Canarium subulatum and Illicium petelotii Essential Oils from Vietnam: Compositions with Concomitant Antimicrobial and Mosquito Larvicidal Effects

Abstract

Objectives

This study aimed to investigate the essential oil extracted from the in continuation of an extensive research on the chemical compositions and biological potentials of essential oils from Vietnamese plants as they are made available, we studied the leaves of Canarium subulatum Guillaumin (Burseraceae) and Illicium petelotii A. C. Smith (Schisandraceae).

Methods

Chemical compounds in essential oils were identified using GC-FID/MS (gas chromatography-flame ionization detection/mass spectrometry) analysis. The broth microdilution method was used for the antimicrobial assay. The larvicidal activity was evaluated against Aedes aegypti and Culex quinquefasciatus larvae, with lethal concentration (LC₅₀ and LC₉₀) values calculated after 24 and 48 h of exposure.

Results

The leaves were collected from Pù Hoạt Natural Reserve, Vietnam. From the mass spectral analysis, (E)-caryophyllene (17.0%), α-pinene (16.9%), limonene (15.1%), and (E)-β-ocimene (13.8%) were the major compounds in C. subulatum. Caryophyllene oxide (26.8%), and (Z)-β-elemene (11.3%) are the significant constituents of I. petelotii. The antimicrobial results showed that I. petelotii exhibited minimum inhibitory concentration (MIC) value of 64.0 µg/mL against three microbes of namely exhibited Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, and Bacillus cereus ATCC 14579. C. subulatum exhibited MIC value of 128.0 µg/mL to the mentioned microbes. Both C. subulatum and I. petelotii showed anti-candidal activity at MIC of 64.0 µg/mL against Candida albicans ATCC 10231. The essential oils of C. subulatum and I. petelotii were toxic against Aedes aegypti with median lethal concentrations (LC₅₀) values of 39.33 μg/mL and 36.88 μg/mL, respectively at 24 h. Both samples. Both oil samples displayed larvicidal activity towards Culex quinquefasciatus with LC₅₀ values of 32.60 μg/mL and 48.94 μg/mL, respectively, at 24 h. This is the first reported data on C. subulatum and I. petelotii volatile contents, antimicrobial and larvicidal activities.

Conclusion

This study provides a comprehensive analysis of the chemical composition and biological activities of Canarium subulatum and Illicium petelotii essential oil. The findings highlight its potent antimicrobial and mosquito larvicidal properties. Further research is warranted to explore its mechanisms of action and potential applications in pharmaceuticals and pest control.

Keywords

Burseraceae Schisandraceae essential oil monoterpenes sesquiterpenes Gram-positive bacteria anti-Candidal activity

Introduction

Canarium a member of the family Burseraceae consist of nearly 78 species, mostly trees. The species C. subulatum Guillaumin is a tree occurring in tropical forests of the world including southern China and Indo-China. In Vietnam, C. subulatum is called trám múi nhọn.¹ The plant has a height of 20–35 m tall and diameter of 30–65 cm. The stipulate leaves have leaflets in 2–5 pairs, while the blades are broadly ovate or lanceolate.² The flowers are 7–11 mm, slender and puberulent with glabrous stamens. The stamens are smaller in female flowers. The upper part of the ovary is glabrous, but are conspicuously absent in male flowers.² Extract of C. subulatum exhibited anti-inflammatory effects,² antioxidative actions³ and skin protective effects.³ The chemical investigation of the bark extract of C. subulatum afforded β-amyrin, (-)-cubebene, scopoletin, scopoline and derivatives of methylellagic acid.⁴ Furthermore, β-amyrin and (-)-cubebene exhibited moderate anti-herpetic activity, whereas 3,4-dihydroxybenzoic acid, and ellagic acid displayed free-radical scavenging potential.⁴ Information on the composition of the volatile oils from C. subulatum were not found in the literature. In the literature, terpene compounds were the predominant compounds in previously known Canarium plant volatiles. The volatiles from resin of C. album were monoterpenoids,⁵ while C. parvum C. tramdenanum has mixture of sesquiterpene and monoterpene hydrocarbons.⁶ The predominant components of C. bengalense leaves from Vietnam were also sesquiterpene and monoterpene hydrocarbons.⁷ The essential oil of C. schweinfurthii analysed from Gabonese comprised mainly monoterpene hydrocarbons constituents.⁸ The essential oil C. schweinfurthii of Gabonese origin displayed wound healing potential.⁸

Illicium is considered as the sole genus of the Schisandraceae family.⁹ Species in this genus are native to the tropical worlds of Asia and America such as southeastern United States, Mexico, and the Caribbean.¹⁰ I. petelotii A. C. Smith, could attained a height of 3–5 m. The leaves occur in clusters of 3–6 at distal nodes with petiole of 0.5–1.5 cm. The leaf blades are oblong-elliptic to narrowly obovate, 6–14 × 1.8–4.2 cm. The flowers are axillary or subterminal and have flower peduncle of 0.5–4.5 cm. The fruits have 5–7 follicles while the seeds are about 6 × 4.5 × 3 mm in dimension. I. petelotii flowers in March and April, and bears fruits in the months of August and September.¹⁰ I. petelotii was not previously investigated upon for both the volatile and non-volatile constituents. In Vietnam the essential oils of other Illicium plants were widely studied. The fruit of I. griffithii contained monoterpenoids such as α-pinene, linalool, and limonene as the main compounds.¹¹ The high contents of phenylpropanoids such as safrole,^12,13 4-methoxysafrole,¹² and methyl eugenol¹³ were also reported from I. griffithii. Sample of essential oil of I. griffithii from Sa Pa (Vietnam) contained high contents of sesquiterpenoids including α-cadinol, γ-muurolol, γ-curcumene and aromadendrene, while large amount of monoterpenoids mainly borneol and 1,8-cineole were found in sample from Tam Đảo National Park sample.¹⁴ I. majus afforded oil whose major constituents were sesquiterpene hydrocarbons (aromadendrene, cuparene and calamenene) and oxygenated monoterpene (1,8-cineole),¹⁵ while abundant of 1,8-cineole was found in I. micranthun.¹⁵ Lastly, sesquiterpenoids were the prominent components in I. tsaii and comprised mainly of (E)-nerolidol, β-caryophyllene, β-cedrene, and calamenene.¹⁵

No study was previously conducted and reported for the essential oils of C. subulatum and I. petelotii. As we continue to investigate and reports our findings on essential oils from Vietnamese plants,^16–18 the current study gave separate information on constituents present in the essential oils, data on antimicrobial study, and larvicidal potentials of C. subulatum and I. petelotii leaves.

Results and Discussion

Although, both essential oils have yellow coloured, however, quantitative and qualitative compositions were observed in their chemical compositions. The yields calculated for the essential were C. subulatum (0.2% v/w) and I. petelotii (0.48% v/w).

Compositions of C. subulatum

The leaves of C. subulatum has fifty-two compounds mainly terpenes with oil contents of 99.5%. The classes of compounds (Table 1) comprised of monoterpenoids (hydrocarbons 59.1%, oxygenated monoterpenes 0.1%), and sesquiterpenoids (hydrocarbons 34.2%, oxygenated sesquiterpenes 4.4%). The minor class of compounds include phenylpropanoid (0.4%), alkenes (0.3%), aldehydes (0.8%) and ketones (0.2%). Present in higher percentages in the essential oil were (E)-caryophyllene (17.0%), α-pinene (16.9%), limonene (15.1%) and (E)-β-ocimene (13.8%). The minor compounds such as myrcene (3.5%), (Z)-β-ocimene (2.8%), germacrene D (2.8%), α-phellandrene (2.7%), α-humulene (2.5%), bicyclogermacrene (2.5%), caryophyllene oxide (1.8%), (E,E)-α-farnesene (1.7%), p-cymene (1.3%), α-copaene (1.2%), and β-pinene (1.1%) were identified above 1%.

Table 1.

Chemical Constituents of Essential Oils from the Leaves of C. subulatum and I. petelotii.

Sr. No	RT (min)	Compounds^a	RI^b	RI^c	Concentration (%)^d
Sr. No	RT (min)	Compounds^a	RI^b	RI^c	C. subulatum	I. petelotii
1	10.51	α-Pinene	932	929	16.9	2.1
2	11.00	Camphene	955	946	0.2	-
3	11.70	Sabinene	978	976	0.2	-
4	11.83	β-Pinene	984	978	1.1	2.5
5	12.10	Myrcene	991	988	3.5	-
6	12.70	α-Phellandrene	1010	990	2.7	-
7	13.10	α-Terpinene	1021	1015	0.1	-
8	13.36	p-Cymene	1029	1021	1.3	0.7
9	13.54	Limonene	1032	1030	15.1	0.6
10	13.60	β-Phellandrene	1034	1032	0.8	-
11	13.65	(Z)-β-Ocimene	1036	1034	2.8	-
12	13.66	1,8-Cineole	1038	1036	-	0.5
13	14.05	(E)-β-Ocimene	1049	1043	13.8	-
14	1452	γ-Terpinene	1063	1056	0.2	-
15	15.58	Terpinolene	1090	1088	0.4	-
16	15.83	Linalool	1101	1100	-	0.4
17	16.40	(E)-4,8-Dimethyl-nona-13,7-triene	1117	1117	0.3	-
18	17.51	(E)-Sabinol	1149	1147	-	0.5
19	18.79	Terpinen-4-ol	1185	1187	0.1	0.6
20	18.98	p-Cymen-8-ol	1191	1191	-	1.0
21	19.23	α-Terpineol	1198	1197	-	0.5
22	19.44	Methyl chavicol	1202	1202	0.4	-
23	19.46	Myrtenol	1204	1204	-	0.6
24	19.53	Decanal	1206	1206	0.7	-
25	19.55	Myrtenal	1207	1207	-	0.3
26	22.55	2-Undecanone	1290	1292	0.2	-
27	22.56	Bornyl acetate	1294	1294	-	0.3
28	22.72	Dihydroedulan	1301	1300	-	0.2
29	24.33	δ-Elemene	1348	1345	0.8	-
30	24.71	α-Copaene	1390	1397	1.2	3.2
31	26.07	β-Bourbonene	1400	1399	-	0.3
32	26.15	(Z)-β-Elemene	1403	1401	0.8	11.3
33	26.39	Dodecanal	1411	1410	0.1	-
34	26.86	(E)-α-Bergamotene	1426	1426	0.3	-
35	27.27	(E)-Caryophyllene	1437	1437	17.0	5.7
36	27.45	γ-Elemene	1444	1443	0.3	-
37	27.51	(Z)–α-Bergamotene	1446	1445	0.3	-
38	27.84	Guaia-4,5-diene	1455	1455	0.2	-
39	27.85	Aromadendrene	1457	1457	-	0.2
40	27.94	(Z)-β-Farnesene	1460	1462	0.2	-
41	28.25	(E)-β-Farnesene	1470	1468	0.3	-
42	28.31	α-Humulene	1472	1472	2.5	1.2
43	28.54	9-epi-(E)-Caryophyllene	1479	1479	0.4	0.5
44	28.88	β-Chamigrene	1488	1488	-	0.6
45	28.89	γ-Muurolene	1490	1489	0.4	-
46	29.01	α-Amorphene	1494	1492	0.3	-
47	29.14	Germacrene D	1498	1500	2.8	-
48	29.18	n-Pentadecane	1500	1500	-	0.3
49	29.33	β-Selinene	1505	1503	0.4	1.9
50	29.54	(E,E)-α-Farnesene	1512	1510	1.7	-
51	29.59	α-Selinene	1513	1513	-	0.8
52	29.61	Bicyclogermacrene	1514	1515	2.5	-
53	29.84	γ-Guaiene	1522	1525	0.2	-
54	30.19	β-Sesquiphellandrene	1533	1535	0.2	-
55	30.28	δ-Cadinene	1537	1537	0.9	-
56	31.08	Elemol	1563	1561	-	0.4
57	31.24	(E)-Nerolidol	1569	1570	0.2	2.1
58	31.51	Germacrene B	1578	1577	0.5	1.5
59	32.07	Spathulenol	1597	1600	0.7	2.0
60	32.30	Caryophyllene oxide	1605	1607	1.8	26.8
61	32.54	Cubeban-11-ol	1613	1611	0.3	0.4
62	32.75	Humulene epoxide I	1620	1622	-	0.4
63	32.91	Copaborneol	1626	1628	-	0.6
64	33.08	Humulene epoxide II	1631	1633	0.2	1.6
65	33.47	1-epi-Cubenol	1646	1650	0.4	0.4
66	33.62	γ-Eudesmol	1651	1653	-	0.6
67	33.80	Caryophylla-3(15),7(14)-dien-6-ol	1657	1657	-	1.4
68	33.84	epi-α-Muurolol	1659	1661	0.2	-
69	33.94	α-Muurolol	1662	1663	0.1	-
70	34.20	α-Cadinol	1670	1670	0.3	0.4
71	34.21	β-Eudesmol	1672	1672	-	0.4
72	34.31	neo-Intermedeol	1676	1678	0.2	3.1
73	34.59	(Z)-Calamenen-10-ol	1686	1690	-	0.5
74	34.71	Acorenone	1690	1692	-	2.9
75	34.85	Cadalene	1694	1695	-	0.8
76	35.75	(2Z,6E)-Farnesol	1728	1722	-	2.1
77	36.96	Cyclocolorenone	1773	1775	-	0.4
78	38.55	Hexahydrofarnesyl acetone	1834	1836	-	3.7
Total					99.5	89.3
Monoterpene hydrocarbons (Sr. No. 1-11, 13-15)					59.1	5.9
Oxygenated monoterpenes (Sr. No. 12,16, 18-20, 23, 25, 27)					0.1	4.7
Sesquiterpene hydrocarbons (Sr. No. 29-32, 34-47, 49-55, 57, 75)					34.2	28.0
Oxygenated sesquiterpenes (Sr. No. 56, 59-74, 76, 78)					4.4	50.2
Alkanes (Sr. No. 48)					-	0.3
Alkenes (Sr. No. 17)					0.3	-
Aldehydes (Sr. No. 24, 33)					0.8	-
Ketones (Sr. No. 26)					0.2	-
Phenylpropanoids (Sr. No. 22)					0.4	-
Epoxides (Sr. No. 28)					-	0.2

Elution order on HP-5MS column; ^bRetention indices on HP-5MS capillary column;

Literature retention index taken from NIST;¹⁹ ^dRelative percentage values are means of three determinations ± SD, which are insignificant and excluded to avoid congestion.

Abbreviations: RT, Retention time on HP-5MS column; -, Not detected.

Since there is no record of essential oil composition of C. subulatum in literature, the present data cannot be compared with any other analysis from this species. Qualitatively, terpene compounds were previously described as main the components from other Canarium plants essential oils. The abundant of β-caryophyllene in C. subulatum was in tandem with data obtained for the various parts of C. parvum,⁶ and C. tramdenanum⁶ from Vietnam. However, there is a large chemical diversity in the compositions and identity of abundant compounds of Canarium essential oils. parvum. For example, o-cymene, one of the main compounds of C. subulatum was not identified in large quantity in C. album,⁵ C. parvum,⁶ C. tramdenanum⁶ and C. bengalense.⁷ Moreover, α-pinene, the main monoterpene compound of C. album occurred in much lower amount in C. subulatum. In addition, prominent terpenes such as sabinene, (E)-β-ocimene, α-phellandrene, germacrene D, α-humulene, α-copaene, β-elemene, γ-elemene, α-amorphene and δ-elemene identified in Canarium samples from Vietnam,^5–7 were present in much lower amount in C. subulatum. Similarly, bulnesol present in C. tramdenanum⁶ and epi-bicyclosesquiphellandrene identified in C. bengalense⁷ were conspicuously absent in study on C. subulatum under study. Interestingly, limonene, one of the main compounds of C. subulatum of Vietnam was also a significant compound of Canarium essential oils from other parts of the world. These includes C. luzonicum from Turkey,²⁰ C. strictum from Pakistan,²¹ and C. pimela from China.²² The main composition of C. subulatum of Vietnam was found also to differed from data obtained from other countries such as C. schweinfurthii from Gabon which has an abundant of terpinolene and α-phellandrene,⁸ C. schweinfurthii dominated by α-terpineol²³ and C. schweinfurthi from Uganda containing high amount α-thujene and β-phellandrene.²⁴

In conclusion, C. subulatum studied leaf of Vietnam has a chemical composition at variant with volatiles of other Canarium plants nationally and internationally. Various authors have deduced factors contributing to these chemical variations as influence of soil and climatic conditions, as well as the nature of each plants and parts used for the experiment. Conclusively, Canarium samples produced varying chemical compositions.

Essential oil Constituents of I. petelotii

I. petelotii essential oil comprised mainly of oxygenated sesquiterpene (50.2%) and hydrocarbon counterparts (28.0%). There are significant quantities of monoterpene hydrocarbons (5.9%) and oxygenated monoterpenes (4.7%). The minor classes of compounds include alkane (0.3%) and epoxide (0.2%). The predominant volatile compounds were caryophyllene oxide (26.8%), (Z)-β-elemene (11.3%) and (E)-caryophyllene (5.7%). All other significant compounds were α-copaene (3.2%), neo-intermedeol (3.1%), acorenone (2.9%), β-pinene (2.5%), α-pinene (2.1%), (E)-nerolidol (2.1%), (2Z,6E)-farnesol (2.1%), and spathulenol (2.0%) (Table 1). No report yet could be seen on the chemical constituents on I. petelotii sample and this was presented here as first analysis. The authors provided a comparison of chemical contents of this study with the main constituents of other Illicium samples.^11–15 The results show an indication of both qualitative and quantitative variations among the terpene constituents of Illicium essential oils. From the reported chemical compositions, caryophyllene oxide, and (E)-β-elemene, the major constituents of I. petelotii are either absent or identified in much lower quantities from some other Illicium essential oils. Moreover, several compounds found in the previously analysed Illicum essential oils were not identified in this reported I. petelotii. The abundant constituents of previous Illicium essential oils including pinene, linalool, limonene and eucalyptol from I. griffithii,^11,14 sabinene and (E)-nerolidol present in I. micranthum^14,15 and I. tsai,¹⁵ as well as aromadendrene found in I. majus,¹⁵ were observed in lower quantity in this studied I. petetlotii. However, safrole,^12,13 4-methoxysafrole,¹² methyl eugenol,¹³ borneol,¹⁴ α-muurolol¹⁴ and α-curcumene¹⁴ which are the main compounds of I. griffithii,^11–14 cuparene, an abundant terpene present in I. majus,¹⁵ as well as calamenene found in I. tsaii,¹⁵ were completely absent in this reported volatiles of I. petelotii.

Performance of the Essential Oils in Microbial Tests

The performance of the essential oils with respect to microbial tests were observed and recorded in Table 2. Notably both essential oils did not display antibacterial action against P. aeruginosa, E. coli and S. enterica. These three mentioned microorganisms are Gram-negative in nature. Both C. subulatum and I. petelotti exhibited anti-candidal activity with MIC of 64.0 μg/mL. The essential oil I. petelotii displayed good activity towards the Gram-positive microbes (S. aureus, B. cereus and E. faecalis) with MIC of 64.0 μg/mL, while C. subulatum displayed moderate action against these microbes at much lower MIC value of MIC value 128.0 μg/mL. The corresponding IC₅₀ values of 23.12 ± 0.10, 22.56 ± 0.00 and 19.45 ± 0.10 µg/mL, respectively, were obtained from I. petelotii for the mentioned microorganisms. However, C. subulatum exhibited IC₅₀ values of 45.23 ± 0.00, 40.45 ± 0.50 and 39.56 ± 0.10 µg/mL, respectively.

Table 2.

Antimicrobial Activity of the Essential Oils from the Leaves of C. subulatum and I. petelotii.^a

Microorganisms	MIC μg/mL^b,c		IC₅₀ μg/mL^a
Microorganisms	C. subulatum	I. petelotii	C. subulatum	I. petelotii
Enterococcus faecalis ATCC29212	128.0 ± 0.00	64.0 ± 0.150	45.23 ± 0.00	23.12 ± 0.10
Staphylococcus aureus ATCC25923	128.0 ± 0.10	64.0 ± 0.10	40.45 ± 0.50	22.56 ± 0.00
Bacillus cereus ATCC14579	128.0 ± 0.05	64.0 ± 0.02	39.56 ± 0.10	19.45 ± 0.10
Pseudomonas aeruginosa ATCC27853	Na	Na	Nt	Nt
Candida albicans ATCC10231	64.0 ± 0.00	64.0 ± 0.01	21.45 ± 0.01	20.35 ± 0.00
Escherichia coli ATCC25922	Na	Na	Nt	Nt
Salmonella enterica ATCC13076	Na	Na	Nt	Nt

Mean value of three replicate assays; ^bStreptomycin, MIC values of 0.21 μg/mL - 2.20 μg/mL; ^cNystasin, MIC of 0.46 μg/mL.

Abbreviations: - No activity; Nt, Not tested.

Majority of the studied essential oils have shown higher activity towards the Gram-positive microorganisms and lesser activity against Gram-positive bacteria.²⁵ Since essential oils comprised of large number of compounds, it is likely that their mode of action involves several targets in the bacterial cells.²⁵ Previous studies have shown that the biological activities of essential oils from different species of plants are dependent of the major compounds of abundance. In some other cases, synergies between the major and some minor constituents have also enhanced the activity of natural products including essential oils.²⁵ These compounds exhibited activity by firstly destroy the microbial cytoplasmic wall to enhance permeability and passage of large protons and ions. Nevertheless, the antibacterial effect can be sum up as cumulative actions of several compounds and not to a specific compound.²⁵

Although it has been reported that the antibacterial activity of essential oils can be attributed to the presence of terpenoids and non-terpenic compounds, the mode of action of these compounds remain largely unknown.²⁵ Previous reports showed that (E)-caryophyllene was selective in its antibacterial activity more specifically against S. aureus, with better anti-fungal activity more than kanamycin.²⁶ This compound also showed activity towards B. cereus.²⁷ (E)-Caryophyllene was able to altered membrane permeability and integrity of both S. aureus and B. cereus, leading to membrane damage and intracellular content leakage, which eventually caused cell death. Both essential oil of Aloysia gratissima and β-caryophyllene exhibited antibacterial activity against S. aureus, in addition to potentiating the action of norfloxacin against S. aureus, P. aeruginosa, and E. coli.²⁸ (E)-Caryophyllene displayed rapid bacterial killing against E. faecalis.²⁹ A report has shown that (E)-caryophyllene has shown synergism in antimicrobial studies.³⁰ Several reports have indicated that α-pinene has wide potential in antimicrobial therapy in order to inhibit the growth of bacteria as an isolated result or as a synergist of antibiotics. Results have shown that among other microbes, α-pinene has inhibited the growth of S. aureus, B. cereus, E. coli, C. albicans.^15,26,31 Caryophyllene oxide, and (E)-nerolidol have exhibited pronounce activity towards presented rapid bacterial killing against E. faecalis, S. aureus and C. albicans.²⁹ It was found that limonene showed antibacterial activity against Gram-positive and Gram-negative microorganisms. These include S. aureus in a synergistic manner³² and E. coli.^33–35 The antimicrobial activities of essential oils from some plants was correlated most strongly to the content of (Z)-β-ocimene, δ-cadinene, camphene, terpinen-4-ol, elemol, β-pinene, β-elemene, γ-cadinene, α-terpineol, among others.³⁴ β-Ocimene has been implicated in the antimicrobial potential of several plants and natural compounds.^19,36 It should be noted that essential oil constituents have been implicated in negative antimicrobial correlation.³⁴ Studied are on-going on the mode of action and interaction between essential oils and individual constituents. Spathulenol, a sesquiterpenoid is known to have once showed killing effect on some microbes.³⁷

A notable observation was the essential oils of C. subulatum showed better activity towards the Gram-positive bacteria than I. petelotii. These differences could be attributed in part to the great complexity of the double membrane-containing cell envelope in Gram-negative bacteria compared to the single membrane structure of the Gram-positive ones which made it harder for essential oils to diffuse through.^38,39

Larvicidal Effect of C. subulatum and I. petelotii on Mosquito larvae

The samples of C. subulatum and I. petelotii possessed larvicidal effects and mortality on Ae. aegypti and Cx. quinquefasciatus (Table 3). In the test against Ae. aegypti, C. subulatum exhibited LC₅₀ values of 39.33 µg/mL (24 h) and 35.36 µg/mL (48 h), while the LC₉₀ values were recorded as 51.50 μg/mL and 49.68 μg/mL. In addition, I. petelotii showed LC₅₀ values of 36.88 µg/mL (24 h) and 69.59 µg/mL at 48 h. The observed LC₉₀ values for I. petelotii were 52.61 µg/mL (24 h) and 148.66 µg/mL (48 h). During the tests for activity against Cx. quinquefasciatus, C. subulatum showed stronger activity with the LC₅₀ values of 32.60 µg/mL and 28.55 µg/mL, at 24 h and 48 h, respectively. The observed LC₉₀ were 69.42 μg/mL and 62.79 μg/mL, respectively. However, I. petelotii had LC₅₀ of 48.94 µg/mL/42.95 µg/mL (24 h vs 48 h), and LC₉₀ of 69.21 μg/mL/75.55 μg/mL (24 h vs 48 h). Based on previous postulations where strong mosquito larvicidal activity are considered best when LC₅₀≤ 50 µg/mL,⁴⁰ both C. subulatum and I. petelotii essential oils showed strong activity against the mosquito vectors because LC₅₀ values ranged between 28.55-48.94 µg/mL. On the whole, I. petelotii essential showed stronger activity towards Ae. Aegypti at 24 h than C. subulatum. However, C. subulatum was more potent against Cx. quinquefasciatus than I. petelotii. This result compares favorably with data reported for many others essential oils in the literature. The biological effects of many compounds both major and minor can be assumed as one of factors elucidating the larvicidal activity of C. subulatum and I. petelotii. Against Ae. Aegypti, the activity (LC₅₀) of monoterpenes α-pinene was 15.4 μg/mL, β-pinene (12.1 μg/mL), and linalool (38.6 μg/mL), while the sesquiterpene β-caryophyllene was active with value of 88.3 μg/mL.^41,42 However, caryophyllene oxide, a sesquiterpenoid, identified in both C. subulatum and I. petelotii was not known to be a good larvicidal agent (LC₅₀> 500 μg/mL).⁴¹ Several other compounds found in C. subulatum and I. petelotii such as β-caryophyllene,⁴¹ bornyl acetate⁴² among others have shown mosquito repellent activity. In effect, this study indicates that both C. subulatum and I. petelotii which possessed LC₅₀ values between 28.55 and 48.94 μg/mL may be considered as strong larvicidal agents towards Cx. quinquefasciatus.

Table 3.

Results of the Larvicidal Activity of the Essential Oils.^a

Essential oils	LC₅₀ (95% limits)	LC₉₀ (95% limits)	χ²	p
A. aegypti
24 h
C. subulaum	39.33 (35.82-42.38)	51.50 (47.71-56.96)	0.0494	0.997
I. petelotii	36.88 (33.89-40.00)	52.61 (47.83-59.83)	0.3113	0.958
48 h
C. subulaum	35.36 (32.55-38.41)	49.68 (45.09-56.67)	0.2196	0.974
I. petelotii	69.59 (62.14-79.03)	148.66 (122.58-198.09)	4.4142	0.220
C. quinquifasciatus
24 h
C. subulaum	32.60 (29.34-36.26)	69.42 (59.82-84.35)	7.6154	0.055
I. petelotii	48.94 (45.57-52.40)	69.21 (62.67-82.30)	1.7326	0.630
48 h
C. subulaum	28.55 (25.63-31.80)	62.79 (54.03-76.30)	7.5723	0.056
I. petelotii	42.95 (39.06-47.21)	75.55 (66.37-90.51)	5.5554	0.135

LC₅₀: 50% lethal concentration; LC₉₀: 90% lethal concentration; ^aPermethrin, the standard drug used as positive control displayed larvicidal activity against Cx. quinquefasciatus and Ae. aegypti with LC₅₀ values in the range of 2.19 - 3.43 μg/mL.

Materials and Methods

Collection of C. subulatum and I. petelotii

Both C. subulatum and I. petelotii plant samples were collected in April 2020. The plant samples were confirmed by Huong, LT with ascension number LTH 919 (C. subulatum) and LTH 891 (I. petelotii). Authors have deposited authenticated samples at University of Vinh.

The Essential Oils

In this experiments, 2.0 kg of C. subulatum and I. petelotii were hydrodistilled accordingly in a Clevenger glass apparatus as described previously.^16–18 The process consists of pulverization of each plant using a grinding machine, and each sample was divided into separate three equal parts for ease of hydrodistillation. By doing this, each separate part of known weighs was packed into distillation flask where the samples were completely immersed on addition of the sample to the flask. Heating was then applied by connecting the flask to the source of heat, for 3 h, with the whole distillation apparatus maintained at normal pressure as conditions mentioned earlier.⁴³ The essential oils were allowed to distilled over that period. Thereafter, the obtained oils of C. subulatum and I. petelotii were drained into separate sample bottles, where they are stored at cool temperature (4 °C) before use after the calculation of each individual yields as done previously.^16–18 Both samples of essential oils were diluted (1% n-hexane) before instrumental analysis by gas chromatography (GC) and mass spectrometry (GC/MS).

The GC Analysis

Here, the apparatus in the GC was a HP 7890A Plus, having HP-5MS column (film thickness of 0.25 µm). The column dimension was 30 m×0.25 mm. The sample was run under GC conditions of injector temperature (250 °C) and detector temperatures (260 °C). Also, 1.0 µL of samples was injected into the GC column at the split ratio of 10:1 and the inlet pressure of 6.1 kPa with Helium under the flow rate of 1 mL/min as the carrier gas. The injection temperature program rises from 40 °C (2 min rest) to 220 °C (hold 10 min). The triplicate analysis of each sample was done. Quantification was done to determine the percentages of the individual compounds of C. subulatum and I. petelotii as reported previously.^16–18

The GC/MS

In the experiment here, the GC (HP 7890A Plus) was coupled with a HP 5973 MSD Mass spectrometer. All conditions for the GC were also obtainable here. The resultant spectral were obtained under set experimental parameters as reported previously.^16–18

Detection of the Components of C. subulatum and I. petelotii Samples

In this last stage was the analysis of mass spectral to identify the various compounds of C. subulatum and I. petelotii essential oils. To accomplish this, the retention indices based on a series of n-alkanes was compared with a set of hydrocarbons. Moreover, the MS of C. subulatum and I. petelotii was compared with the compounds documented earlier⁴⁴ as done previously.^16–18

The Test to Determine Antimicrobial Efficacy of C. subulatum and I. petelotii

The microbial tests on C. subulatum and I. petelotii was determined using a set of Gram-positive, Gram-negative, and yeast (Candida albicans). The strains of microbes used were from American Type Culture Collection (ATCC). All microbial strains were subjected to culture for 24 h. This was done with tryptic soil agar (bacteria) and potato dextrose agar (yeasts). The inoculum of bacteria was adjusted to 5 × 10⁵ CFU/mL, while yeast was standardized to 1 × 10³ CFU/mL. C. subulatum and I. petelotii were dissolved in ethanol and diluted in a culture medium to achieve concentrations from 16384 μg/mL to 2 μg/mL. Inoculated wells with and without antimicrobial agents were assayed to control the adequacy of the broth for microorganism growth and medium sterility, respectively. The incubation of the microplates was bacteria was done at 37 °C while 35 °C was used for yeasts. The incubation was monitored under 24 h period. The lowest concentration that allowed no discernible growth of the tested microorganism was identified as the MIC. Streptomycin served as the positive control for bacteria, and Nystatin was used for C. albicans. Sterile distilled water was used as a negative control. Each experiment was performed 3 times as described previously.^16–18

The Study on the Killing Effects of Mosquitoes

Larvae of A. aegypti and C. quinquefasciatus were collected from a mosquito colony maintained at the Laboratory of Parasitology and Entomology of Duy Tan University, Da Nang Vietnam. In this study, aliquots of the essential oils of C. subulatum and I. petelotii dissolved in dimethylsulfoxide (DMSO) (1% stock solution) were placed in a separate beaker (500 mL capacity). The solution was added to water in separate beakers which has 25 larvae each of A. aegypti and C. quinquefasciatus. In each experiment, a set of controls using DMSO was also run for comparison. The killing effects of the essential oils (mortality) was recorded after 24 h. After 48 h of exposure during which no nutritional supplement was added, the mortality was recorded again. The experiments were carried out 25 ± 2 °C. Each test was conducted with three replicates of eight different concentrations (100, 50, 25, 12.5, 6.0, 3.0, 1.5, and 0.75 µg/mL). The larvicidal effects and LC₅₀ for each sample were determined accordingly.^16–18

Statistical Analysis

All experiments were conducted in triplicate. Data from larvicidal activity were evaluated through log-probit analysis using Minitab 19 (Minitab LLC, State College, PA, USA) to determine the LC₅₀ and LC₉₀, representing the concentration in µg/mL that caused 50% and 90% mortality along with 95% confidence intervals. The differences between the mean values obtained for experimental groups in the chemical constituents and antimicrobial studies were calculated as a mean of standard deviation (SD) of three independent measurements using Microsoft Excel program 2003.

Footnotes

Acknowledgments

The authors would like to thank the editor and anonymous reviewers for their thoughtful comments and efforts toward improving our manuscript.

Declaration of Conflicting Interests

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

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Do Ngoc Dai

Nguyen Huy Hung

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