Essential oil profiles of seeds, peels, and leaves obtained from Limnocitrus littoralis (Miq.) swingle species, in the Southcentral coast of Vietnam

Abstract

The essential oils of seeds, peels, and leaves from Limnocitrus littoralis (Miq.) Swingle growing wildly on the southcentral coast of Vietnam were extracted by hydrodistillation method. The yields of the oils were recorded. The organoleptic and physicochemical properties of the oils were defined and evaluated according to international standards at 25 °C. A total of forty-three, fifty, and sixty-five components of these oils were analysed and identified, respectively. Major constituents in the seed oil were β-pinene (3.7%, Quang Ngai; 24.0%, Ninh Thuan), β-myrcene (11.1%, Quang Ngai; 59.2%, Ninh Thuan), o-cymene (57.7%, Quang Ngai), and limonene (18.5%, Quang Ngai; 94.5%, Phu Yen; 8.5%, Ninh Thuan). Major constituents in the peel oil included α-pinene (11.3%, Ninh Thuan), β-pinene (3.9%, Quang Ngai; 62.6%, Ninh Thuan), and limonene (54.0%, Quang Ngai; 94.0%, Phu Yen; 3.5%, Ninh Thuan). Major constituents in the leaf oils consisted of β-pinene (36.4%, Ninh Thuan), β-myrcene (38.0%, Quang Ngai; 28.9%, Ninh Thuan), and limonene (17.3%, Quang Ngai; 93.2%, Phu Yen). This research highlighted the aromatic value of bearing oil plants growing wildly on the Southcentral coast of Vietnam.

KEYWORDS:

• Limnocitrus littoralis
• aromatic plant
• essential oils
• Vietnamese southcentral coast

Introduction

The name Limnocitrus littoralis (Miq.) Swingle was accepted as a botanical name of a species in the genus Limnocitrus (family Rutaceae) reported on Tropicos page (data supplied on 2012 April 18th, record 50263295). (Swingle 1940; The Plant List 2021; World Flora Online 2021) L. littoralis is classified as an endangered species in the Red List of IUCN. (International Union for Conservation of Nature v.3. The IUCN Red List of Threatened Species 2021) In Vietnam, L. littoralis species named ‘cam đu$\stackrel{`}{o}$ng’ (sweet orange), ‘c$\stackrel{`}{\stackrel{ˆ}{a}}$m đàng’, or ‘đa tu bi$\hat{e}$n’ is distributed in Southcentral coastal areas, especially along the beach. (Cong 2021) Besides being valuable medicinal plants, L. littoralis trees growing wildly on Ly Son island (Quang Ngai province) are exploited as one of the tourism attractions. L. littoralis fruits are always attracted by almost tourists because of their nice flavor. (Cong 2021) (Sauvan et al. 2008a; Sauvan et al. 2008b; Sauvan et al. 2009) reported the invention of using alcohol extract of L. littoralis leaves as an active agent in the cosmetic composition.

Because of the shortage of plant resources, the scientific research on L. littoralis species has been therefore limited. Until now, only one project (Nguyen 2012) and two aricles (Doan et al. 2019; Le et al. 2020) reported relating to the essential oil of L. littoralis species. Furthermore, these reports almost show the study on species growing in Vietnam including Quang Ngai province and Ninh Thuan province. Nguyen (2012) analyzed the chemical composition of essential oils of L. littoralis species growing in Ninh Thuan province (Vietnam). In this work, the contents of peel and leaf essential oils extracted by hydrodistillation were 2.2% and 1.4% respectively. By GC combined with the GC/MS method, the chemical composition of those essential oils was also reported. The eight constituents of peel essential oils dominated by 1,3,6-octatriene (46.8%) and limonene (15.2%) and the twelve constituents of leaf essential oils dominated by β-panasinsene (28.8%) and β-myrcene (21.7%) were reported. Doan et al. (2019) hydrodistillated L. littoralis leaves gathered in the Quang Ngai region. A yield of 1.0% (v/w) of oil was obtained. The forty components in leaf essential oils identified by GC and GC/MS methods were subjugated by monoterpene hydrocarbons (27.7%), sesquiterpene hydrocarbons (32.3%), and oxygenated sesquiterpenes (4.6%). The major constituents were represented by myrcene (24.9%), γ-muurolene (11.0%), and oleic acid (10.3%). In addition, the anti-inflammatory activity of the essential oil was also proven by evaluating capacity against the nitric oxide (NO) generation with IC50 value (12.50 ± 1.19 mg/L). (Doan et al. 2019; Le et al. 2020) determined the bioactivities of essential oils extracted from the leaves of L. littoralis, including cytotoxicity, antiviral, antibacterial, antimycotic, and antitrichomonas effects. The report indicated that L. littoralis oils displayed the strongest inhibition against Candida tropicalis and Candida parapsilosis strains.

Building the volatile essential oil profile is needed in elucidating high aromatic value from wildly growing L. littoralis species. In this present study, the essential oils obtained from the seeds, the peels, and the leaves of three L.littoralis varieties (Figure 1) such as Quang Ngai, Phu Yen, and Ninh Thuan were investigated on extraction yields, organoleptic/physicochemical properties, and chemical compositions.

Figure 1. L. littoralis trees of three varieties: A. Quang Ngai, B. Phu Yen, and C. Ninh Thuan.

Materials and methods

Materials

The L. littoralis materials growing wildly on beaches were collected every September from 2017 to 2020 on Ly Son island (15°25'44.72"N, 109° 5'11.41"E at an altitude of 11 m a.s.l., Quang Ngai province), Tuy Hoa (13° 2'30.79"N, 109°21'12.29"E at an altitude of 6 m a.s.l., Phu Yen province), and Nui Chua National Park (11°40'44.31"N, 109°10'55.43"E at an altitude of 41 m a.s.l., Ninh Thuan province). The fresh fruits including seeds (Figure 2) and fresh leaves were washed, cleaned, and stored for extraction. Seeds and rinds were separated by hands. All fresh materials were ground before hydrodistillation.

Figure 2. Fruits and fruit cross-sections including seeds of three L. littoralis varieties: A. Quang Ngai, B. Phu Yen, and C. Ninh Thuan.

The collections of plant samples were carried out following Decree No.59/2017/ND-CP and Decree No.06/2019/ND-CP issued from the Office of the Government of Vietnam on ‘Management of access to genetic resources and the sharing of benefits arising from their utilization’ and on ‘Management of endangered, precious and rare species of forest fauna and flora and observation of convention on international trade in endangered species of wild fauna and flora’, respectively. In addition, we also worked under the permission of the local direct management agencies.

All plant materials, as well as all voucher specimens (VHOANG1106/Ly Son-Quang Ngai, VHOANG1107/Tuy Hoa-Phu Yen, VHOANG1108/Nui Chua-Ninh Thuan), were authenticated and deposited by Dr. Hoang Viet in the herbarium, Department of Ecology-Evolutionary Biology, University of Science, Vietnam National University Ho Chi Minh City.

Isolation of essential oils

100 g of each sample of fresh ground materials of seeds, peels, and leaves along with 900, 500, and 800 ml of water respectively was submitted to a 2000ml round bottom flask. The Clevenger apparatus was set up for hydrodistillation adapting the method reported by (Nguyen et al. 2016). Seed and leaf oils were distilled for 6 h and peel oils for 4 h. Each of the distillate oil samples was extracted with 15 mL of diethyl ether (3 × 15 ml). Ethereal solutions were dried with anhydrous sodium sulfate (Na₂SO₄). Remove Na₂SO₄ by a filter. The anhydrous ether was recovered from the filtrate by rotary evaporation. The pure essential oil was collected and stored in the fridge for the next experiments (Nguyen et al. 2016).

The percentage content of essential oil was calculated by the following Equation 1(2) $\begin{array}{rl}LRI& =100\ast (n+(N-n)\ast (R{T}_i-R{T}_n)/\\ & \left(R{T}_N+1-R{T}_n\right))\end{array}$(2) : (1) $\begin{array}{rl}Content\left(\mathrm{\%}\right)& =\left(Essentialoil\right(g)/\\ & freshplantmaterial\left(g\right){)}^{\ast }100\end{array}$(1) All experiments were performed in triplicate.

Organoleptic and physicochemical evaluation of essential oils

Organoleptic properties and physical properties were determined by AFNOR (French Association of Normalization) standards (AFNOR 1992) or ISO (International Organization for Standardization) standards (specific gravity/ISO 279:1998, refractive index/ISO 280:1998, optical rotation/ISO 279:1998), at 25 °C. The chemical properties including the acid value and saponification value were carried out based on ISO 1242:1999 and ISO 3657:2020, respectively.

All the experiments were done in triplicate.

Chromatographic analysis of essential oils

Gas chromatography (GC)

Analyses were performed on an Agilent gas chromatography 6890N GC, equipped with a Pheno-menex 7HG-G010-11 Zebron ZB-5 GC capillarycolumn (30 m x 0.32 mm x 0.25 µm). Nitrogen was used as the carrier gas at the flow rate of 1.74 ml/ min. The constant pressure mode at 9.32 psi was used and the oven temperature was programed from 60 °C to 240 °C at the rate of 3 °C/min. The injector and detector temperature were set at 250 °C. Oil samples of 0.1 μl were injected with splitless mode.

Gas chromatography-mass spectrometry (GC/MS)

The oil was analysed by an Agilent gas chromatography 7890A, equipped with a Phenomenex 7HG-G010-11 Zebron ZB-5 GC capillary column (30 m x 0.32 mm x 0.25 µm), coupled to an Agilent Mass Selective Detector 5975C VL MSD Triple-Axis. Helium was used as the carrier gas at a flow rate of 1.20 mL/min. The constant pressure mode at 13.209 psi was chosen on the GC program. The injection temperature was 250 °C; the injection volume was 1.0 µL; the split ratio was 1:25, and the ionization voltage was 70 eV. The oven temperature was programed from 60 °C to 240 °C at the rate of 3 °C/min. The detector was set at 250 °C.

Identification of essential oil constituents

Constituents of the oil analyzed by GC were compared their mass spectra with those of standard compounds registered in the (NIST 2014) and (NIST 2020) libraries. Moreover, the identification was also confirmed by collating the observed LRI (linear retention index) of each constituent with those reported in the compilation of retention indices published by (Adams 2007). The observed LRI was calculated by using Equation 2 propounded by van den Dool and Kratz (1963) to the homologous series of C₉-C₂₀ n-alkanes (Sigma-Aldrich). The relative percentage of each constituent in the oil was determined by peak area normalization. No response factors were calculated. (2) $\begin{array}{rl}LRI& =100\ast (n+(N-n)\ast (R{T}_i-R{T}_n)/\\ & \left(R{T}_N+1-R{T}_n\right))\end{array}$(2)

Results and discussion

Extraction of essential oils

Hydrodistillation of materials of seeds, peels, and leaves of L. littoralis from different areas was done for 6, 4, and 6 h, respectively. The results of extraction were presented in Table 1.

Table 1. Contents of essential oils of seeds, peels, and leaves of L. littoralis varieties from the Vietnamese Southcentral coast.

		Material/water	Content of essential oils
Part of plant	Location	(g/ml)	(%, w/w)
Seeds	Quang Ngai	100/900	1.4 ± 0.01
	Phu Yen	100/500	0.7 ± 0.04
	Ninh Thuan	100/800	0.5 ± 0.03
Peels	Quang Ngai	100/900	0.1 ± 0.01
	Phu Yen	100/500	0.1 ± 0.01
	Ninh Thuan	100/800	0.2 ± 0.01
Leaves	Quang Ngai	100/900	0.8 ± 0.01
	Phu Yen	100/500	0.8 ± 0.04
	Ninh Thuan	100/800	0.8 ± 0.02

As shown in Table 1, except for the seeds from Quang Ngai, leaves gave the highest content of the oil. The peel gave the lowest oil content. There were no significant differences in the oil yield obtained from the peel and leaf materials collected in various regions on the Vietnamese Southcentral coast. However, the peel oil contents extracted from the Ninh Thuan variety (0.2 ± 0.01%) were less than those reported by Thuong (2.2%) (Nguyen 2012) a factor of 11.0. Similarly, the leaf oil content obtained from the Quang Ngai variety (0.8 ± 0.01%) was less than the one reported by Doan et al. (2019) (1.0%) a factor of 1.3. The leaf oil content obtained from the Ninh Thuan variety (0.8%) was less than the one reported by Thuong (1.4%) (Nguyen 2012) a factor of 1.8. The influences of fruit/seed maturity (Figure 2) and sample collecting time could contribute significantly to variations of quantitative seed oils and peel oils obtained from the same location. In addition, in the present study, the experimental materials of L. littoralis were collected in September, the time the climate changed strongly by a lot of wind, while those were collected by Doan et al. (2019) in October, the month of the fully ripe fruit season and rainy season (Đ$\text{ặ}$ng et al. 2003) and by Thuong (Nguyen 2012) in July, the last month of the sunny season (Nam et al. 2018).

Evaluation of essential oil properties

The organoleptic and physical properties of L. littoralis essential oils were determined and presented in Table 2. All of the essential oils were mobile transparent liquids. The seed oils were clear, pleasant, and fresh. The peel oils were yellow and spicy. The leaf gave a golden yellow oil with a strong and spicy smell. The specific gravities of all essential oils were around 0.85 g/cm³. There were no significant differences among all of the oil samples. The optical rotatory of the seed and peel oils from Quang Ngai and Phu Yen varieties gave the dextrorotatory value while the others gave the levorotatory value. This could reveal the significant difference in the chemical composition of the oils.

Table 2. Organoleptic and physical properties of essential oils of seeds, peels, and leaves of L. littoralis varieties from Vietnamese Southcentral coast.

	Organoleptic properties, 25 °C			Physical properties, 25 °C
Essential oils	Appearance	Colour	Odour	Specific gravity (g/cm^3)	Refractive index ${\mathit{n}}_{\mathit{D}}^{25}$	Optical rotation ${\mathit{\alpha }}_{\mathit{D}}^{25}$
Seeds
Quang Ngai	Transparent liquid	Clear	Pleasant, fresh	0.849 ± 0.0014	1.4685 ± 0.00055	+ 9.213 ± 0.1778
Phu Yen			Pleasant, slight sweet, citrusy, fresh	0.844 ± 0.0005	1.4433 ± 0.00055	+ 91.450 ± 0.1277
Ninh Thuan			Pleasant, warm, green	0.846 ± 0.0004	1.4464 ± 0.00031	- 1.176 ± 0.0981
Peels
Quang Ngai	Transparent liquid	Pale yellow	Pleasant, sweet, fresh	0.848 ± 0.0002	1.4814 ± 0.00062	+ 42.998 ± 0.3238
Phu Yen			Pleasant, slight sweet, fresh	0.848 ± 0.0007	1.4683 ± 0.00038	+ 87.485 ± 0.4262
Ninh Thuan			Pleasant, warm, green	0.853 ± 0.0006	1.4532 ± 0.00046	- 11.973 ± 0.0820
Leaves
Quang Ngai	Transparent liquid	Golden yellow	Strong, spicy	0.845 ± 0.0008	1.4782 ± 0.00097	- 1.485 ± 0.0025
Phu Yen				0.844 ± 0.0007	1.4570 ± 0.00021	−29.166 ± 0.0943
Ninh Thuan				0.856 ± 0.0013	1.4831 ± 0.00035	−2.235 ± 0.0137

The chemical properties of L. littoralis essential oils were evaluated and shown in Table 3. Acid values of essential oils from L. littoralis varieties were low, 023-9.15 mg KOH/g. These results indicated that the oil was rich in hydrocarbon and had an excellent storage life. The low saponification values (4.89-20.19 mg KOH/g) of oils proved the small proportion of shorter carbon chain lengths of fatty acids.

Table 3. Chemical properties of essential oils of seeds, peels, and leaves of L. littoralis varieties from Vietnamese Southcentral coast.

	Acid value	Saponification value
Essential oils	(mg KOH/g)	(mg KOH/g)
Seeds
Quang Ngai	3.32 ± 0.187	9.37 ± 0.1795
Phu Yen	0.23 ± 0.025	6.59 ± 0.117
Ninh Thuan	0.42 ± 0.030	5.41 ± 0.021
Peels
Quang Ngai	5.06 ± 0.117	10.57 ± 0.194
Phu Yen	2.11 ± 0.080	4.89 ± 0.119
Ninh Thuan	7.22 ± 0.031	12.90 ± 0.031
Leaves
Quang Ngai	1.21 ± 0.151	7.10 ± 0.074
Phu Yen	0.61 ± 0.040	7.13 ± 0.097
Ninh Thuan	9.15 ± 0.074	20.19 ± 0.025

Analysis of chemical compositions

By using GC methods and comparing LRI, one hundred constituents (Table 4) in the essential oil from seeds, peels, and leaves of three L. littoralis varieties were identified.

Table 4. Chemical composition of essential oils from the seeds, peels, and leaves of three L. littoralis varieties on Vietnamese Southcentral coast.

				%GC
		LRI		Seed essential oils			Peel essential oils			Leaf essential oils
		Ref.											Method of
No	Compound name	(Adams 2007)	Obs.	S-QN	S-PY	S-NT	P-QN	P-PY	P-NT	L-QN	L-PY	L-NT	identification
1	(E)−2-Hexenal	846	846							0.1			LRI, MS
2	(2E,4E)-Hexadienol	912	912	0.1				0.1					LRI, MS
3	α-Thujene	924	924	0.5		0.2						0.1	LRI, MS
4	α-Pinene	932	931	2.4	0.2	1.5	0.6	0.3	11.3	0.1	0.5	1.8	LRI, MS
5	Camphene	946	947			0.1			0.8			0.2	LRI, MS
6	cis-Sabinene	969	970	0.4							0.1		LRI, MS
7	β-Pinene	974	976	3.7	0.9	24.0	3.9	0.1	62.6	0.2	0.2	36.4	LRI, MS
8	β-Myrcene	988	987	11.1	1.4	59.2	9.6	0.4		38.5	1.1	28.9	LRI, MS
9	α-Phellandrene	1002	1006			0.1							LRI, MS
10	α-Terpiene	1014	1016								Trace	0.1	LRI, MS
11	p-Cymene	1020	1023	57.7	1.3		0.9		3.8			0.1	LRI, MS
12	Limonene	1024	1027	18.5	94.5	8.5	54.0	94.0	3.5	17.5	0.1	0.1	LRI, MS
13	β-Phellandrene	1025	1033								93.2	2.5	LRI, MS
14	(Z)-β-Ocimene	1032	1033				0.3			0.2	2.0	0.9	LRI, MS
15	(E)-β-Ocimene	1044	1046				1.2				0.1	0.3	LRI, MS
16	γ-Terpinene	1054	1055	0.2	0.2	2.8					Trace	0.2	LRI, MS
17	exo-5-Norbornene-2-methanol	1058	1058	0.1									LRI, MS
18	cis-Linalool oxide (furanoid)	1067	1069	0.1			0.2	0.1	0.3	Trace		Trace	LRI, MS
19	trans-Linalool oxide (furanoid)	1084	1084	0.2	Trace	0.2	2.1				0.09	0.1	LRI, MS
20	Terpinolene	1086	1085	0.5		0.3							LRI, MS
21	Linalool	1095	1099				0.2	0.1	0.1	1.2		0.9	LRI, MS
22	(2E,4E)-Octadienol	1113	1114				1.7						LRI, MS
23	Linalyl butyrate		1117				0.2						MS
24	1-Terpineol	1130	1130	0.1	0.1			0.1	0.1				LRI, MS
25	Methyl 4-methylvalerate		1133							0.3			MS
26	trans-Pinocarveol	1137	1135	0.2	0.2			0.1	4.5				LRI, MS
27	Camphor	1141	1142							0.5		0.1	LRI, MS
28	Citronellal	1148	1148							0.1		0.1	LRI, MS
29	(3Z)-Nonen-1-ol	1152	1153	0.1									LRI, MS
30	cis-Linalool oxide (pyranoid)	1170	1173					0.2	0.4				LRI, MS
31	Terpinen-4-ol	1174	1179	0.6	0.1	0.1		0.2	0.5	0.1		0.3	LRI, MS
32	α-Terpineol	1186	1194							0.1		0.3	LRI, MS
33	Dihydrocarveol	1192	1194	0.3	0.1		0.1						LRI, MS
34	γ-Terpineol	1199	1199	0.3									LRI, MS
35	iso-Dihydrocarveol	1212	1210				0.3						LRI, MS
36	trans-Carveol	1215	1214	0.1				0.6	1.6				LRI, MS
37	cis-Carveol	1226	1225	0.4	0.1								LRI, MS
38	Tetrahydrocitronellyl acetate	1231	1229				0.1						LRI, MS
39	Pulegone	1233	1236					0.3	0.4				LRI, MS
40	Carvone	1239	1242	0.2				0.8	0.5				LRI, MS
41	cis-Carvone oxide	1259	1260	0.2									LRI, MS
42	Geranial	1264	1269	0.1				0.1	0.2	Trace		Trace	LRI, MS
43	Citronellyl formate	1271	1274	0.1									LRI, MS
44	Geranyl formate	1298	1297	Trace									LRI, MS
45	Methyl geranate	1322	1321	0.4	0.2								LRI, MS
46	δ-Elemene	1335	1337		0.1					3.6			LRI, MS
47	α-Cubebene	1345	1344					0.4		Trace			LRI, MS
48	α-Terpinyl acetate	1346	1346				0.1						LRI, MS
49	α-Longipinene	1350	1351		0.1					Trace		Trace	LRI, MS
50	Hydroxycitronellol	1359	1359				0.1						LRI, MS
51	Cyclosativene	1369	1368							0.1			LRI, MS
52	α-Copaene	1374	1374							0.2			LRI, MS
53	β-Cubebene	1387	1386				0.9			0.2			LRI, MS
54	iso-Longifolene	1389	1390							0.9	Trace	0.6	LRI, MS
55	Cyprene	1398	1397							Trace			LRI, MS
56	β-Longipinene	1400	1401	0.2			0.1						LRI, MS
57	Longifolene	1407	1406	0.2									LRI, MS
58	α-Cedrene	1410	1411							Trace			LRI, MS
59	α-Santalene	1416	1415			0.74		0.1		Trace	0.4	2.0	LRI, MS
60	(E)-Caryophyllene	1417	1416				0.1			4.8		2.0	LRI, MS
61	β-Duprezianene	1424	1425							1.0		0.1	LRI, MS
62	α-trans-Bergamortene	1432	1429				4.0	0.2		0.5		0.3	LRI, MS
63	β-Humulene	1439	1438				0.8			0.6	0.2	0.6	LRI, MS
64	epi-Cedrane	1447	1449	0.1	0.2	0.22	0.6			0.5		0.2	LRI, MS
65	α-Humulene	1452	1453			0.19	0.9			2.7	0.1	0.6	LRI, MS
66	(E)-β-Farnesene	1454	1455				0.1						LRI, MS
67	β-Santalene	1457	1456				0.2						LRI, MS
68	allo-Aromadendrene	1458	1459							1.1	0.2	1.9	LRI, MS
69	9-epi-(E)-Caryophyllene	1464	1462				1.6						LRI, MS
70	β-Acoradinene	1469	1468							0.1		0.1	LRI, MS
71	β-Chamigrene	1476	1477							0.4			LRI, MS
72	9-epi-(Z)-Caryophyllene		1479				0.2						MS
73	Germacrene D	1484	1485				0.5	0.2	0.2	8.0	0.1	1.0	LRI, MS
74	β-Selinene	1489	1488							4.2	0.1		LRI, MS
75	α-Salinene	1498	1497				1.0						LRI, MS
76	α-Muurolene	1500	1500				0.9						LRI, MS
77	β-Bisabolene	1505	1504	0.4	0.3	0.7	1.0				Trace	0.1	LRI, MS
78	(Z)-α-Bisabolene	1506	1507								Trace	Trace	LRI, MS
79	δ-Amorphene	1511	1511								0.1	0.2	LRI, MS
80	BHT	1514	1516				0.6			0.2			LRI, MS
81	7-epi-α-Selinene	1520	1519				1.3			4.4			LRI, MS
82	(E)-iso-γ-Bisabolene	1528	1528				1.0			0.6			LRI, MS
83	α-Cadinene	1537	1536							0.6			LRI, MS
84	δ-Cuprenene	1542	1543				0.1			0.1			LRI, MS
85	Selina-3,7(11)-diene	1545	1547							Trace			LRI, MS
86	β-Vetivenene	1554	1555							0.1			LRI, MS
87	Germacrene B	1559	1559							Trace			LRI, MS
88	α-Cedrene epoxide	1574	1577					0.9	0.7				LRI, MS
89	Spathulenol	1577	1578							0.4			LRI, MS
90	Ledol	1602	1602							Trace			LRI, MS
91	γ-Eudesmol	1630	1632							0.2			LRI, MS
92	epi-α-Muurolol	1640	1641							0.5			LRI, MS
93	Selin-11-en-4α-ol	1658	1658							0.4	0.7	15.7	LRI, MS
94	7-epi-α-Eudesmol	1662	1663							2.4	Trace	0.1	LRI, MS
95	α-Bisabolone oxide A	1684	1686							0.1			LRI, MS
96	(Z)-α-trans-Bergamotol	1690	1691							0.1			LRI, MS
97	(2E,6E)-Farnesol	1742	1740							0.1			LRI, MS
98	Phytol	1942	1946	0.3									LRI, MS
99	Geranyllinalool	1960	1959							0.1		0.2	LRI, MS
100	iso-Bergapten	2033	2034							0.7			LRI, MS
	Total of identified compounds			99.6	100.0	98.7	91.2	99.2	91.5	98.3	99.4	99.2
	Hydrocarbons			95.8	99.2	98.4	85.5	95.6	82.1	91.1	98.6	81.4
	- Monoterpene			94.9	98.5	96.6	70.4	94.7	81.9	56.5	97.4	71.4
	- Sesquiterpene			0.9	0.7	1.8	15.8	0.5	0.2	34.6	1.2	10.0
	Oxygenated compounds			3.8	0.8	0.3	5.7	3.6	9.4	7.3	0.8	17.8
	- Monoterpenoids			2.7	0.6	0.3	2.9	2.7	8.7	1.9	0.1	1.7
	- Esters of monoterpenoids			0.5	0.2		0.4			0.1		0.2
	- Sesquiterpenoids							0.9	0.7	4.1	0.7	15.8
	- Diterpenoids			0.3
	- Furanocoumarin									0.7
	- Aliphatic alcohols/aldehydes			0.2			1.7			0.1
	- BHT						0.6			0.2
	- Others			0.1

S: Seed essential oils; Peel essential oils; L: Leaf essential oils; QN: Quang Ngai; PY: Phu Yen; NT: Ninh Thuan

Trace < 0.05%. Percentage by FID peak area normalization with the use of the response factor only for main components (≥ 10%). LRI (linear retention index) on ZB-5 [Ref.: reference Adams (2007)], Obs.: observed].

Among them, totals of forty-three, fifty, and sixty-five components belonged to the seed oil, the peel oil, and the leaf oil, respectively.

Seed essential oils

Among forty-three volatile compounds identified, thirty-seven were found in Quang Ngai, seventeen in Phu Yen, and fifteen in Ninh Thuan oils.

The seed oils from Quang Ngai, Phu Yen, and Ninh Thuan were dominated respectively by 95.8%, 99.2%, and 98.4% of hydrocarbons, especially in monoterpene hydrocarbons. The most abundant components in the Quang Ngai, Phu Yen, and Ninh Thuan seed oils were o-cymene (57.7%), limonene (94.5%), and β-myrcene (59.2%), respectively. The other prominent main components in seed oils of three varieties were β-pinene (24.0% of Ninh Thuan), β-myrcene (11.1% of Quang Ngai), and limonene (18.5% of Quang Ngai). Four sesquiterpene hydrocarbons in a total of eight different compounds were found in each variety, accounting for 0.9%, 0.7%, and 1.8% from Quang Ngai, Phu Yen, and Ninh Thuan respectively.

A total of nineteen oxygenated compounds were found in Quang Ngai, seven in Phu Yen, and two in Ninh Thuan seed oils, amounting to 3.8%, 0.8%, and 0.3%, respectively. These compounds were categorized into monoterpenoids (alcohols, carbonyls, oxides/epoxides, esters, and oxides/epoxides), diterpenoids (phytol); esters of monoterpenoids; and aliphatic alcohols.

The occurrence of many significant oxygenated compounds including alcohols (exo-5-norbornene-2-methanol, γ-terpineol, trans-carveol), ketone (carv-one), aldehyde (geranial), oxides/epoxide (cis-linalool oxide, cis-carvone), esters (citronellyl formate, geranyl formate, methyl geranate), aliphatic alcohols ((2E,4E)-hexadienol, (3Z)-nonen-1-ol), and diterpene (phytol) in the seed oils from Quang Ngai variety. Those could be used to distinguish the two other oils besides the high limonene concentration of the Phu Yen variety.

Peel essential oils

Among constituents in the chemical compositions of the peel oil, there were thirty-six in Quang Ngai, nineteen in Phu Yen, and seventeen in Ninh Thuan.

As seed oils were, the peel oils of three varieties from Quang Ngai, Phu Yen, and Ninh Thuan were contributed mainly by hydrocarbons, accounting for 86.1%, 95.2%, and 82.1%, respectively. Among hydrocarbons, monoterpene compounds dominated the chemical composition of the peel oils with high concentrations, 70.4% of Quang Ngai, 94.7% of Phu Yen, and 81.9% of Ninh Thuan. The sesquiterpene hydrocarbons amounted to 15.8%, 0.5%, and 0.2% of Quang Ngai, Phu Yen, and Ninh Thuan peel oils, respectively. Among three varieties, the high concentration of sesquiterpene hydrocarbons (15.8%) in Quang Ngai was relatively notable. The oxygenated compounds presented in the oils at minor levels include 5.0% of Quang Ngai, 3.9% of Phu Yen, and 9.35% of Ninh Thuan.

Limonene was recorded as the most abundant component in the chemical composition of peel oils from Quang Ngai and Phu Yen varieties, amounting to 54.0% and 94.0%, respectively. The Ninh Thuan peel oil was distinguished from two other oils by its possession of β-pinene (62.6%) as the most abundant component. The other compound that contributed to the main components in the chemical composition of peel oils of the Ninh Thuan variety was a monoterpene hydrocarbon, α-pinene (11.3%). A total of three esters, linalyl butyrate, tetrahydrocitronellyl acetate, α-terpinyl acetate constituted 0.4% in both the Quang Ngai and Phu Yen peel oils. The ester was not detected in the Ninh Thuan oil.

The results recorded that the peel oils of L. littoralis variety Ninh Thuan were not in agreement completedly with the previous study published by Thuong et al (Nguyen 2012), with a total of thirty-six components compared to eight components and β-pinene (62.6%) as the most abundant component comparing 1,3,6-octatriene (46.8%).

The variations in contents and quantitative levels of the volatile compounds in seed and peel essential oils in these studies and previous studies could be due to differences in varieties, environmental factors, fruit maturity, and sample collecting time.

Leaf essential oils

Among constituents identified in the leaf oil, fourty-eight were found in Quang Ngai, twenty-three in Phu Yen, and thirty-seven in Ninh Thuan oils. The chemical compositions of Quang Ngai, Phu Yen, and Ninh Thuan oils dominated by hydrocarbons were 91.1%, 98.6%, and 81.4%, respectively. Monoterpene hydrocarbons contributed at a high concentration, 56.5% in Quang Ngai, 97.4% in Phu Yen, and 71.4% in Ninh Thuan oils. Except for the oils of the Quang Ngai variety contained 34.6% of sesquiterpene hydrocarbons, two other oils were contributed at minor levels, 1.2% to the Phu Yen variety and 10.0% to the Ninh Thuan variety. To oxygenated compounds, their concentrations were 7.3%, 0.8%, and 17.8% in Quang Ngai, Phu Yen, and Ninh Thuan, respectively.

Whilst the main abundant components in Quang Ngai leaf oils being β-myrcene (38.0%) and limonene (17.3%), and in Ninh Thuan were β-pinene (36.4%), β-myrcene (28.9%), and selin-11-en-4α-ol (15.7%), those in Phu Yen was only limonene at very high concentrations (93.2%).

Forty components identified in leaf oils of the Quang Ngai variety published by (Doan et al. 2019) contained 27.7% of monoterpene hydrocarbons, 32.3% of sesquiterpene hydrocarbons, and 4.6% of oxygenated sesquiterpenes (4.6%). These results could be considered in agreement with results obtained in this study, 56.5% of monoterpene hydrocarbons, 34.6% of sesquiterpene hydrocarbons, and 4.1% of oxygenated sesquiterpenes. However, the major constituents were not like completely, except for β-myrcene (24.9% vs 38.0%). In this study, γ-muurolene and oleic acid were not detected in the chemical composition of leaf oils from the Quang Ngai variety.

As the previous study reported by Thuong et al (Nguyen 2012), a total of twelve constituents, dominated by β-panasinsene (28.8%) and β-myrcene (21.7%), of leaf essential oils from L. littoralis variety Ninh Thuan were identified whilst thirty-seven constituents of those dominated by β-pinene (36.4%), β-myrcene (28.9%), and selin-11-en-4α-ol (15.7%) were identified in this study.

The components of essential oils from seeds, peels, and leaves of L. littoralis variety Quang Ngai were more abundant than in two other varieties. As mentioned above on the variations in contents and quantitative levels of the volatile compounds in seed and peel essential oils, those in the leaf oils were also affected particularly by environmental factors and sample collecting time.

Several factors including habitat, salinity, temperature, altitude, seasonality, plant age and development, and water availability have been reported to influence the chemical compositions and the bioactive compounds of essential oil (Al-Rowaily et al. 2020). Recently, soil fertility affected the yield of oil palm in Ghana (Obeng et al. 2020). Notably, the soil nutrient and climate conditions also drive the chemical composition of essential oil of the Siparuna muricata in Ecuador (Burneo et al. 2021). In our study, the content of essential oil contained in seeds of L. Littoralis from Quang Ngai and Phu Yen was higher than Ninh Thuan. In contrast, the essential oils of peels and leaves from Ninh Thuan were the highest values. These differences may come from the physical composition of the soils in places where collected samples were. At the Ly Son island, Quang Ngai, the geomorphology forms have a wide variety of soils: modern marine deposition, marine sediment, basalt flow, volcanic eruptions, and other geo-features (Hoang et al. 2018). Among that, basaltic flows were deficient nutrients for leaves and peel of Citrus trees. Therefore, this affected the content of essential oils in leaves and peels. Whilst, sandy soils were distributed mainly in the central regions of Vietnam including Ninh Thuan and Phu Yen. In 2015, one study showed the relationship between soil rich in sand and aromadendrene (Rapposelli et al. 2015). Despite, our study hasn’t analyzed the soil's chemical and physical properties, the results presented that the content and chemical composition of essential oils varied between seeds vs. peels, and leaves.

Furthermore, for the first time showed the constituent of essential oils was different in the three locations which were impacted by environmental factors. These contributed to developing suitably the plant conservation policy for each region as well as the strategy for growth and raising awareness of the aroma value of this red list plant.

Fragmentation patterns of main components of essential oils

The main components of essential oils from seeds, peels, and leaves included α-pinene, β-pinene, β-myrcene, o-cymene, limonene, and selin-7(11)-en-4α-ol (Figure 3).

Figure 3. Mass spectra of main components from the analysis of essential oils from three L. littoralis varieties compared to the standard compound. A: α-pinene; B: β-pinene; C: β-myrcene; D: o-cymene; E: limonene; F: selin-7(11)-en-4α-ol.

α-Pinene was one of the main components in peel oils of L. littoralis Ninh Thuan variety. The fragmentation of α-pinene (Figure 3A) began with the formation of a molecular ion at m/z 136. The peak at m/z 121 resulted from loss of a methyl group. The base peak assigned 100% abundance appeared at m/z 93 because of losing the isopropyl group. Especially, the formation of an ion at m/z 105 was due to the loss of a methyl group and a proton. The ion m/z 77 appeared by the formation of ion phenyl from the loss of a methyl group and a proton of the ion at m/z 93. The ion signal at m/z 53 was formed by the loss of two carbons of the ion at m/z 77. The fragmentation of α-pinene compound was presented by Ansory et al. (2020) and compared with this one provided by mass spectra libraries.

β-Pinene was one of the main components in the seed, peel, and leaf oils of L. littoralis Ninh Thuan variety. The fragmentation of β-pinene (Figure 3B) was almost similar to the reference spectra of α-pinene. However, the formation of ion m/z 107 instead of ion m/z 105 presented the difference in fragmentation of β-pinene and α-pinene. Fragmentation patterns of β-pinene compound in the mass spectral interpretation were presented by many previous studies (Adams 2007; Yahaya et al. 2019).

β-Myrcene was one of the main components in the seed and leaf oils of two L. littoralis varieties, Quang Ngai and Ninh Thuan. For most monoterpene hydrocarbons, the fragmentation often gives molecular ion at m/z 136 and base peak at m/z 93, accounting for 100% of relative intensity in the total ion chromatogram. The appearance of the ion signal at m/z 69 in β-myrcene’s fragmentation (Figure 3C) lower 50% of relative intensity than the one of the total ion signal showed the difference from all the monoterpenes under normal experimental conditions. This was proved by many previous studies (Adams 2007; Tani 2013).

o-Cymene was one of the main components in the seed oils of L. littoralis Quang Nai variety. The fragmentation of o-cymene (Figure 3D) was similar to the authentic standard obtained from the NIST library search. The fragmentation began with the formation of the signal at m/z 134 as a molecular ion. The loss of the methyl group gave an ion signal at m/z 119 as a base peak. Simultaneously, the formation of the ion at m/z 91 was due to the loss of the isopropyl group of o-cymene molecular.

Limonene, a typical monoterpene hydrocarbon presenting as the main component in seeds, peels, and leaves of essential oils obtained from two L. littoralis varieties, Quang Ngai and Phu Yen. With the fragmentation of limonene molecular (Figure 3E) as well as other monoterpene hydrocarbons, the molecular ion peak of limonene appeared at m/z 136. However, unlike others, the base peak of limonene was at m/z 68 instead of 93. The fragmentation pattern of limonene was presented in detail by Adams and Tashev (Adams 2007; Adams and Tashev 2019; Ansory et al. 2020).

Selin-7(11)-en-4α-ol, sesquiterpene alcohol, was one of the main components in the leaf oils of L. littoralis Ninh Thuan variety. The fragmentation of selin-7(11)-en-4α-ol (Figure 3F) began with the formation of the molecular ion at m/z 222. The comparison with reference spectra obtained by the NIST library search and the collation of the observed LRI with that of one authentic standard reported in the compilation of retention could be used to identify the final structure of this compound (Carbonell et al. 2000; Adams 2007).

Conclusions

Essential oils profiles of seeds, peels, and leaves obtained from L. littoralis species, especially on the Southcentral coast of Vietnam were built. The quantity and quality of the oils were affected strongly by environmental factors, fruit maturity, and sample collecting time. Although the essential oils from different plant parts and different varieties gave different chemical compositions and different major components, these essential oils were found to be volatile compounds, especially in monoterpene hydrocarbons and valuable oxygenated compounds. Identification of main compounds in the essential oils was elucidated by mass spectra. These results gave important information for seeking aromatic plant resources as well as bearing oil plant resources. L. littoralis is a wild species of plant that is needed to protect and develop sustainably because of its valuable essential oils, its resistance to harsh environments, and its protection from coastal erosion.

Author contributions

Conceptualization and design: Thanh-Tu Thi Nguyen, Anh Viet Nguyen, Thao-Tran Thi Nguyen

Analysis and interpretation of the data: Thanh-Tu Thi Nguyen, Anh Viet Nguyen, Nhuan Ngoc Doan, Thao-Tran Thi Nguyen.

Drafting of the paper: Thanh-Tu Thi Nguyen, Tai The Diep, Thao-Tran Thi Nguyen.

Funding acquisition: Nhuan Ngoc Doan, Thao-Tran Thi Nguyen.

Investigation: Thanh-Tu Thi Nguyen, Anh Viet Nguyen, Thao-Tran Thi Nguyen.

Methodology: Thanh-Tu Thi Nguyen, Tai The Diep, Thao-Tran Thi Nguyen.

Project administration: Thao-Tran Thi Nguyen.

Supervision: Thao-Tran Thi Nguyen.

Writing – review & editing: Tai The Diep, Thao-Tran Thi Nguyen

Statement of appropriate permissions

The authors did the collection of plant samples following Decree No.59/2017/ND-CP and Decree No.06/2019/ND-CP and the permission of the local direct management agencies (Ly Son commune, Ministry of National Defence at Tuy Hoa, and Nui Chua National Park Administration).

Acknowledgments

The authors thank Prof. Le Ngoc Thach, Assoc. Prof. Nguyen Tien Cong, and Assoc. Prof. Tran Hoang Phuong for their thoughtful comments on improving our report. The authors also thank the People’s Committee of the Ly Son Commune (Ly Son island, Quang Ngai Province), the Ministry of National Defence at Tuy Hoan (Phu Yen province), and the Nui Chua National Park Administration (Ninh Thuan Province) for licensing, supporting sample collection.

Data availability

The authors confirm that the data supporting the findings of this study are available in the Mendeley repository, Mendeley Data at https://data.mendeley.com/datasets/pv2f97jyv5/2.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by University of Science, Vietnam National University at Hochiminh City [grant no HH2021-15].