albopictus, and Ae. aegypti, and for antibacterial activity against a selection of Gram-positive and Gram-negative bacteria, and for activity against Candida albicans. Zingiber essential oils rich in α-pinene and β-pinene showed the best larvicidal activity. Zingiber nudicarpum rhizome essential oil showed excellent antibacterial activity against Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus, with minimum inhibitory concentrations (MIC) of 2, 8, and 1 μg/mL, respectively. However, the major components, α-pinene and β-pinene, cannot explain the antibacterial activities obtained.

1. Introduction

Vietnam is located in the tropics of Southeast Asia, and several mosquito-borne diseases are endemic, including Japanese encephalitis [1], dengue fever [2], and Zika [3]. Culex species are considered to be important vectors of Japanese encephalitis virus (JEV), including Culex quinquefasciatus Say (Diptera: Culicidae) [4], but other mosquito genera may also serve as competent vectors of the virus [5,6]. Dengue fever is hyperendemic to Vietnam with all four serotypes of the virus in circulation, resulting in periodic acute epidemics of both dengue fever and dengue hemorrhagic fever [7,8]. Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) mosquitoes are the principal vectors of dengue fever virus (DFV) in Vietnam [9]. Zika virus (ZIKV) first appeared in Vietnam in 2016, where the primary transmission vector is Aedes mosquitoes [10]. Exacerbating this problem is the increasing insecticide resistance in Aedes [11,12,13] and Culex mosquitoes [14,15].

As observed throughout the world, antimicrobial resistance is an increasing problem in Vietnam [16]. Particularly noteworthy are antibiotic-resistant organisms in hospital settings, including Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa [17], Streptococcus pneumoniae, Haemophilus influenzae [18], Escherichia coli, and Staphylococcus aureus [19].

Zingiber Mill. is a species-rich genus within the subfamily Zingireroideae of Zingiberaceae, which are native to Southeast Asia [20]. The Plant List currently has 146 accepted names for Zingiber species [21]. The phytochemistry, particularly essential oil chemistry, and the pharmacology of Zingiber have been reviewed [22]. Currently, at least thirty-six species of Zingiber have been reported in Vietnam [23,24,25].

There is a need to discover new and alternative insect-control agents and antimicrobial agents. In this work, essential oils from seven species of Zingiber growing in Vietnam were collected and analyzed by gas chromatographic methods. Six species were screened for mosquito larvicidal activity and antibacterial and antifungal activity.

Zingiber cornubracteatum Triboun & K. Larsen was first recorded in northern Thailand (Mae Hong Son) [26], but has since been collected in northern Vietnam (Thanh Hoa, Nghe An, and Quang Binh provinces) [25]. There have been no reports on the phytochemistry of this plant. Zingiber neotruncatum T.L. Wu, K. Larsen & Turland has been recorded from southern and western Yunnan province, China [27] and Nghệ An province, Vietnam [28]. There have been no previous reports on the essential oil of this species. Zingiber nitens M.F. Newman is known from Bolikhamsai Province, Laos [29], and Nghệ An Province, Vietnam [30]. The essential oil composition of Z. nitens from Vietnam has been previously published [31]. Zingiber nudicarpum D. Fang has been recorded in Guangxi Province, China [27], Laos [32], and Vietnam [33]. The essential oil composition of Z. nudicarpum from Vietnam has been previously published [34]. Zingiber ottensii Valeton is native to Southeast Asia, including Thailand, Indonesia, Malaysia, Laos, Myanmar, and Vietnam [35,36]. Essential oil compositions of Z. ottensii have been reported from Malaysia [37,38], Thailand [39,40], and Indonesia [41]. Zingiber recurvatum S.Q. Tong & Y.M. Xia has been recorded in southern Yunnan Province, China [42], northern Laos [32], and Vietnam [43,44]. There are apparently no reports on the volatile phytochemistry of this plant. Zingiber vuquangensis Lý N.S., Lê T.H., Trịnh T.H., Nguyễn V.H., Đỗ N.Đ. is a new species, only recently recorded in Vietnam [45]. The essential oil composition of Z. vuquangensis has been reported [46].

2. Results and Discussion

2.1. Essential Oil Compositions

The Zingiber plant tissues (leaves, stems, or rhizomes) were collected from north-central Vietnam and the plant tissues subjected to hydrodistillation to obtain the respective essential oils (Table 1, Figure 1). Gas chromatographic–mass spectral (GC-MS) and gas chromatographic–flame ionization detection (GC-FID) were used to analyze the essential oil compositions, which are compiled in Table 2.

Table 1.

Plant collection and hydrodistillation details of Zingiber species from north-central Vietnam.

Zingiber Species	Vietnamese Name	Collection Site Coordinates; Elevation	Collection Month Year	Voucher Number	Plant Part	% Yield (v/w)
Zingiber cornubraceatum Triboun & K. Larsen	Gừng lá bắc cựa	Khe Kèm water fall, Pù Mát National Park 19°58′14″ N, 104°48′20″ E; 280 m	September 2019	830	Leaf Rhizome	0.12 0.15
		Xuân Lý commune, Như Thanh District, Bến En National Park 19°39′09″ N, 105°30′24″ E; 80 m	October 2019	832	Leaf Stems Rhizome	0.14 0.10 0.17
		Tri Lễ commune, Quế Phong District, Pù Hoạt Nature Reserve 19°38′57″ N, 104°40′55″ E; 660 m	September 2018	735	Leaf Rhizome	0.10 0.12
Zingiber neotruncatum T.L. Wu, K. Larsen & Turland	Gừng lá mới	Đồng Văn commune, Quế Phong district, Pù Hoạt Nature Reserve 19°53′58″ N, 105°07′45″ E; 315 m	October 2018	746	Rhizome	0.26
Zingiber nitens M.F. Newman	Gừng lá sáng bóng	Vũ Quang National Park 18°17′13″ N, 105°26′12″ E; 180 m	September 2018	738	Leaf Stems	0.21 0.45
Zingiber nudicarpum D.Fang	Gừng quả trần	Thông Thụ commune, Quế Phong district, Pù Hoạt Nature Reserve 19°49′5″ N, 104°55′35″ E; 460 m	April 2019	760	Leaf Stems Rhizome	0.12 0.08 0.15
		Bạch Mã National Park 16°13′44″ N, 107°53′54″ E; 350 m	April 2018	778	Leaf Stems	0.15 0.10
		Nam Đông District, Thừa Thiên Huế province Nam Đông District, Thừa Thiên Huế province 16°13′9″ N, 107°43′28″ E; 110 m	July 2019	777	Leaf Stems Rhizome	0.14 0.09 0.18
Zingiber ottensii Valeton	Gừng ottensi	Tri Lễ commune, Quế Phong district, Pù Hoạt Nature Reserve 19°39′05″ N, 105°40′59″ E; 640 m	April 2019	772	Leaf Roots	0.18 0.25
Zingiber recurvatum S.Q. Tong & Y.M. Xia	Gừng lá bắc cong	Tri Lễ commune, Quế Phong district, Pù Hoạt Nature Reserve 19°39′37″ N, 104°40′29″ E; 640 m	July 2019	792	Leaf Rhizome	0.14 0.19
Zingiber vuquangensis Ly N.S., Le T.H., Trinh T.H., Nguyen V.H., Do N.D.	Gừng vũ quang	Nậm Nhong commune, Quế Phong district, Pù Hoạt Nature Reserve 19°30′31″ N, 104°43′13″ E; 590 m	April 2019	771	Leaf Stems Rhizome	0.15 0.10 0.14

Figure 1.

Distribution map of Zingiber species from the north-central Vietnam. (A): Zingiber cornubraceatum Triboun & K. Larsen (red squares); Z. neotruncatum Triboun & K. Larsen (blue triangle), Z. nitens M.F. Newman (orange circle), Z. nudicarpum D.Fang (green stars). (B): Zingiber ottensii Valeton (red square), Z. recurvatum S.Q. Tong & Y.M. Xia (blue circle), Z. vuquangensis Ly N.S., Le T.H., Trinh T.H., Nguyen V.H., Do N.D. (orange star).

Table 2.

Chemical compositions of essential oils of Zingiber species from north-central Vietnam.

RIcalc	RIdb	Compounds	Z. cornubracteatum (Pù Hoạt) Leaf		Z. cornubracteatum (Pù Hoạt) Rhizome	Z. cornubracteatum (Pù Mát) Leaf	Z. cornubracteatum (Pù Mát) Rhizome	Z. cornubracteatum (Bến En) leaf	Z. Cornubracteatum (Bến En) Stem	Z. cornubracteatum (Bến En) Rhizome	Z. neotruncatum Rhizome	Z. nitens Leaf	Z. nitens Rhizome	Z. ottensii Leaf	Z. ottensii Rhizome
899	894	2-Heptanol	---		---	---	---	---	---	---	---	---	---	---	---
921	923	Tricyclene	---		---	---	---	---	---	---	---	---	0.9	---	---
925	927	α-Thujene	---		---	---	2.1	---	---	2.2	---	---	---	---	0.9
934	933	α-Pinene	5.1		14.5	2.7	8.2	10.1	9.9	9.8	1.9	11.2	10.5	4.0	3.4
943	945	α-Fenchene	---		---	---	0.3	---	---	0.3	---	---	0.9	---	---
951	953	Camphene	---		1.2	---	3.2	0.3	0.3	1.3	0.2	0.3	40.4	---	0.3
952	953	Thuja-2,4(10)diene	---		---	---	---	---	---	---	---	---	---	---	---
971	972	Sabinene	0.8		0.5	0.9	0.7	1.4	1.4	11.9	0.4	1.7	0.2	0.4	21.6
978	978	β-Pinene	20.1		14.8	18.8	8.8	67.3	66.8	33.1	3.1	6.0	1.1	17.1	11.7
981	985	6-Methylhept-5-en-2-one	---		---	---	---	---	---	---	0.3	---	---	---	---
988	991	Myrcene	0.4		0.4	1.8	7.7	1.0	1.0	4.2	0.3	0.4	2.0	0.4	1.3
989	988	Dehydro-1,8-cineole	---		---	---	---	---	---	---	---	---	---	---	---
989	984	2-Pentylfuran	---		---	---	---	---	---	---	---	---	---	---	---
1002	998	Octanal	---		---	---	---	---	---	---	---	---	---	---	---
1004	1108	1,3,8-p-Menthatriene	---		---	---	---	---	---	---	---	---	---	---	---
1006	1007	α-Phellandrene	0.8		0.4	1.1	0.4	---	---	1.6	---	---	0.2	---	0.6
1008	1009	δ-3-Carene	0.2		0.1	0.2	5.3	---	---	2.1	---	---	---	---	---
1016	1018	α-Terpinene	---		---	0.2	0.2	---	---	0.8	---	---	---	---	3.5
1018	1022	m-Cymene	---		---	---	0.1	---	---	---	---	---	---	---	---
1023	1025	p-Cymene	0.4		0.5	0.3	3.9	---	---	2.7	---	---	---	---	2.1
1034	1028	Limonene	0.6		1.0	1.2	5.1	1.2	1.2	3.4	0.2	0.9	4.6	0.8	0.9
1035	1031	β-Phellandrene	0.5		0.9	0.8	2.2	0.3	0.3	0.5	---	0.2	---	0.2	0.3
1035	1030	1,8-Cineole	---		10.4	---	6.1	0.2	0.2	2.5	0.7	---	---	---	4.3
1036	1034	(Z)-β-Ocimene	0.3		---	---	---	---	---	---	---	0.2	0.9	0.3	---
1046	1045	(E)-β-Ocimene	0.6		0.4	0.4	0.4	0.1	0.1	0.8	1.2	2.1	12.7	2.1	0.9
1057	1057	γ-Terpinene	0.3		0.4	0.4	0.7	0.2	0.2	2.4	---	---	0.1	---	5.5
1067	1063	1-Octanol	---		---	---	---	---	---	---	---	---	---	---	---
1068	1067	cis-Linalool oxide (furanoid)	---		0.3	---	---	---	---	---	---	---	---	---	---
1072	1065	cis-Sabinene hydrate	---		---	---	---	---	---	---	---	---	---	---	0.3
1084	1086	Terpinolene	0.1		---	0.3	---	---	---	0.7	---	---	0.8	---	1.1
1084	1084	trans-Linalool oxide (furanoid)	---		0.3	---	---	---	---	---	---	---	---	---	---
1087	1083	Fenchone	---		---	---	---	---	---	---	---	---	0.2	---	---
1088	1089	p-Cymenene	---		---	---	---	---	---	---	---	---	---	---	---
1099	1102	Perillene	---		---	---	0.3	0.1	0.1	---	51.3	---	---	---	---
1099	1097	2-Nonanol	---		---	---	0.3	---	---	---	---	---	---	---	---
1100	1101	Linalool	0.5		31.0	-	11.6	0.2	0.2	0.4	---	0.4	---	---	0.2
1100	1104	Rosefuran	---		---	---	---	---	---	---	0.2	---	---	---	---
1104	1098	trans-Sabinene hydrate	---		0.3	---	---	---	---	---	---	---	---	---	0.2
1105	1107	Nonanal	---		---	---	---	---	---	---	---	---	---	---	---
1112	1116	(E)-4,8-Dimethylnona-1,3,7-triene	---		---	0.1	0.2	---	---	---	---	---	---	---	---
1118	1114	endo-Fenchol	---		---	---	0.3	---	---	0.2	---	---	---	---	---
1118	1118	cis-p-Menth-2-en-1-ol	---		---	---	---	---	---	---	---	---	---	---	0.4
1120	1128	allo-Ocimene	---		---	---	---	---	---	---	---	---	---	---	---
1124	1118	exo-Fenchol	---		0.3	---	---	---	---	---	---	---	---	---	---
1128	1122	α-Campholenal	---		---	---	0.1	---	---	---	---	---	---	---	---
1135	1136	trans-p-Menth-2-en-1-ol	---		---	---	---	---	---	---	---	---	---	---	0.3
1137	1135	Nopinone	---		---	---	---	---	---	---	---	---	---	---	---
1141	1135	trans-Pinocarveol	---		---	---	---	---	---	0.2	---	---	---	---	---
1144	1140	trans-Verbenol	---		0.8	---	---	---	---	---	---	---	---	---	---
1146	1149	Camphor	---		---	---	---	---	---	---	---	---	---	---	---
1148	1165	Lavandulol	---		---	---	---	---	---	---	0.2	---	---	---	---
1159	1167	Benzyl acetate	---		---	---	---	---	---	---	---	---	---	---	---
1162	1160	Pinocarvone	---		---	---	---	---	---	---	---	---	---	---	---
1167	1165	iso-Neral	---		---	---	---	---	---	---	0.7	---	---	---	---
1169	1162	δ-Terpineol	---		---	---	---	---	---	---	---	---	---	---	---
1170	1166	p-Mentha-1,5-dien-8-ol	---		---	---	---	---	---	---	---	---	---	---	---
1171	1165	Borneol	---		3.2	---	0.7	---	---	0.1	---	---	0.8	---	0.5
1180	1180	Terpinen-4-ol	0.1		0.7	---	0.5	---	---	1.4	0.1	---	---	---	17.1
1185	1179	p-Cymen-8-ol	---		---	---	---	---	---	---	---	---	---	---	---
1185	1185	iso-Geranial	---		---	---	---	---	---	---	1.1	---	---	---	---
1194	1195	α-Terpineol	---		0.6	---	0.2	---	---	0.1	---	---	---	---	0.9
1194	1195	Myrtenal	---		---	---	---	---	---	---	---	---	---	---	---
1194	1193	(4Z)-Decenal	---		---	---	---	---	---	---	---	---	---	---	---
1195	1194	Myrtenol	1.0		0.2	---	---	---	---	---	---	---	---	---	---
1198	1195	cis-Piperitol	---		---	---	---	---	---	---	---	---	---	---	0.1
1205	1204	Verbenone	---		---	---	---	---	---	---	---	---	---	---	---
1206	1206	Decanal	---		---	---	---	---	---	---	---	---	---	---	---
1209	1207	trans-Piperitol	---		---	---	---	---	---	---	---	---	---	---	0.2
1216	1218	endo-Fenchyl acetate	---		---	---	0.6	---	---	0.5	---	---	3.3	---	---
1217	1215	trans-Carveol	---		---	---	---	---	---	---	---	---	---	---	---
1220	1217	β-Cyclocitral	---		---	---	---	---	---	---	---	---	---	---	---
1222	1227	Nerol	---		---	---	---	---	---	---	---	---	---	---	---
1224	1223	Citronellol	---		---	---	---	---	---	---	---	---	---	---	---
1227	1232	Thymyl methyl ether	---		---	---	---	---	---	---	---	---	---	---	---
1236	1235	Neral	---		---	---	---	---	---	---	12.3	---	---	---	---
1241	1238	Cuminal	---		---	---	---	---	---	---	---	---	---	---	---
1242	1239	Carvone	---		---	---	---	---	---	---	---	---	---	---	---
1248	1249	Geraniol	---		---	---	0.2	---	---	---	0.3	---	---	---	---
1265	1264	Geranial	---		---	---	---	---	---	---	17.0	---	---	---	---
1271	1266	1-Decanol	---		---	---	0.2	---	---	---	---	---	---	---	---
1281	1287	iso-Bornyl acetate	---		---	---	---	---	---	---	---	---	---	---	---
1284	1285	Bornyl acetate	0.2		2.8	---	7.9	---	---	2.5	---	---	14.5	---	0.3
1285	1291	Safrole	---		---	---	---	---	---	---	---	---	---	---	---
1293	1290	Dihydroedulan IIA	---		---	---	---	---	0.2	---	---	---	---	0.3	---
1295	1298	Carvacrol	---		---	---	---	---	---	---	---	---	---	---	---
1295	1293	Methyl myrtenate	---		---	---	---	---	---	---	---	---	---	---	---
1307	1295	trans-Sabinyl acetate	---		---	---	---	---	---	0.2	---	---	---	---	---
1330	1324	Methyl geranate	---		---	---	0.3	---	---	---	0.2	---	---	---	---
1330	1332	Bicycloelemene	---		---	---	---	---	---	---	---	---	---	---	---
1330	1335	δ-Elemene	1.4		---	1.5	---	0.4	0.5	---	---	---	---	0.2	---
1333	1325	Myrtenyl acetate	0.1		0.5	---	2.0	---	---	0.7	---	---	---	---	---
1342	1349	α-Cubebene	0.1		---	---	---	---	---	---	---	---	---	---	---
1345	1346	α-Terpinyl acetate	---		---	---	---	---	---	---	---	---	---	---	---
1364	1367	Cyclosativene	---		---	---	---	---	---	---	---	---	---	---	---
1364	1372	iso-Ledene	---		---	---	---	---	---	---	---	---	---	---	---
1368	1373	α-Ylangene	---		---	---	---	---	---	---	---	---	---	---	---
1374	1375	α-Copaene	0.2		---	0.4	---	---	---	---	---	---	---	3.2	---
1375	1380	Geranyl acetate	---		---	---	---	---	---	---	0.6	---	---	---	---
1377	1383	cis-β-Elemene	---		---	---	---	---	---	---	---	---	---	---	---
1377	1365	Eugenol	---		---	---	---	---	---	---	---	---	---	---	---
1382	1382	β-Bourbonene	---		---	---	---	---	---	---	---	---	---	---	---
1386	1387	β-Cubebene	0.4		---	0.5	---	---	---	---	---	---	---	---	---
1387	1390	trans-β-Elemene	0.2		---	0.5	---	3.9	4.4	0.7	---	0.8	0.2	17.0	0.3
1400	1403	Methyl eugenol	---		---	---	---	---	---	---	---	---	---	---	---
1403	1405	Sesquithujene	---		---	---	---	---	---	---	---	0.1	---	---	---
1403	1408	(Z)-Caryophyllene	---		---	---	---	---	---	---	---	---	---	---	---
1405	1406	α-Gurjunene	---		---	0.3	---	---	---	0.2	---	---	---	0.6	---
1405	1415	β-Maaliene	---		---	---	---	---	---	---	---	---	---	---	---
1415	1416	cis-α-Bergamotene	---		---	---	---	---	---	---	---	---	---	---	---
1419	1417	(E)-Caryophyllene	8.9		0.4	13.9	1.7	1.8	2.1	0.6	1.5	---	---	28.0	0.7
1427	1432	γ-Elemene	0.3		---	---	---	0.2	0.3	0.1	---	---	---	---	---
1428	1424	2,5-Dimethoxy-p-cymene	---		---	---	---	---	---	---	---	---	---	---	---
1428	1430	β-Copaene	---		---	---	---	---	---	---	---	---	---	---	---
1428	1438	α-Maaliene	---		---	---	---	---	---	---	---	---	---	---	---
1431	1416	α-Santalene	---		---	---	---	---	---	---	---	---	---	---	---
1431	1432	trans-α-Bergamotene	---		---	---	---	---	---	---	---	---	---	---	---
1433	1437	α-Guaiene	---		---	---	---	---	---	---	---	---	---	---	---
1437	1438	Aromadendrene	0.3		---	0.3	---	0.2	---	---	---	---	---	0.1	---
1440	1440	(Z)-β-Farnesene	---		---	---	---	---	---	---	---	0.3	---	---	---
1444	1431	β-Gurjunene	0.3		---	0.6	---	---	---	---	---	---	---	---	---
1445	1445	Myltayl-4(12)-ene	---		---	---	---	---	---	---	---	---	---	---	---
1446	1453	Geranyl acetone	---		---	---	---	---	---	---	---	---	---	---	---
1447	1453	trans-Muurola-3,5-diene	---		---	---	---	---	---	---	---	---	---	---	---
1450	1452	(E)-β-Farnesene	---		---	---	---	---	---	---	---	0.2	---	---	---
1452	1457	Prezizaene	---		---	---	---	---	---	---	---	---	---	---	---
1454	1454	α-Humulene	0.7		---	1.0	0.3	0.2	0.3	---	---	---	---	3.0	3.8
1458	1457	allo-Aromadendrene	---		---	---	---	---	---	---	---	---	---	---	---
1460	1461	cis-Cadina-1(6),4-diene	---		---	---	---	---	---	---	---	---	---	---	---
1470	1472	trans-Cadina-1(6),4-diene	---		---	0.7	---	---	---	---	---	---	---	---	---
1471	1476	β-Chamigrene	---		---	---	---	---	---	---	---	---	---	---	---
1471	1475	γ-Gurjunene	---		---	---	---	---	---	---	---	---	---	---	---
1472	1482	γ-Curcumene	---		---	---	---	---	---	---	---	---	---	---	---
1473	1478	γ-Muurolene	0.3		---	0.5	0.1	---	---	---	---	---	---	---	---
1474	1476	Selina-4,11-diene	---		---	---	---	---	---	---	---	---	---	---	---
1477	1482	α-Amorphene	---		---	---	---	---	---	---	---	---	---	---	---
1477	1479	ar-Curcumene	---		---	---	---	---	---	---	---	5.2	0.2	---	---
1477	1487	(E)-β-Ionone	---		---	---	---	---	---	---	---	---	---	---	---
1478	1483	trans-β-Bergamotene	---		---	---	---	---	---	---	---	---	---	---	---
1479	1464	9-epi-(E)-Caryophyllene	0.4		---	0.8	---	0.1	0.2	---	---	0.2	---	0.2	---
1480	1480	Germacrene D	1.6		---	13.7	0.8	0.7	0.8	0.2	---	0.7	---	2.5	---
1481	1488	δ-Selinene	---		---	---	---	---	---	---	---	---	---	---	---
1482	1492	cis-β-Guaiene	---		---	---	---	---	---	---	---	---	---	---	---
1483	1487	Aristolochene	---		---	---	---	---	---	---	---	---	0.3	---	---
1488	1492	β-Selinene	0.3		---	0.4	---	0.5	0.6	---	---	---	---	0.8	---
1489	1496	Valencene	---		---	---	---	---	---	---	---	---	---	0.4	---
1490	1490	γ-Amorphene	---		---	---	---	---	---	---	---	---	---	---	---
1491	1493	α-Zingiberene	---		---	---	---	---	---	---	---	17.4	1.6	---	---
1494	1498	α-Selinene	---		---	---	---	---	---	---	---	---	---	---	---
1494	1497	Bicyclogermacrene	7.4		---	18.9	0.5	2.7	3.0	0.4	0.9	1.5	---	1.2	---
1496	1500	α-Muurolene	---		---	---	---	---	---	---	---	---	---	---	---
1496	1506	α-Bulnesene	---		---	---	---	---	---	---	---	---	---	---	---
1497	1502	trans-β-Guiaene	---		---	---	---	---	---	---	---	---	---	---	---
1500	1500	n-Pentadecane	---		---	---	---	---	---	---	0.2	---	---	---	---
1501	1505	(E,E)-α-Farnesene	---		---	0.9	0.2	0.5	0.6	0.2	---	---	---	1.1	---
1501	1495	γ-Amorphene	---		---	---	---	---	---	---	---	---	---	---	---
1502	1496	Viridiflorene	---		---	---	---	---	---	---	---	---	---	---	---
1503	1509	β-Curcumene	---		---	---	---	---	---	---	---	---	---	---	---
1506	1508	β-Bisabolene	---		---	---	0.2	---	---	0.2	1.2	3.7	0.2	---	---
1511	1512	γ-Cadinene	0.3		---	0.5	---	---	0.2	---	---	---	---	---	---
1511	1511	δ-Amorphene	---		---	0.4	---	0.2	0.2	---	---	---	---	---	---
1513	1519	Cubebol	---		---	---	---	---	---	---	---	---	---	---	---
1516	1518	δ-Cadinene	0.6		-	0.9	0.2	0.2	0.2	---	---	---	---	2.2	---
1517	1520	7-epi-α-Selinene	---		---	---	---	---	---	---	---	---	---	---	---
1517	1521	Zonarene	---		---	---	---	---	---	---	---	---	---	---	---
1519	1521	trans-Calamenene	---		---	---	---	---	---	---	---	---	---	---	---
1520	1528	(E)-γ-Bisabolene	---		---	---	---	---	---	---	---	0.2	---	---	---
1523	1521	Eugenyl acetate	---		---	---	---	---	---	---	---	---	---	0.2	0.1
1524	1521	β-Sesquiphellandrene	---		---	---	---	---	---	---	---	10.1	0.6	---	---
1529	1528	cis-Calamene	---		---	---	---	---	---	---	---	---	---	---	---
1530	1536	trans-Cadina-1,4-diene	---		---	---	---	---	---	---	---	---	---	---	---
1535	1537	α-Cadinene	---		---	---	---	---	---	---	---	---	---	---	---
1539	1544	α-Calacorene	---		---	---	---	---	---	---	---	---	---	---	---
1546	1549	α-Elemol	0.2		---	0.2	---	---	---	0.2	---	---	---	0.2	0.4
1557	1559	Germacrene B	0.3		---	0.5	---	0.2	0.3	0.3	---	---	---	---	---
1558	1561	(E)-Nerolidol	23.0		---	2.7	8.4	0.9	1.1	0.3	0.1	10.0	1.1	0.5	---
1560	1564	β-Calacorene	---		---	---	---	---	---	---	---	---	---	---	---
1564	1568	Palustrol	---		---	---	---	---	---	---	---	---	---	---	---
1568	1571	Maaliol	---		---	---	---	---	---	---	---	---	---	---	---
1570	1573	(3E,7E)-4,8,12-Trimethyltrideca-1,3,7,11-tetraene	---		---	---	---	---	---	---	---	---	---	---	---
1575	1576	Spathulenol	2.5		---	1.4	---	0.1	0.2	---	0.4	0.6	---	0.2	---
1575	1582	Neryl isovalerate	---		---	---	---	---	---	---	---	---	---	---	---
1575	1574	Germacrene D-4-ol	0.2		---	---	---	---	---	---	---	0.9	---	---	---
1582	1577	Caryophyllene oxide	2.2		---	2.0	0.6	---	0.2	0.3	0.3	---	0.1	1.2	0.4
1582	1582	ar-Turmerol	---		---	---	---	---	---	---	---	0.2	---	---	---
1583	1570	Dendrolasin	---		---	---	---	---	---	---	---	---	---	---	---
1583	1585	epi-Globulol	---		---	---	---	---	---	---	---	---	---	---	---
1587	1590	Globulol	---		---	---	---	---	---	---	---	---	---	---	---
1590	1581	Clovenol	---		---	---	---	---	---	---	---	---	---	---	---
1592	1594	Salvial-4(14)en-1-one	---		---	---	---	---	---	---	---	---	---	---	---
1593	1592	Viridiflorol	---		---	---	---	---	---	---	---	---	---	---	---
1593	1593	Scapanol g	---		---	0.3	---	---	---	---	---	---	---	---	---
1594	1600	Guaiol	0.5		---	0.2	---	0.1	0.2	0.3	---	---	---	---	---
1594	1601	trans-β-Elemenone	---		---	---	---	---	---	---	---	---	---	---	---
1596	1599	Cubeban-11-ol	---		---	---	---	---	---	---	---	---	---	---	---
1599	1592	Humulene epoxide I	---		---	---	---	---	---	---	---	---	---	---	1.2
1600	1609	Rosifoliol	---		---	---	---	---	---	---	---	---	---	---	---
1604	1607	5,7-di-epi-Eudesmol	---		---	---	---	---	---	---	---	---	---	---	---
1604	1602	Ledol	0.3		---	0.3	---	---	---	---	---	---	---	---	---
1613	1618	1,10-di-epi-Cubenol	---		---	---	---	---	---	---	---	---	---	---	---
1616	1613	Humulene epoxide II	---		---	---	0.2	---	---	---	---	---	---	---	0.6
1616	1611	Zingiberenol	---		---	---	---	---	---	---	---	7.2	0.2	---	---
1618	1623	Humulane-1,6-dien-3-ol	---		---	---	---	---	---	---	---	---	---	---	---
1620	1622	10-epi-γ-Eudesmol	---		---	0.3	---	---	---	---	---	---	---	---	---
1625	1631	1-epi-Cubenol	---		---	0.2	---	---	---	---	---	---	---	---	---
1627	1618	epi-Cedrol	---		---	---	---	---	---	---	---	---	---	---	---
1628	1622	Alismol	0.4		---	---	---	---	---	---	---	---	---	---	---
1630	1630	γ-Eudesmol	---		---	---	---	---	---	---	---	---	---	---	0.2
1632	1631	Caryophylla-4(12),8(13)-dien-5α-ol	---		---	---	---	---	---	---	---	---	---	---	---
1634	1632	α-Acorenol	---		---	---	---	---	---	---	---	3.3	0.2	---	---
1636	1639	Caryophylla-4(12),8(13)-dien-5β-ol	---		---	---	---	---	---	---	---	---	---	---	---
1640	1638	τ-Cadinol	0.9		---	0.2	---	---	---	---	---	---	---	---	---
1640	1643	Cubenol	---		---	---	---	---	---	---	---	---	---	---	---
1642	1644	τ-Murrolol	---		---	0.4	---	---	---	---	---	---	---	---	---
1645	1644	α-Muurolol (=δ-Cadinol)	---		---	0.2	---	---	---	---	---	---	---	---	---
1651	1649	β-Eudesmol	---		---	0.3	0.3	---	---	---	---	---	---	---	0.3
1652	1652	α-Cadinol	0.5		---	1.0	---	---	0.1	---	---	0.4	---	0.3	---
1652	1647	1,2-Diacetoxy-4-allylbenzene	---		---	---	---	---	---	---	---	---	---	---	---
1653	1652	α-Eudesmol	---		---	---	---	---	---	---	---	---	---	---	0.3
1657	1658	Selin-11-en-4α-ol	---		---	---	---	---	---	---	---	---	---	---	---
1662	1658	neo-Intermedeol	0.5		---	---	---	---	0.1	-	---	0.7	0.2	0.2	---
1668	1668	14-Hydroxy-9-epi-(E)-caryophyllene	---		---	0.2	---	---	---	---	---	---	---	---	---
1670	1675	Cadalene	---		---	---	---	---	---	---	---	---	---	---	---
1671	1676	Mustakone	---		---	---	---	---	---	---	---	---	---	---	---
1679	1681	γ-Bicyclofarnesal	---		---	---	0.5	---	---	0.5	---	---	---	---	---
1681	1685	α-Bisabolol	---		---	---	---	---	---	---	---	4.1	---	---	---
1684	1685	Germacra-4(15),5,10(14)-trien-1α-ol	---		---	---	---	---	---	---	---	---	---	---	---
1684	1683	epi-α-Bisabolol	---		---	---	0.1	---	---	---	---	---	---	---	---
1690	1693	Germacrone	---		---	---	---	---	---	---	---	---	---	---	---
1691	1694	Germacra-4(15),5,10(14)-trien-1β-ol	---		---	---	---	---	---	---	---	---	---	---	---
1696	1689	6α-Hydroxygermacra-1(10),4-diene	---		---	---	---	---	---	---	---	0.7	---	---	---
1701	1698	(2Z,6Z)-Farnesol	---		---	---	---	---	---	---	---	0.9	---	---	---
1702	1690	(Z)-trans-α-Bergamotol	---		---	---	---	---	---	---	---	---	---	---	---
1707	1714	Nootkatol	---		---	---	---	---	---	---	---	---	---	---	---
1713	1713	Pentadecanal	0.2		---	---	---	---	---	0.2	---	---	---	---	---
1738	1740	Mint sulfide	1.5		---	1.9	---	---	0.1	---	---	---	---	---	---
1739	1732	Zerumbone	---		---	---	---	---	---	---	---	---	---	---	12.5
1761	1759	Benzyl benzoate	---		---	---	---	---	---	---	---	---	---	---	---
1774	1760	(Z)-Lanceol	---		---	---	---	---	---	---	0.3	---	---	---	---
1799	1809	Ambrial	0.1		---	---	0.9	---	---	3.6	---	---	---	0.3	---
1838	1838	Phytone	---		---	---	---	---	---	---	---	---	---	---	---
1848	1845	6,10,14-Trimethylpentadecan-2-one	1.1		---	---	---	0.1	0.2	---	---	---	---	---	---
1862	1864	Benzyl salicylate	---		---	---	---	---	---	---	---	---	---	---	---
2002	---	(E)-15,16-Bisnorlabda-8(17),11-dien-13-one	---		---	---	0.3	---	---	0.2	---	---	---	---	---
2104	2105	(E)-Phytol	---		---	0.3	---	0.3	0.4	---	---	---	---	0.2	---
		Monoterpene hydrocarbons	30.2		35.1	29.1	49.3	81.9	81.2	77.8	7.3	23.0	75.3	25.3	54.1
		Oxygenated monoterpenoids	1.9		51.4	0.0	30.8	0.5	0.5	8.8	84.7	0.4	18.8	0.0	24.8
		Sesquiterpene hydrocarbons	24.0		0.4	57.2	4.0	11.8	13.7	2.9	3.6	40.4	3.1	60.5	4.8
		Oxygenated sesquiterpenoids	31.2		0.0	9.9	10.1	1.1	1.9	1.6	1.1	29.0	1.8	2.6	15.9
		Others	2.9		0.0	2.3	1.9	0.4	0.9	4.0	0.5	0.0	0.0	1.0	0.1
		Total identified	90.2		86.9	98.5	96.1	95.7	98.2	95.1	97.2	92.8	99.0	89.4	99.7
RIcalc	RIdb	Compounds	Z. nudicarpum (Pù Hoạt) leaf	Z. nudicarpum (Pù Hoạt) stem	Z. nudicarpum (Pù Hoạt) rhizome	Z. nudicarpum (Nam Đông) leaf	Z. nudicarpum (Nam Đông) stem	Z. nudicarpum (Nam Đông) rhizome	Z. nudicarpum (Bạch Mã) leaf	Z. nudicarpum (Bạch Mã) stem	Z. recurvatum leaf	Z. recurvatum rhizome	Z. vuquangensis leaf	Z. vuquangensis stem	Z. vuquangensis rhizome
899	894	2-Heptanol	---	---	---	---	0.2	---	---	---	---	---	---	---	---
921	923	Tricyclene	---	---	---	---	---	tr	tr	tr	---	---	---	---	---
925	927	α-Thujene	0.1	---	0.1	0.8	0.1	tr	0.1	tr	0.3	0.3	---	---	---
934	933	α-Pinene	10.9	---	18.7	5.0	10.6	4.0	6.5	6.1	16.3	1.9	11.3	0.5	9.8
943	945	α-Fenchene	---	---	---	---	---	---	---	---	0.2	---	---	---	---
951	953	Camphene	1.6	---	1.3	0.1	0.3	2.1	0.6	0.8	0.4	1.9	0.2	---	0.3
952	953	Thuja-2,4(10)diene	---	---	---	tr	0.2	---	---	---	---	---	---	---	---
971	972	Sabinene	---	---	1.4	2.3	0.1	tr	0.2	0.1	1.2	0.3	1.0	---	0.7
978	978	β-Pinene	34.0	0.5	58.3	26.6	9.0	9.8	0.7	5.6	71.6	4.7	38.5	3.1	45.0
981	985	6-Methylhept-5-en-2-one	---	---	---	---	0.1	---	---	---	---	---	---	---	---
988	991	Myrcene	0.8	---	0.9	0.8	0.4	0.6	0.7	1.2	1.0	2.1	0.5	---	0.7
989	988	Dehydro-1,8-cineole	---	---	---	---	0.1	---	---	---	---	---	---	---	---
989	984	2-Pentylfuran	---	---	---	tr	---	---	---	---	---	---	---	---	---
1002	998	Octanal	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1004	1108	1,3,8-p-Menthatriene	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1006	1007	α-Phellandrene	---	---	---	tr	0.1	0.2	0.9	0.6	0.2	0.6	---	---	---
1008	1009	δ-3-Carene	---	---	0.2	---	0.2	3.1	3.7	1.8	---	1.5	---	---	---
1016	1018	α-Terpinene	0.1	---	---	0.2	tr	tr	0.1	tr	---	---	---	---	0.1
1018	1022	m-Cymene	---	---	---	---	tr	tr	---	---	---	---	---	---	---
1023	1025	p-Cymene	0.2	---	0.4	0.4	6.0	0.9	0.2	0.1	---	0.6	---	---	0.2
1034	1028	Limonene	1.5	---	1.8	2.0	2.1	1.8	1.7	6.0	1.8	2.4	0.9	---	1.9
1035	1031	β-Phellandrene	0.2	---	0.4	0.2	---	0.7	0.3	0.3	0.3	0.6	0.3	---	2.3
1035	1030	1,8-Cineole	0.9	---	0.3	0.9	21.2	6.8	tr	tr	---	0.6	---	---	---
1036	1034	(Z)-β-Ocimene	---	---	---	tr	---	---	0.1	0.2	0.2	---	---	---	---
1046	1045	(E)-β-Ocimene	---	---	0.7	tr	0.1	---	0.1	0.2	0.6	0.4	---	---	---
1057	1057	γ-Terpinene	0.5	---	0.3	0.8	0.1	0.4	0.1	0.1	0.1	0.6	---	---	0.4
1067	1063	1-Octanol	---	---	---	---	0.3	---	---	---	---	---	---	---	---
1068	1067	cis-Linalool oxide (furanoid)	---	---	---	---	---	---	---	---	---	---	---	---	---
1072	1065	cis-Sabinene hydrate	---	---	---	---	---	---	---	---	---	---	---	---	---
1084	1086	Terpinolene	0.1	---	0.3	0.2	0.1	0.3	0.1	0.1	---	0.4	---	---	0.4
1084	1084	trans-Linalool oxide (furanoid)	---	---	---	---	---	---	---	---	---	---	---	---	---
1087	1083	Fenchone	---	---	---	---	---	---	---	---	---	---	---	---	---
1088	1089	p-Cymenene	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1099	1102	Perillene	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1099	1097	2-Nonanol	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1100	1101	Linalool	2.4	11.0	0.5	0.6	0.3	0.5	0.7	1.0	---	3.7	---	---	---
1100	1104	Rosefuran	---	---	---	---	---	---	---	---	---	---	---	---	---
1104	1098	trans-Sabinene hydrate	---	---	---	---	---	---	---	---	---	---	---	---	---
1105	1107	Nonanal	---	---	---	0.1	---	---	tr	tr	---	---	---	---	---
1112	1116	(E)-4,8-Dimethylnona-1,3,7-triene	0.1	---	---	tr	---	---	---	---	---	0.4	---	---	---
1118	1114	endo-Fenchol	---	---	---	---	0.1	0.7	---	---	---	0.2	---	---	---
1118	1118	cis-p-Menth-2-en-1-ol	---	---	---	---	---	---	---	---	---	---	---	---	---
1120	1128	allo-Ocimene	0.3	---	---	---	---	---	---	---	---	---	---	---	---
1124	1118	exo-Fenchol	---	---	---	---	---	---	---	---	---	---	---	---	---
1128	1122	α-Campholenal	---	---	---	tr	0.2	---	---	---	---	---	---	---	---
1135	1136	trans-p-Menth-2-en-1-ol	---	---	---	---	---	---	---	---	---	---	---	---	---
1137	1135	Nopinone	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1141	1135	trans-Pinocarveol	---	---	---	tr	2.0	0.2	---	---	---	---	---	---	---
1144	1140	trans-Verbenol	---	---	---	---	0.4	---	---	---	---	---	---	---	---
1146	1149	Camphor	---	---	---	---	0.1	0.3	0.1	tr	---	---	---	---	---
1148	1165	Lavandulol	---	---	---	---	---	---	---	---	---	---	---	---	---
1159	1167	Benzyl acetate	---	---	---	---	---	---	0.1	tr	---	---	---	---	---
1162	1160	Pinocarvone	---	---	0.2	tr	1.5	0.2	---	---	---	---	0.2	---	0.3
1167	1165	iso-Neral	---	---	---	---	---	---	---	---	---	---	---	---	---
1169	1162	δ-Terpineol	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1170	1166	p-Mentha-1,5-dien-8-ol	---	---	---	---	0.6	---	---	---	---	---	---	---	---
1171	1165	Borneol	0.2	0.6	---	tr	0.2	4.0	---	---	---	---	---	---	---
1180	1180	Terpinen-4-ol	---	0.2	---	0.1	0.4	0.4	0.1	0.1	0.1	0.1	---	---	---
1185	1179	p-Cymen-8-ol	---	---	---	tr	0.3	---	---	---	---	---	---	---	---
1185	1185	iso-Geranial	---	---	---	---	---	---	---	---	---	---	---	---	---
1194	1195	α-Terpineol	---	0.2	---	0.1	---	1.6	tr	0.1	---	---	---	---	0.2
1194	1195	Myrtenal	0.2	---	0.3	0.1	2.6	---	---	---	---	---	0.2	---	0.3
1194	1193	(4Z)-Decenal	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1195	1194	Myrtenol	---	---	---	---	0.9	---	---	---	---	---	---	---	---
1198	1195	cis-Piperitol	---	---	---	---	---	---	---	---	---	---	---	---	---
1205	1204	Verbenone	---	---	---	---	0.3	---	---	---	---	---	---	---	---
1206	1206	Decanal	---	---	---	0.1	---	---	---	0.1	---	0.2	---	---	---
1209	1207	trans-Piperitol	---	---	---	---	---	---	---	---	---	---	---	---	---
1216	1218	endo-Fenchyl acetate	---	---	0.1	tr	0.5	6.5	---	---	---	0.2	---	---	---
1217	1215	trans-Carveol	---	---	---	---	0.2	---	---	---	---	---	---	---	---
1220	1217	β-Cyclocitral	---	---	---	tr	---	---	---	---	---	---	---	---	---
1222	1227	Nerol	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1224	1223	Citronellol	---	---	---	---	0.3	0.4	---	---	---	---	---	---	---
1227	1232	Thymyl methyl ether	---	---	---	---	---	0.4	---	---	---	---	---	---	---
1236	1235	Neral	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1241	1238	Cuminal	---	---	---	---	tr	---	---	---	---	---	---	---	---
1242	1239	Carvone	---	---	---	---	0.2	---	---	---	---	---	---	---	---
1248	1249	Geraniol	---	---	---	---	1.0	0.8	---	---	---	0.3	---	---	---
1265	1264	Geranial	---	---	---	---	0.2	tr	---	---	---	---	---	---	---
1271	1266	1-Decanol	---	---	---	---	---	---	---	---	---	---	---	---	---
1281	1287	iso-Bornyl acetate	---	---	---	---	---	---	tr	0.4	---	0.2	---	---	---
1284	1285	Bornyl acetate	---	---	0.3	---	0.4	1.2	0.2	1.1	---	10.4	---	---	---
1285	1291	Safrole	---	---	---	---	---	---	tr	0.2	---	---	---	---	---
1293	1290	Dihydroedulan IIA	---	---	---	1.1	---	---	---	---	---	---	2.6	0.8	0.1
1295	1298	Carvacrol	---	---	---	---	0.3	---	---	---	---	---	---	---	---
1295	1293	Methyl myrtenate	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1307	1295	trans-Sabinyl acetate	---	---	---	---	---	---	---	---	---	---	---	---	---
1330	1324	Methyl geranate	---	---	---	---	---	---	---	---	---	---	---	---	---
1330	1332	Bicycloelemene	---	---	---	0.2	---	---	1.1	0.5	---	---	---	---	---
1330	1335	δ-Elemene	0.2	1.1	---	---	---	---	0.9	0.4	---	0.2	1.0	1.4	0.5
1333	1325	Myrtenyl acetate	---	---	---	---	---	---	---	---	---	---	---	---	---
1342	1349	α-Cubebene	---	---	---	---	---	---	0.7	0.4	---	---	---	---	---
1345	1346	α-Terpinyl acetate	---	---	---	0.1	---	1.3	---	---	---	---	---	---	---
1364	1367	Cyclosativene	---	---	---	---	---	---	0.3	0.1	---	---	---	---	---
1364	1372	iso-Ledene	---	---	---	---	---	---	---	tr	---	---	---	---	---
1368	1373	α-Ylangene	---	---	---	tr	0.2	0.1	---	---	---	---	---	---	---
1374	1375	α-Copaene	---	---	---	0.1	3.1	0.7	3.2	1.5	---	0.2	---	0.3	0.2
1375	1380	Geranyl acetate	---	---	---	---	---	---	---	0.2	---	0.5	---	---	---
1377	1383	cis-β-Elemene	---	---	---	---	---	---	0.2	0.2	---	---	---	---	---
1377	1365	Eugenol	---	---	---	---	---	---	---	---	---	---	---	0.9	---
1382	1382	β-Bourbonene	---	---	---	tr	---	---	0.4	0.1	---	---	---	---	---
1386	1387	β-Cubebene	---	---	---	---	0.1	---	1.7	0.6	---	---	---	---	---
1387	1390	trans-β-Elemene	1.1	1.1	0.4	2.3	0.4	0.8	4.7	3.7	---	0.8	5.9	10.0	2.5
1400	1403	Methyl eugenol	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1403	1405	Sesquithujene	---	---	---	---	---	---	---	---	---	---	---	---	---
1403	1408	(Z)-Caryophyllene	---	---	---	---	---	---	---	---	---	---	---	0.4	---
1405	1406	α-Gurjunene	---	---	---	0.1	---	---	0.1	0.1	---	0.3	---	---	---
1405	1415	β-Maaliene	---	---	---	---	---	---	0.1	---	---	---	---	---	---
1415	1416	cis-α-Bergamotene	---	0.4	---	0.2	---	---	0.1	0.1	---	---	0.2	0.3	---
1419	1417	(E)-Caryophyllene	13.9	52.6	1.9	24.3	1.6	4.9	6.4	6.3	1.7	11.3	12.2	14.4	2.3
1427	1432	γ-Elemene	---	---	---	---	---	0.3	0.7	---	---	---	---	---	---
1428	1424	2,5-Dimethoxy-p-cymene	0.1	---	---	---	---	---	---	---	---	---	---	---	---
1428	1430	β-Copaene	---	---	---	0.2	---	---	---	0.5	---	---	---	---	---
1428	1438	α-Maaliene	---	---	---	---	---	---	---	0.1	---	---	---	---	---
1431	1416	α-Santalene	---	---	0.1	---	---	---	---	---	---	---	---	---	---
1431	1432	trans-α-Bergamotene	0.2	0.1	---	tr	---	---	0.3	0.1	---	---	---	---	---
1433	1437	α-Guaiene	---	---	---	tr	---	0.2	---	---	---	---	---	---	---
1437	1438	Aromadendrene	0.2	0.2	---	0.1	---	---	0.6	0.5	---	---	0.2	0.4	0.4
1440	1440	(Z)-β-Farnesene	0.2	0.2	---	---	---	---	---	---	---	1.1	---	0.5	---
1444	1431	β-Gurjunene	---	0.1	---	---	---	---	---	---	---	---	---	0.9	0.2
1445	1445	Myltayl-4(12)-ene	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1446	1453	Geranyl acetone	---	---	---	0.3	---	---	---	---	---	---	---	---	---
1447	1453	trans-Muurola-3,5-diene	---	---	---	---	0.1	0.4	0.2	0.1	---	---	---	---	---
1450	1452	(E)-β-Farnesene	---	---	0.3	0.2	---	0.1	0.3	0.2	---	---	---	---	---
1452	1457	Prezizaene	---	---	---	---	---	---	---	0.4	---	---	---	---	---
1454	1454	α-Humulene	2.1	5.9	0.3	3.2	1.1	4.8	6.4	8.8	0.2	6.9	1.1	1.6	0.5
1458	1457	allo-Aromadendrene	---	---	---	0.9	0.7	0.3	1.9	0.1	---	---	---	---	---
1460	1461	cis-Cadina-1(6),4-diene	---	---	---	---	0.1	0.1	---	---	---	---	---	---	---
1470	1472	trans-Cadina-1(6),4-diene	---	---	---	---	0.2	---	0.4	0.2	---	---	---	---	---
1471	1476	β-Chamigrene	---	---	---	0.1	---	---	---	---	---	---	0.6	2.6	0.7
1471	1475	γ-Gurjunene	---	---	---	---	---	---	---	---	---	---	---	1.5	---
1472	1482	γ-Curcumene	---	---	---	---	---	---	0.3	0.3	---	---	---	---	---
1473	1478	γ-Muurolene	0.2	---	---	0.1	0.3	0.1	0.3	0.3	---	0.4	---	---	---
1474	1476	Selina-4,11-diene	---	---	---	---	---	1.8	---	0.1	---	---	---	---	---
1477	1482	α-Amorphene	---	---	---	---	tr	0.2	---	0.1	---	---	---	---	---
1477	1479	ar-Curcumene	---	---	---	---	---	---	---	---	---	---	1.4	2.8	0.8
1477	1487	(E)-β-Ionone	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1478	1483	trans-β-Bergamotene	---	---	---	---	---	---	0.2	---	---	---	---	---	---
1479	1464	9-epi-(E)-Caryophyllene	1.7	0.5	---	---	---	---	---	---	---	0.3	---	0.2	-
1480	1480	Germacrene D	0.6	0.4	---	3.6	tr	0.3	6.5	4.2	0.8	2.7	2.0	1.4	2.2
1481	1488	δ-Selinene	---	---	---	---	---	---	---	0.2	---	---	---	---	---
1482	1492	cis-β-Guaiene	---	---	---	---	---	---	0.3	---	---	---	---	---	---
1483	1487	Aristolochene	---	---	---	0.5	0.8	---	---	---	---	---	---	---	---
1488	1492	β-Selinene	0.6	0.3	0.3	0.8	5.4	2.4	1.4	1.8	---	0.3	---	2.0	0.6
1489	1496	Valencene	---	---	---	---	---	---	---	---	---	---	---	---	---
1490	1490	γ-Amorphene	---	---	---	0.1	0.1	0.4	1.3	0.8	---	---	0.8	1.6	---
1491	1493	α-Zingiberene	---	---	---	---	---	---	---	---	---	---	3.6	1.7	0.6
1494	1498	α-Selinene	---	---	---	---	1.4	2.2	---	---	---	---	1.0	---	---
1494	1497	Bicyclogermacrene	9.6	1.1	0.3	3.3	---	---	16.1	9.8	2.0	5.1	0.7	---	1.3
1496	1500	α-Muurolene	---	---	---	0.3	0.2	0.2	---	---	---	---	---	---	---
1496	1506	α-Bulnesene	---	---	---	---	---	---	0.5	0.3	---	---	---	---	---
1497	1502	trans-β-Guiaene	---	---	---	---	---	---	0.1	---	---	---	---	---	---
1500	1500	n-Pentadecane	---	---	---	---	---	---	---	---	---	---	---	---	---
1501	1505	(E,E)-α-Farnesene	1.6	0.7	0.4	---	0.4	0.3	---	---	---	0.7	---	---	---
1501	1495	γ-Amorphene	---	0.2	---	---	---	---	---	---	---	---	---	---	---
1502	1496	Viridiflorene	---	---	---	---	---	---	---	---	---	---	---	---	0.4
1503	1509	β-Curcumene	---	---	---	---	---	---	0.1	0.1	---	---	---	---	---
1506	1508	β-Bisabolene	---	---	---	0.1	0.1	0.6	0.7	0.3	---	---	1.4	2.0	0.4
1511	1512	γ-Cadinene	0.1	---	---	0.2	1.7	3.1	0.2	0.2	---	0.2	---	0.3	0.2
1511	1511	δ-Amorphene	---	---	---	---	---	---	---	---	---	---	---	---	---
1513	1519	Cubebol	---	---	---	---	0.1	---	0.4	0.2	---	---	---	---	---
1516	1518	δ-Cadinene	0.6	0.2	---	0.4	1.0	1.7	2.9	2.4	---	0.5	---	0.9	1.4
1517	1520	7-epi-α-Selinene	---	---	---	---	tr	1.1	---	---	---	---	1.0	1.1	---
1517	1521	Zonarene	---	---	---	---	---	---	0.1	0.3	---	---	---	---	---
1519	1521	trans-Calamenene	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1520	1528	(E)-γ-Bisabolene	---	---	---	---	---	---	0.5	0.2	---	---	---	---	---
1523	1521	Eugenyl acetate	---	---	---	---	---	---	---	---	---	---	---	1.0	0.6
1524	1521	β-Sesquiphellandrene	---	---	---	---	---	---	---	---	---	---	0.7	0.5	---
1529	1528	cis-Calamene	---	---	---	---	---	---	---	---	---	---	---	0.3	---
1530	1536	trans-Cadina-1,4-diene	---	---	---	---	0.1	---	0.3	0.2	---	---	---	---	---
1535	1537	α-Cadinene	---	---	---	---	tr	0.2	---	---	---	---	---	---	---
1539	1544	α-Calacorene	---	---	---	---	0.5	0.4	---	---	---	---	---	---	---
1546	1549	α-Elemol	---	0.2	---	---	0.1	0.4	0.8	0.9	---	0.2	---	0.6	---
1557	1559	Germacrene B	---	---	---	---	---	0.7	0.6	0.2	---	---	0.3	---	0.2
1558	1561	(E)-Nerolidol	1.1	1.2	0.2	0.2	1.1	---	---	---	0.3	3.3	0.8	3.7	0.9
1560	1564	β-Calacorene	---	---	---	---	0.2	0.1	---	---	---	---	---	---	---
1564	1568	Palustrol	---	---	---	---	---	---	tr	0.1	---	---	---	---	---
1568	1571	Maaliol	---	---	---	---	---	0.2	tr	0.3	---	---	---	---	---
1570	1573	(3E,7E)-4,8,12-Trimethyltrideca-1,3,7,11-tetraene	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1575	1576	Spathulenol	0.9	0.7	0.1	2.1	tr	0.2	1.2	0.9	0.2	1.3	---	1.2	0.2
1575	1582	Neryl isovalerate	---	---	---	---	---	0.2	---	---	---	---	---	---	---
1575	1574	Germacrene D-4-ol	---	---	---	---	---	---	---	---	---	---	---	---	---
1582	1577	Caryophyllene oxide	1.3	4.4	0.4	3.6	8.0	2.4	tr	0.3	0.2	4.1	2.0	4.5	1.6
1582	1582	ar-Turmerol	---	---	---	---	---	---	---	---	---	---	---	---	---
1583	1570	Dendrolasin	0.2	---	---	---	---	---	---	---	---	---	---	---	---
1583	1585	epi-Globulol	---	---	---	0.8	tr	0.9	---	---	---	---	---	---	---
1587	1590	Globulol	---	---	---	---	tr	0.4	1.0	1.6	---	---	---	---	---
1590	1581	Clovenol	---	---	---	---	---	---	---	---	---	---	---	0.2	---
1592	1594	Salvial-4(14)en-1-one	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1593	1592	Viridiflorol	---	---	---	0.2	---	0.5	0.7	1.2	---	---	---	---	---
1593	1593	Scapanol g	---	---	---	---	---	---	---	---	---	---	---	---	---
1594	1600	Guaiol	0.4	0.6	0.1	---	0.2	0.5	---	---	---	0.2	---	0.7	---
1594	1601	trans-β-Elemenone	---	---	---	---	tr	0.4	---	---	---	---	---	---	---
1596	1599	Cubeban-11-ol	---	---	---	0.1	---	---	tr	0.3	---	---	---	---	---
1599	1592	Humulene epoxide I	---	---	---	---	---	---	---	---	---	0.4	---	0.9	---
1600	1609	Rosifoliol	---	---	---	---	---	---	0.2	0.4	---	---	---	---	---
1604	1607	5,7-di-epi-Eudesmol	---	---	---	---	---	0.2	---	---	---	---	---	---	---
1604	1602	Ledol	---	---	---	0.3	---	---	---	---	---	0.2	---	---	---
1613	1618	1,10-di-epi-Cubenol	---	---	---	---	0.2	0.7	---	---	---	---	---	---	---
1616	1613	Humulene epoxide II	0.2	0.8	---	0.4	1.8	1.3	tr	0.2	---	2.8	---	0.6	0.6
1616	1611	Zingiberenol	---	---	---	---	---	---	---	---	---	---	---	0.3	---
1618	1623	Humulane-1,6-dien-3-ol	---	---	---	---	---	---	8.2	9.9	---	---	---	---	---
1620	1622	10-epi-γ-Eudesmol	---	---	---	---	tr	0.4	---	---	---	2.9	---	---	---
1625	1631	1-epi-Cubenol	0.2	0.3	---	---	0.2	0.3	---	1.3	---	---	---	---	---
1627	1618	epi-Cedrol	---	---	0.2	---	---	---	---	---	---	---	---	---	---
1628	1622	Alismol	---	---	---	---	---	---	---	---	---	---	---	---	0.4
1630	1630	γ-Eudesmol	---	---	---	---	0.3	1.7	0.5	0.7	---	---	---	---	---
1632	1631	Caryophylla-4(12),8(13)-dien-5α-ol	---	---	---	0.7	0.1	---	---	---	---	---	---	---	---
1634	1632	α-Acorenol	---	---	---	---	---	---	---	---	---	---	---	---	---
1636	1639	Caryophylla-4(12),8(13)-dien-5β-ol	---	---	---	0.9	0.3	0.4	---	---	---	---	---	---	---
1640	1638	τ-Cadinol	0.9	2.2	---	0.1	---	0.3	---	---	---	---	---	1.7	0.6
1640	1643	Cubenol	---	---	---	---	0.1	---	0.6	0.7	---	---	---	---	---
1642	1644	τ-Murrolol	---	---	---	0.2	0.1	0.4	---	0.4	---	0.2	---	0.6	---
1645	1644	α-Muurolol (= δ-Cadinol)	0.2	---	---	0.1	tr	0.1	0.2	0.7	---	---	---	---	---
1651	1649	β-Eudesmol	---	---	---	---	---	---	---	---	---	---	---	---	---
1652	1652	α-Cadinol	0.5	0.5	---	0.7	tr	1.3	1.0	1.7	---	0.3	0.2	2.0	0.5
1652	1647	1,2-Diacetoxy-4-allylbenzene	0.1	1.9	---	---	---	---	---	---	---	---	---	---	---
1653	1652	α-Eudesmol	---	---	---	---	0.6	1.1	---	---	---	---	---	1.0	---
1657	1658	Selin-11-en-4α-ol	---	---	---	0.2	tr	0.5	---	---	---	---	---	---	---
1662	1658	neo-Intermedeol	0.7	1.2	---	---	0.2	4.4	0.2	0.6	---	0.2	0.4	2.6	---
1668	1668	14-Hydroxy-9-epi-(E)-caryophyllene	---	0.6	---	0.6	0.3	0.5	---	---	---	0.4	0.2	1.0	---
1670	1675	Cadalene	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1671	1676	Mustakone	---	---	---	---	0.1	---	---	---	---	---	---	---	---
1679	1681	γ-Bicyclofarnesal	---	0.9	0.6	---	---	---	---	---	---	1.2	---	0.6	0.4
1681	1685	α-Bisabolol	---	---	---	---	---	---	---	---	---	---	---	---	---
1684	1685	Germacra-4(15),5,10(14)-trien-1α-ol	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1684	1683	epi-α-Bisabolol	---	---	---	---	---	---	---	---	---	---	---	---	---
1690	1693	Germacrone	---	---	---	---	0.2	1.6	---	---	---	---	---	---	---
1691	1694	Germacra-4(15),5,10(14)-trien-1β-ol	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1696	1689	6α-Hydroxygermacra-1(10),4-diene	---	---	---	---	---	---	---	---	---	---	---	---	---
1701	1698	(2Z,6Z)-Farnesol	---	---	---	---	---	---	---	---	---	---	---	---	---
1702	1690	(Z)-trans-α-Bergamotol	---	0.3	---	---	---	---	---	---	---	---	---	---	---
1707	1714	Nootkatol	---	---	---	0.1	---	---	---	---	---	---	---	---	---
1713	1713	Pentadecanal	---	---	---	0.1	---	---	---	---	---	0.3	---	---	0.9
1738	1740	Mint sulfide	---	---	---	0.1	---	---	---	---	0.2	0.9	0.6	2.6	---
1739	1732	Zerumbone	---	---	---	---	---	---	---	---	0.2	3.9	---	---	---
1761	1759	Benzyl benzoate	---	---	---	---	---	---	2.5	3.0	---	0.3	---	0.3	---
1774	1760	(Z)-Lanceol	---	---	---	---	---	---	---	---	---	---	---	---	---
1799	1809	Ambrial	---	---	---	0.2	tr	0.5	---	---	---	---	0.4	2.8	2.4
1838	1838	Phytone	---	---	---	0.7	---	---	---	---	---	---	0.2	0.2	---
1848	1845	6,10,14-Trimethylpentadecan-2-one	---	---	---	---	---	---	---	---	---	---	---	---	---
1862	1864	Benzyl salicylate	---	---	---	---	---	---	0.5	0.8	---	---	---	---	---
2002	---	(E)-15,16-Bisnorlabda-8(17),11-dien-13-one	---	0.8	---	---	---	---	---	---	---	---	---	0.3	---
2104	2105	(E)-Phytol	---	0.2	---	0.9	---	---	---	---	---	0.2	---	0.6	---
		Monoterpene hydrocarbons	50.3	0.5	84.8	39.4	29.6	23.9	16.0	23.4	94.2	18.3	52.7	3.6	61.8
		Oxygenated monoterpenoids	3.8	12.0	1.7	2.0	34.8	25.3	1.1	2.9	0.1	16.2	0.4	0.0	0.8
		Sesquiterpene hydrocarbons	32.9	65.1	4.0	41.4	20.0	28.5	63.3	46.6	4.7	31.0	34.1	49.1	15.4
		Oxygenated sesquiterpenoids	6.6	13.9	1.6	11.6	14.0	21.3	14.8	22.2	0.9	21.6	3.6	22.2	5.2
		Others	0.2	2.9	0.0	4.0	0.8	0.5	3.1	4.1	0.2	2.3	3.8	9.5	4.0
		Total identified	93.8	94.4	92.1	98.4	99.2	99.5	98.3	99.2	100.0	89.4	94.6	84.4	87.2

RI_calc = Retention Indices determined with respect to a homologous series of n-alkanes on an HP-5ms column; RI_db = Retention Indices from the databases (NIST 17 and Wiley version 10); tr = trace (< 0.05%).

The rhizome essential oils of Z. cornubracteatum were predominantly composed of monoterpene hydrocarbons and oxygenated monoterpenoids. The major components were α-pinene (8.2–14.5%), β-pinene (8.8–33.1%), limonene (1.0–5.1%), 1,8-cineole (2.5–10.4%), and linalool (0.4–31.0%). Both α-pinene and β-pinene were major components in the leaf essential oils of Z. cornubracteatum (2.7–10.1% and 18.8–67.3%, respectively). However, sesquiterpene hydrocarbons, including (E)-caryophyllene (1.8–13.9%), germacrene D (0.7–13.7%), bicyclogermacrene (2.7–18.9%), as well as the sesquiterpenoid (E)-nerolidol (0.9–23.0%), were also abundant in the leaf essential oils.

The leaf essential oils of Z. nudicarpum were also rich in α-pinene (5.0–10.9%), β-pinene (0.7–34.0%), in addition to the sesquiterpene hydrocarbons (E)-caryophyllene (6.4–24.3%), α-humulene (2.1–6.4%), germacrene D (0.6–6.5%), and bicyclogermacrene (3.3–16.1%). The leaf essential oil of Z. nudicarpum from Pù Hoạt Nature Reserve previously reported also showed α- and β-pinenes (2.4% and 11.7%, respectively) [34]. Important differences are apparent between the previously reported essential oil and those from the present study. The previous report found no (E)-caryophyllene or germacrene D, but large concentrations of cedrol (14.8%) and β-eudesmol (13.8%), which were not observed in this current study. Interestingly, the stem essential oils of Z. nudicarpum showed wide variation in monoterpene hydrocarbon concentrations, with the sample from Pù Hoạt Nature Reserve showing only low concentrations of monoterpene hydrocarbons compared to samples from Nam Đông or Bạch Mã National Park. For example, the concentrations were: α-pinene (0.0, 10.6, 6.1%), β-pinene (0.5, 9.0, 5.6%), p-cymene (0.0, 6.0, 0.1%), and limonene (0.0, 2.1, 6.0%). The Pù Hoạt stem essential oil had a high concentration of (E)-caryophyllene (52.6%). The rhizome essential oil from Pù Hoạt Nature Reserve had α-pinene (18.7%) and β-pinene (58.3%) as dominant constituents, and is qualitatively similar to a sample earlier reported from that collection site [34]. α-Pinene and β-pinene concentrations were lower in the rhizome essential oil sample from Nam Đông (4.0% and 9.8%, respectively).

The most abundant constituents in the rhizome essential oil of Z. neotruncatum were perillene (51.3%), neral (12.3%), and geranial (17.0%). Perillene is a major component of Perilla frutescens (perillene chemotype) [47] and Elsholtzia polystachya (perillene chemotype) [48], but has been found in Zingiber essential oils in small concentrations, e.g., Z. officinale rhizome oil (0.1–0.6%) [40,49,50] and Z. zerumbet leaf oil (1.2%) [51]. Neral and geranial are also major components of Z. officinale rhizome oil [49,50].

Camphene (40.4%) dominated the rhizome essential oil composition of Z. nitens, followed by bornyl acetate (14.5%), (E)-β-ocimene (12.7%), and α-pinene (10.5%). In comparison, the rhizome essential oil of Z. nitens from Pù Mát National Park previously reported contained bornyl acetate (11.8%), (E)-β-ocimene (1.1%), and α-pinene (7.3%), along with β-pinene (21.0%), and δ-elemene (12.8%) [31]. In contrast, the leaf essential oil of Z. nitens was composed largely of α-zingiberene (17.4%), α-pinene (11.2%), β-sesquiphellandrene (10.1%), (E)-nerolidol (10.0%), zingiberenol (7.2%), β-pinene (6.0%), and ar-curcumene (5.2%). The previously reported Z. nitens leaf essential oil was devoid of α-zingiberene, β-sesquiphellandrene, zingiberenol, and ar-curcumene, but contained large concentrations of δ-elemene (17.0%) and ledol (8.1%), which were not observed in the present sample. In addition, concentrations of trans-β-elemene, germacrene D, and bicyclogermacrene were high in the previous report (8.8%, 8.2%, and 8.3%, respectively), but low in the present sample (0.8%, 0.7%, and 1.5%, respectively). The variations in chemical constituents can likely be attributed to the different geographical collection sites as well as climatic factors. The Pù Mát sample was collected in May, 2014 (beginning of the rainy season), while the sample from Vũ Quang National Park (this work) was collected in September, 2018 (height of the rainy season).

The essential oil from the leaves of Z. ottensii had abundant sesquiterpene hydrocarbons, including (E)-caryophyllene (28.0%) and trans-β-elemene (17.0%), along with the monoterpene β-pinene (17.1%). (E)-Caryophyllene (19.6%) and trans-β-elemene (12.3%) were also found to be key constituents in Z. ottensii leaf oil from Bandung, West Java, Indonesia [41]. However, zerumbone was a major component (11.4%) in the leaf oil from Indonesia, but was not detected in the sample from Vietnam. The rhizome essential oil from Vietnam had monoterpenes as the major components, such as sabinene (21.6%), β-pinene (11.7%), γ-terpinene (5.5%), and terpinen-4-ol (17.1%), in addition to the sesquiterpenoid zerumbone (12.5%). Rhizome essential oils of Z. ottensii from other geographical locations have been reported; the major components are presented in Table 3. A hierarchical cluster analysis using the eight major constituents (Figure 2) reveals the similarities between these rhizome essential oils. The clusters are largely defined by the zerumbone concentrations.

Table 3.

Comparison of the major components of Zingiber ottensii rhizome essential oils from different geographical locations.

Major Components	Collection Location
	Johor, Malaysia [37]	Petchaburi, Thailand [39]	Sabah, Malaysia [38]	Chiang Mai, Thailand [40]	Bandung, Indonesia [41]	Pù Hoạt Nature Reserve, Vietnam (This Work)
Sabinene	7.2	6.5	4.1	21.1	8.6	21.6
β-Pinene	5.1	4.3	1.3	6.8	3.5	11.7
p-Cymene	0.0	6.9	0.0	0.0	0.0	2.1
1,8-Cineole	3.3	1.2	0.6	3.9	5.8	4.3
γ-Terpinene	5.1	0.2	0.4	4.7	4.8	5.5
Terpinen-4-ol	16.8	11.2	3.2	19.7	16.6	17.1
α-Humulene	10.9	5.6	18.3	15.6	3.3	3.8
Zerumbone	25.6	40.1	36.7	0.0	14.2	12.5

Figure 2.

Dendrogram obtained from agglomerative hierarchical cluster analysis of the rhizome essential oils from Zingiber ottensii from different geographical locations.

The monoterpenes α-pinene (16.3%) and β-pinene (71.6%) dominated the leaf essential oil composition of Z. recurvatum. The major components in the rhizome essential oil of Z. recurvatum were (E)-caryophyllene (11.3%), bornyl acetate (10.4%), α-humulene (6.9%), and bicyclogermacrene (5.1%).

Zingiber vuquangensis leaf and rhizome essential oils were both rich in α-pinene (11.3% and 9.8%, respectively) and β-pinene (38.5% and 45.0%, respectively). The sesquiterpene hydrocarbons trans-β-elemene (5.9% and 10.0%), and (E)-caryophyllene (12.2% and 14.4%) were major components in the leaf and stem essential oils, respectively. The leaf, stem, and rhizome essential oils from Z. vuquangensis from Vu Quang National Park, Ha Tinh Province, Vietnam, have been previously published [46]. A comparison of the major components is summarized in Table 4. Although there are qualitative similarities in the essential oil compositions from these two collections (α-pinene, β-pinene, and (E)-caryophyllene are major components), there are some notable differences. Bornyl acetate and zerumbone were major components in the rhizome essential oil from the Vu Quang collection, but were not observed in the Pù Hoạt sample; trans-β-elemene was observed in relatively small concentrations in the sample from Vu Quang, but was a major component in the leaf and stem essential oils from Pù Hoạt. The differences in chemical composition can be attributed to the geographical locations of the two collections and/or the season when the samples were collected. The Vu Quang sample was collected in August, 2014 (rainy season), while the Pù Hoạt sample was collected in April, 2019 (dry season).

Table 4.

Major components in the leaf, stem, and rhizome essential oils of Zingiber vuquangensis.

Major Components	Vu Quang National Park [46]			Pù Hoạt Nature Reserve
	Leaf	Stem	Rhizome	Leaf	Stem	Rhizome
α-Pinene	6.9	5.2	2.2	11.3	0.5	9.8
β-Pinene	24.7	26.1	8.0	38.5	3.1	45.0
Bornyl acetate	2.5	0.0	20.9	0.0	0.0	0.0
trans-β-Elemene	0.8	0.5	0.6	5.9	10.0	2.5
(E)-Caryophyllene	12.3	13.9	4.5	12.2	14.4	2.3
α-Humulene	7.7	8.2	9.6	1.1	1.6	0.5
Zerumbone	3.0	3.6	14.1	0.0	0.0	0.0

2.2. Mosquito Larvicidal Activity

Several of the Zingiber essential oils (depending on availability) were assayed for insecticidal activity against larvae of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquitoes. The 24- and 48-h larvicidal activities are presented in Table 5.

Table 5.

Larvicidal activities of Zingiber essential oils from north-central Vietnam.

Zingiber Essential Oil	LC50 (95% Confidence Levels)	LC90 (95% Confidence Levels)	χ2	p
	Twenty-four-hour
	Aedes aegypti
Z. cornubracteatum (BE) rhizome	16.97 (15.49–18.42)	24.56 (22.65–27.31)	1.7 × 10−6	1.000
Z. neotruncatum rhizome	34.95 (32.49–37.37)	51.49 (48.32–55.56)	6.625	0.036
Z. nudicarpum (PH) leaf	19.30 (17.95–20.56)	23.74 (22.40–25.40)	0.0	1.000
Z. nudicarpum (PH) rhizome	23.44 (21.94–25.17)	31.81 (29.07–36.31)	1.1 × 10−3	0.999
Z. ottensii rhizome	38.16 (35.50–40.89)	57.87 (53.60–63.62)	3.286	0.193
Z. recurvatum rhizome	20.90 (18.45–23.30)	36.35 (32.79–41.63)	3.697	0.157
Permethrin (control)	0.0094 (0.0082–0.0107)	0.0211 (0.0185–0.0249)	57.6	0.000
	Aedes albopictus
Z. cornubracteatum (BE) rhizome	12.72 (10.43–14.41)	21.56 (19.43–25.10)	4.6 × 10−6	1.000
Z. neotruncatum rhizome	21.50 (19.71–23.52)	31.99 (28.97–36.94)	0.0402	0.980
Z. nudicarpum (PH) leaf	22.33 (20.93–23.86)	31.12 (27.52–36.51)	0.2454	0.970
Z. nudicarpum (PH) rhizome	28.05 (25.80–30.41)	42.09 (38.92–46.29)	3.205	0.201
Z. ottensii rhizome	19.79 (17.91–21.75)	30.81 (27.81–35.76)	0.03531	0.983
Z. recurvatum rhizome	45.58 (43.24–47.49)	58.58 (55.18–63.22)	0.01253	0.994
Permethrin (control)	0.0024 (0.0021–0.0026)	0.0042 (0.0038–0.0049)	4.64	0.031
	Culex quinquefasciatus
Z. cornubracteatum (BE) rhizome	24.31 (21.79–26.84)	41.34 (37.72–46.34)	25.99	0.000
Z. neotruncatum rhizome	33.58 (31.39–36.00)	42.76 (39.94–46.48)	6.675	0.036
Z. nudicarpum (PH) leaf	12.44 (8.78–15.67)	44.29 (39.05–51.64)	34.20	0.000
Z. nudicarpum (PH) rhizome	11.50 (9.65–13.36)	27.15 (23.92–31.87)	30.33	0.000
Z. ottensii rhizome	27.19 (25.04–29.58)	40.09 (36.82–44.64)	8.377	0.015
Z. recurvatum rhizome	31.67 (29.30–34.05)	47.02 (43.94–50.94)	1.300	0.522
Permethrin (control)	0.0188 (0.0173–0.0206)	0.0294 (0.0270–0.0326)	24.1	0.000
	Forty-eight-hour
	Aedes aegypti
Z. cornubracteatum (BE) rhizome	16.32 (14.72–17.84)	24.52 (22.50–27.54)	9.8 × 10−6	1.000
Z. neotruncatum rhizome	33.91 (31.45–36.33)	50.42 (47.26–54.46)	8.156	0.017
Z. nudicarpum (PH) leaf	16.97 (15.76–18.32)	22.22 (20.59–24.53)	0	1.000
Z. nudicarpum (PH) rhizome	22.73 (21.09–24.63)	31.31 (28.88–35.35)	8.8 × 10−3	0.996
Z. ottensii rhizome	36.59 (33.77–39.48)	57.61 (53.69–62.84)	7.113	0.029
Z. recurvatum rhizome	17.16 (14.00–19.79)	34.57 (30.87–40.26)	3.687	0.158
	Aedes albopictus
Z. cornubracteatum (BE) rhizome	12.32 (10.36–13.78)	19.57 (17.61–23.23)	0	1.000
Z. neotruncatum rhizome	19.97 (18.07–21.98)	31.24 (28.16–36.33)	0.0528	0.974
Z. nudicarpum (PH) leaf	14.31 (12.03–16.52)	29.76 (27.71–32.79)	2.548	0.467
Z. nudicarpum (PH) rhizome	23.80 (21.40–26.25)	39.59 (36.06–44.53)	12.33	0.002
Z. ottensii rhizome	17.30 (15.15–19.24)	28.94 (25.97–33.94)	0.02453	0.988
Z. recurvatum rhizome	42.47 (40.04–44.88)	57.289 (54.63–61.53)	1.031	0.597
	Culex quinquefasciatus
Z. cornubracteatum (BE) rhizome	11.48 (5.78–15.28)	33.79 (29.71–40.16)	9.085	0.011
Z. neotruncatum rhizome	29.91 (27.77–32.33)	41.42 (38.31–45.66)	13.46	0.001
Z. nudicarpum (PH) leaf	4.901 (3.982–5.817)	18.31 (14.98–23.84)	83.59	0.000
Z. nudicarpum (PH) rhizome	6.687 (4.942–8.197)	18.85 (16.52–22.30)	49.77	0.000
Z. ottensii rhizome	24.64 (22.58–26.98)	36.94 (33.58–41.98)	1.512	0.470
Z. recurvatum rhizome	7.965 (4.368–10.991)	33.52 (26.87–46.73)	2.905	0.234

LC₅₀ = medial lethal concentration, LC₉₀ = 90% lethal concentration, χ² and p = goodness-of-fit chi-square value and p-value, BE = Bến En National Park, PH = Pù Hoạt Nature Reserve.

The essential oils showing the best larvicidal activity against Ae. aegypti were Z. cornubracteatum rhizome essential oil from Bến En National Park (24-h LC₅₀ = 17.0 μg/mL) and Z. nudicarpum leaf essential oil from Pù Hoạt Nature Reserve (24-h LC₅₀ = 19.3 μg/mL). The rhizome essential oil of Z. cornubracteatum also demonstrated remarkable activity against Ae. albopictus (24-h LC₅₀ = 12.7 μg/mL). Both Z. nudicarpum leaf essential oil and rhizome essential oil were very active against Cx. quinquefasciatus larvae, with LC₅₀ values of 12.4 and 11.5 μg/mL, respectively.

Multivariate analysis of the concentrations of the major components in the essential oils that were used for larvicidal activity screening (α-pinene, sabinene, β-pinene, perillene, terpinene-4-ol, neral, geranial, bornyl acetate, (E)-caryophyllene, α-humulene, bicyclogermacrene, and zerumbone), along with their 24-h larvicidal activities, reveals the correlation between activity and composition. The agglomerative hierarchical cluster (AHC) analysis (Figure 3) shows a cluster of largely active essential oils (Z. nudicarpum rhizome, Z. cornubracteatum rhizome, and Z. nudicarpum leaf), along with a marginally active essential oil (Z. ottensii rhizome), and two less active essential oils (Z. recurvatum rhizome and Z. neotruncatum rhizome). The concentrations of β-pinene and, to some extent, α-pinene, are largely responsible for the larvicidal activities of Zingiber essential oils, as seen in the principal component analysis (PCA) (Figure 4). Consistent with this correlation, β-pinene has shown larvicidal activity against Ae. Aegypti, with LC₅₀ values ranging from 12.1 to 42.5 μg/mL [52]. β-Pinene has also shown larvicidal activities against Ae. albopictus (LC₅₀ of 47.33 and 42.39 μg/mL for (+)-β-pinene and (−)-β-pinene, respectively) [53] and Cx. quinquefasciatus (LC₅₀ = 19.6 μg/mL) [54]. α-Pinene and sabinene have also shown larvicidal activity against Ae. aegypti [52], Ae. albopictus [53,55,56], and Cx. quinquefasciatus [57,58].

Figure 3.

Dendrogram obtained from agglomerative hierarchical cluster analysis of the Zingiber essential oils screened for larvicidal activity.

Figure 4.

Principal component biplot of PC1 and PC2 scores and loadings indicating the correlations between Zingiber essential oil major components and larvicidal activities.

Several Zingiber essential oils have been screened for mosquito larvicidal activity. Consistent with the correlation of α- and β-pinene with Zingiber essential oil larvicidal activities, Z. nimmonii rhizome essential oil, with no α-pinene and only low β-pinene (0.8%), showed relatively marginal larvicidal activity against Ae. aegypti and Cx. quinquefasciatus (LC₅₀ values of 44.5 and 48.3 μg/mL, respectively) [59]. Similarly, Z. cernuum rhizome essential oil, with 1.6% α-pinene and 1.2% β-pinene, showed relatively marginal larvicidal activities against Ae. aegypti (LC₅₀ 44.9 μg/mL), Ae. albopictus (LC₅₀ 55.8 μg/mL), or Cx. quinquefasciatus (LC₅₀ 48.4 μg/mL) [60], and the larvicidal activity of Z. zerumbet rhizome essential oil (0.8% α-pinene, 0.1% β-pinene) showed larvicidal activities against Ae. albopictus and Cx. quinquefasciatus with LC₅₀ = 55.8 and 33.3 μg/mL, respectively [61]. Finally, Z. officinale rhizome oil showed relatively weak activity on Cx. quinquefasciatus larvae (LC₅₀ = 50.8 μg/mL) [62]. Although the Z. officinale rhizome essential oil composition was not determined in this study, commercial Z. officinale oil (doTERRA International) contains 4.0% α-pinene and 0.4% β-pinene. In contrast, Z. collinsii rhizome essential oil, with 9.0% α-pinene and 16.3% β-pinene, demonstrated more effective larvicidal activity against Ae. albopictus with an LC₅₀ of 25.5 μg/mL [63].

2.3. Antimicrobial Activity

Several of the essential oils from Zingiber species were tested for antibacterial activity against a panel of Gram-positive (Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus), and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica) bacteria, and for anticandidal activity against Candida albicans (Table 6). The essential oils generally showed good to excellent activity against the Gram-positive organisms compared to Gram-negative. It has frequently been noted that Gram-positive bacteria demonstrate a higher susceptibility to essential oils than do Gram-negative organisms [64,65,66]. This phenomenon has been attributed to the existence of cell wall lipopolysaccharides in the Gram-negative bacteria, which can inhibit the hydrophobic essential oil constituents from diffusing into the cells [67,68]. Candida albicans was also relatively sensitive to the Zingiber essential oils.

Table 6.

Antibacterial and antifungal activities of Zingiber essential oils from north-central Vietnam.

Zingiber Essential Oil (EO)	Gram-Positive Bacteria			Gram-Negative Bacteria			Yeast
	Enterococcus faecalis ATCC 299212	Staphylococcus aureus ATCC 25923	Bacillus cereus ATCC 14579	Escherichia coli ATCC 25922	Pseudomonas aeruginosa ATCC 27853	Salmonella enterica ATCC 13076	Candida albicans ATCC 10231
	MIC (μg/mL)
Z. cornubracteatum (BE) leaf EO	128	128	na	64	na	na	na
Z. cornubracteatum (BE) rhizome EO	32	32	64	256	na	na	256
Z. cornubracteatum (BE) stem EO	128	128	na	64	na	na	na
Z. cornubracteatum (PM) leaf EO	64	64	128	128	na	na	16
Z. cornubracteatum (PM) rhizome EO	32	64	128	64	256	na	16
Z. nudicarpum (PH) leaf EO	32	16	16	na	128	na	16
Z. nudicarpum (PH) rhizome EO	2	8	1	64	16	na	16
Z. ottensii leaf EO	64	64	64	na	16	na	64
Z. ottensii rhizome EO	8	64	32	na	na	na	8
Z. recurvatum leaf EO	16	64	128	na	na	na	128
Z. recurvatum rhizome EO	16	128	64	na	256	256	64
Z. vuquangensis leaf EO	16	16	16	na	na	na	32
Streptomycin	256	256	128	32	256	128	nt
Nistatin	nt	nt	nt	nt	nt	nt	8
Cyclohexamide	nt	nt	nt	nt	nt	nt	32
	IC50 (μg/mL)
Z. cornubracteatum (BE) leaf EO	45.67	45.67	na	18.78	na	na	na
Z. cornubracteatum (BE) rhizome EO	10.45	9.34	18.79	68.99	na	na	99.34
Z. cornubracteatum (BE) stem EO	46.78	19.78	na	67.89	na	na	na
Z. cornubracteatum (PM) leaf EO	20.78	9.34	45.67	45.67	na	na	5.67
Z. cornubracteatum (PM) rhizome EO	9.56	13.56	43.67	20.45	100.34	na	7.89
Z. nudicarpum leaf EO	16.33	8.54	8.57	na	65.44	na	8.67
Z. nudicarpum rhizome EO	1.33	4.35	0.567	33.22	8.66	na	8.99
Z. ottensii leaf EO	32.66	32.33	33.77	na	7.99	na	32.33
Z. ottensii rhizome EO	2.57	17.89	15.56	na	na	na	3.56
Z. recurvatum leaf EO	3.99	13.67	55.89	na	na	na	67.74
Z. recurvatum rhizome EO	6.46	36.87	32.33	na	108.99	112.67	25.67
Z. vuquangensis leaf EO	15.66	8.56	7.33	na	na	na	16.33

BE = Bến En National Park, PM = Pù Mát National Park, MIC = minimum inhibitory concentration, IC₅₀ = median inhibitory concentration, na = not active, nt = not tested.

The essential oil with the best overall antimicrobial activity was Z. nudicarpum rhizome essential oil from Pù Hoạt Nature Reserve with MIC < 10 μg/mL against all three Gram-positive organisms and MIC = 16 μg/mL against P. aeruginosa and C. albicans. It is difficult to correlate essential oil composition with antimicrobial activity, however. The rhizome essential oil of Z. nudicarpum was rich in α-pinene (18.7%) and β-pinene (58.3%). The antimicrobial activities of α-pinene and β-pinene have ranged from excellent to inactive against E. faecalis, S. aureus, B. cereus, or C. albicans [69,70,71]. However, the presence of these two compounds as major components is not enough to impart good antimicrobial activity. The leaf essential oil of Z. recurvatum and the leaf and stem essential oils of Z. cornubracteatum from Bến En National Park were also rich in α-pinene (16.3%, 10.1%, and 9.9%, respectively) and β-pinene (71.6%, 67.3%, and 66.8%), but these essential oils showed significantly lower antimicrobial activity. There are likely synergistic and/or antagonistic effects of minor components responsible for the activities.

3. Materials and Methods

3.1. Plant Material

Zingiber plants were collected from several locations in north-central Vietnam (Table 1, Figure 1). The fresh plant materials (leaves, stems, and/or rhizomes; 2.0 kg each) were immediately chopped and hydrodistilled using a Clevenger apparatus for 4 h to give the essential oils.

3.2. Gas Chromatographic Analysis

Gas chromatography with flame ionization detection (GC-FID) was carried out as previously described [72]: Agilent Technologies HP 7890A Plus Gas chromatograph (Santa Clara, CA, USA), flame ionization detector (FID), HP-5ms column (30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies), H₂ carrier gas (1 mL/min), injector temperature = 250 °C, detector temperature = 260 °C, column temperature program: 60 °C (2 min hold), increase to 220 °C (4 °C /min), 220 °C (10 min hold), inlet pressure = 6.1 kPa, split mode injection (10:1) split ratio), 1.0 μL injection volume.

Gas chromatography–mass spectrometry (GC-MS) was carried out as previously described [72]: Agilent Technologies HP 7890A Plus Chromatograph (Santa Clara, CA, USA), HP-5ms (30 m × 0.25 mm, film thickness 0.25 μm) column, HP 5973 MSD mass detector, He carrier gas (1 mL/min), MS ionization voltage = 70 eV, emission current = 40 mA, acquisitions range = 35–350 amu, sampling rate = 1.0 scan/s. The GC operating conditions were the same as those used for GC-FID. The chemical components of the essential oils were identified based on their retention indices (RI) based on a series of n-alkanes, co-injection with pure compounds when available (Sigma-Aldrich, St. Louis, MO, USA) or identified essential oil components, MS library search (NIST 17 and Wiley Version 10) and by comparing with the literature MS fragmentation [73]. The relative concentrations (%) of the components were calculated based on the GC peak area (FID response) without correction factors. The measurements were carried out in triplicate.

3.3. Mosquito Larvicidal Screening

Larvae of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus were raised in the laboratory as previously described [74]. Aedes aegypti larvae were reared from eggs (Institute of Biotechnology, Vietnam Academy of Science and Technology). Adults of Culex quinquefasciatus and Aedes albopictus were collected in Hoa Khanh Nam ward, Lien Chieu district, Da Nang city (16°03′14.9″ N, 108°09′31.2″ E) and were maintained as described previously [74]. Eggs were hatched and the larvae reared as previously described [74].

Fourth instar larvae of each mosquito species were used for the larvicidal assays, which were carried out as previously described [74]: 250-mL beakers, 150 mL of water, and 20 larvae, aliquots of the Zingiber essential oils dissolved in EtOH (1% stock solution) were added to give final concentrations of 100, 50, 25, 12.5, 6, and 3 μg/mL; EtOH only was the negative control, permethrin was the positive control, mortality was recorded after 24 and 48 h of exposure, experiments were carried out at 25 ± 2 °C, assays were carried out in quadruplicate. The larvicidal data were subjected to log-probit analysis [75] to obtain LC₅₀ values, LC₉₀ values and 95% confidence limits using Minitab^® 19.2020.1 (Minitab, LLC, State College, PA, USA).

All procedures involving vertebrates (mice, chicks) were carried out in accordance with the “Guideline for the Care and Use of Laboratory Animals” which was approved by the Medical-Biological Research Ethics Committee of Duy Tan University (DTU/REC2020/NHH01), Vietnam.

3.4. Antimicrobial Screening

Antimicrobial activity of Zingiber essential oils was carried out on three Gram-negative organisms, Salmonella enterica (ATCC 13076), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922); three Gram-positive organisms, Bacillus cereus (ATCC 14579), Enterococcus faecalis (ATCC 299212), and Staphylococcus aureus (ATCC 25923); and the pathogenic yeast, Candida albicans (ATCC 10231), using the microbroth dilution assay as previously described [72]. Dilutions were formulated from 16,384 to 2 μg/mL in sterile distilled water and pipetted into 96-well microplates. Bacteria were grown in tryptic soy broth or Mueller–Hinton broth (double-strength), fungi were grown in Sabouraud dextrose broth (double-strength). Bacteria and fungi were standardized to 5 × 10⁵ CFU/mL for bacteria and 1 × 10³ CFU/mL for the yeast. The final lane, containing only serial dilutions of the essential oil without bacteria or yeast, was treated as the positive control. Sterile water (no sample) and media with microorganisms were the negative controls; streptomycin was the positive antibiotic standard; cycloheximide and nystatin served as positive antifungal standards. The plates were incubated at 37 °C for 24 h and the minimum inhibitory concentrations were established as the well with the lowest concentration completely inhibiting microbial growth based on turbidity. The IC₅₀ values were determined spectrophotometrically (EPOCH2C spectrophotometer, BioTeK Instruments, Inc Highland Park Winooski, VT, USA) and computed according to the following

$$\%\mathrm{inhibition}=\frac{{\mathrm{OD}}_{\mathrm{control}(-)}-{\mathrm{OD}}_{\mathrm{test}\mathrm{agent}}}{{\mathrm{OD}}_{\mathrm{control}(-)}-{\mathrm{OD}}_{\mathrm{control}(+)}}\times 100\%$$

$${\mathrm{IC}}_{50}={\mathrm{High}}_{\mathrm{conc}}-\frac{\left({\mathrm{High}}_{\mathrm{inh}\%}-50\%\right)\times \left({\mathrm{High}}_{\mathrm{conc}}-{\mathrm{Low}}_{\mathrm{conc}}\right)}{\left({\mathrm{High}}_{\mathrm{inh}\%}-{\mathrm{Low}}_{\mathrm{inh}\%}\right)}$$

where OD = optical density, control(−) = cells with medium but no antimicrobial agent, test agent is a known concentration of antimicrobial agent, control(+) = culture medium without cells, High_conc/Low_conc = concentration of test agent at high concentration/low concentration and High_inh%/Low_inh% = %inhibition at high concentration/% inhibition at low concentration). The antimicrobial assays were carried out in triplicate.

4. Conclusions

There are wide variations in essential oil compositions from the Zingiber species in this study, not only between species and tissues, as expected, but also between essential oils from the same species and tissues collected from different locations. This is an important consideration if the essential oils are to be used for agricultural or medicinal uses, but also if commercialization is considered. The monoterpenes α-pinene and β-pinene seem to be largely responsible for the mosquito larvicidal activities observed. It is worth investigating whether Zingiber or other essential oils rich in these components are viable alternatives for vector control. The presence of α-pinene and β-pinene cannot explain the antimicrobial activities of Zingiber essential oils, and synergistic or antagonistic interactions likely contribute. Nevertheless, several Zingiber essential oils have shown excellent antimicrobial activity and should be investigated further for controlling Gram-positive bacterial and yeast infections.

Author Contributions

Conceptualization, L.T.H. and W.N.S.; methodology, L.T.H. and W.N.S.; software, L.T.H, D.N.D, and W.N.S.; validation, L.T.H., D.N.D., and W.N.S.; formal analysis, D.N.D. and W.N.S.; investigation, L.T.H., N.T.C., L.N.S., T.T.H., L.D.L., I.A.O., and N.H.H.; resources, L.T.H.; data curation, W.N.S.; writing—original draft preparation, L.T.H. and W.N.S.; writing—review and editing, L.T.H., W.N.S., and D.N.D.; visualization, L.T.H.; supervision, L.T.H.; project administration, L.T.H.; funding acquisition, L.T.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED), grant number 106.03-2017.328.

Conflicts of Interest

The authors declare no conflict of interest.