Chemical Composition and Larvicidal Activity of the Essential oil of Alpinia breviligulata (Gagnep.) Gagnep. Against Two Species of Aedes Mosquitoes

Abstract

Background: Essential oils are promising candidates for the development of next-generation biopesticides, offering a sustainable alternative to traditional synthetic pesticides. Methods: In this study, essential oils from various parts of Alpinia breviligulata were subjected to chemical composition analysis using GC/MS and evaluated for their larvicidal activities against Aedes aegypti and Aedes albopictus mosquitoes following WHO guidelines. Results: The leaf essential oil is primarily characterized by the main components α-pinene (19.4%), caryophyllene oxide (15.1%), and α-copaene (8.5%). Essential oil from the roots demonstrated α-humulene (12.3%), camphor (10.3%), endo-fenchyl acetate (9.4%), (E)-β-caryophyllene (7.0%) and α-pinene (5.5%) as its main components. The essential oil of the stems is primarily characterized by the main components caryophyllene oxide (27.7%), humulene epoxide II (6.4%), and α-copaene (5.4%). The compounds β-pinene (18.1%), α-pinene (10.4%), camphene (6.7%) and atractylone (5.0%) are the main components of essential oil extracted from rhizomes. Essential oils of leaves, roots, and rhizomes demonstrated larvicidal activities against Aedes mosquitoes, with 24-h LC₅₀ values ranging from 14.34 to 21.79 µg/mL. In contrast, the stem essential oil showed 24-h LC₅₀ values greater than 77.30 µg/mL. Conclusion: The powerful larvicidal activities of the essential oil, coupled with the relative abundance of the plant, suggest that essential oils of A. breviligulata should be considered as a potential source of raw materials for the production of biopesticides to control Aedes mosquitoes.

Keywords

arboviruses biopesticide dengue

Introduction

Aedes aegypti (Diptera: Culicidae) and Aedes albopictus (Diptera: Culicidae) are major vectors of diseases caused by arboviruses such as dengue, yellow fever, chikungunya, and Zika.^1,2 Dengue fever is a leading health problem, with an estimated 400 million new cases globally every year, of which Asia accounts for 75%, followed by Latin America and Africa.³ Vietnam is a hyperendemic country for dengue, with all four serotypes circulating year-round and a median annual incidence of 232 cases per 100 000 people.⁴ Chikungunya is also projected to become a significant health threat in the near future.^4,5 For decades, synthetic pesticides have played an important role in mosquito control programs. However, the increasing global disease burden^6,7 and the expanding distribution of mosquito species⁸ demonstrate that traditional synthetic pesticides are gradually becoming less effective. Furthermore, traditional pesticides have been reported to have negative impacts on the environment and human health. Recently, essential oils are being considered as a source of biopesticides due to their many advantages, such as broad-spectrum effectiveness, low toxicity to non-target organisms, difficulty for target organisms to develop resistance, and rapid decomposition.^9–11

Alpinia breviligulata (Gagnep.) Gagnep. (Zingiberaceae) is a native species of Vietnam, commonly found growing along streams in secondary forests and plain areas. In Vietnamese folklore, the rhizome of A. breviligulata is often used to treat stomachaches by decocting water to drink.¹² Several previous studies have demonstrated that all different parts of this plant contain essential oils.^13–16 The abundance and availability of A. breviligulata as a raw material for biopesticides prompted us to carry out research on the chemical composition and larvicidal activities of essential oils from different parts of this plant. To our knowledge, this is the first report of the examination of A. breviligulata essential oil as a mosquito larvicidal agent.

Materials and Methods

Plant Material

Leaves, stems, rhizomes, and roots of Alpinia breviligulata were collected from Khanh Hoa Province, Vietnam, in February, 2024, and identified by Dr Dinh Quang Diep (Table 1).

Table 1.

Collection and Hydrodistillation Details of Alpinia breviligulata from Vietnam.

Plant part	Collection date and site	Mass of plant material	Essential oil yield (%, w/w)	Code
Leaves	February, 2024, Khanh Hiep, Khanh Vinh, Khanh Hoa Province (12°24'26.3"N 108°52'47.6"E)	3 kg	0.31	NHH-1 L
Steams		3 kg	0.12	NHH-1 S
Rhizomes		3 kg	0.10	NHH-1 Rh
Roots		3 kg	0.11	NHH-1 R

Extraction of Essential Oils

To determine the yield of essential oils, fresh materials were used. Each extraction process utilized 50 grams of fresh material, and the procedure was repeated three times. The essential oil was extracted through hydrodistillation using a Clevenger apparatus from Witeg Labortechnik, located in Wertheim, Germany. The distillation process lasted for 6 h.

Gas Chromatographic – Mass Spectral Analysis

Gas chromatography–mass spectral analyses (GC/MS) of essential oils were carried out using a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA): with a ZB-5 ms fuzed silica capillary column (60 m length, 0.25 mm diameter, and 0.25 μm film thickness) (Phenomenex, Torrance, CA, USA), He carrier gas, 2.0 mL/min flow rate, injection and ion source temperatures of 260 °C, and a GC oven program of 50 to 260 °C at 2.0 °C/min was used. A 0.1 μL amount of a 5% (w/v) sample of essential oil in CH₂Cl₂ was injected in split mode with a 24.5:1 split ratio. Only one injection for each essential oil was carried out. Retention indices (RI) were determined by the method of Van den Dool and Kratz, calibrating the instrument with a homologous series of n-alkanes (C₈-C₂₈) acquired on the same day using the same instrument under the same conditions.¹⁷ Identification of the essential oil components was carried out with a comparison of MS fragmentation and RI with those available in the databases.^18–21

Larvicidal Biassays

Aedes aegypti and Ae. albopictus mosquitoes are continuously maintained at Duy Tan University. Mosquito eggs were allowed to hatch in tap water overnight. The larvae were then fed a mixture of cat food and yeast in a ratio of 3:1 (w/w). All developmental stages of mosquito species were kept under laboratory conditions with a temperature of 25 °C, relative humidity of 75%, and a 12-h light/12-h dark cycle.

Larvicidal activities were conducted following the guidelines outlined by the World Health Organization with modifications.²² Third instar and early fourth instar larvae were selected for the larvicidal activity assays. Twenty-five larvae were transferred into 250-mL beakers containing 150 mL of distilled water. Essential oils were dissolved in ethanol (Sigma-Aldrich) to prepare 1% stock solutions. Various volumes of the stock solutions were added to the test beakers containing mosquito larvae to achieve desired concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL (n = 100), respectively. Ethanol served as the negative control (0.5 mL ethanol/150 mL H₂O), while permethrin (Sigma-Aldrich) was used as the positive control. Each concentration of agents was tested in quadruplicate. The number of dead larvae was recorded after 24 and 48 h of exposure. Larvicidal activity assays were conducted under the same mosquito rearing conditions. There were no mortalities in the negative (ethanol) controls.

Statistical Analysis

The mortality data were analyzed using log-probit analysis²³ to obtain LC₅₀ and LC₉₀ values, along with 95% confidence limits, utilizing Minitab^® version 19.2020.1 (Minitab, LLC, State College, PA, USA).

Results

Chemical Profiles of Essential Oils

The leaves showed the highest essential oil yield compared to other parts (0.31%) (Table 1). Details of the chemical composition of essential oils from different parts (leaves, root, stem, and rhizome) of A. breviligulata are presented in Table 2. The main components of leaf essential oil were α-pinene (19.4%), caryophyllene oxide (15.1%), and α-copaene (8.5%). Essential oil from the roots included the main components α-humulene (12.3%), camphene (10.3%), endo-fenchyl acetate (9.4%), (E)-β-caryophyllene (7.0%), and α-pinene (5.5%). The essential oil of the stem is characterized by the main components caryophyllene oxide (27.7%), humulene epoxide II (6.4%), and α-copaene (5.4%). The main components of rhizome essential oil are characterized by monoterpenes such as β-pinene (18.1%), α-pinene (10.4%), camphene (6.7%), and in addition, there is a sesquiterpenoid content of 5.0%, which is atractylone.

Table 2.

Essential oil Compositions of Alpinia breviligulata.

RI_calc	RI_db	Compounds	Leaves	Root	Stem	Rhizome
924	923	Tricyclene	tr	0.4	-	0.2
926	925	α-Thujene	-	0.1	-	0.1
934	932	α-Pinene	19.4	5.5	2.1	10.4
948	948	α-Fenchene	-	0.2	-	0.2
950	950	Camphene	0.5	10.3	0.1	6.7
953	953	Thuja-2,4(10)diene	1.3	tr	0.1	0.1
973	971	Sabinene	tr	tr	-	0.1
978	978	β-Pinene	0.2	0.5	0.1	18.1
984	984	6-Methylhept-5-en-2-one	tr	-	-	-
989	989	Myrcene	tr	1.5	0.1	1.3
1006	1006	3-Ethenyl-1,2-dimethyl-1,4-cyclohexadiene	0.2	-	-	-
1008	1007	α-Phellandrene	-	0.1	-	0.1
1010	1009	δ-3-Carene	-	4.1	-	2.8
1018	1018	α-Terpinene	-	tr	-	0.1
1020	1022	m-Cymene	-	tr	-	tr
1025	1025	p-Cymene	0.5	0.9	0.3	0.7
1030	1030	Limonene	1.0	4.5	0.7	4.0
1031	1031	β-Phellandrene	tr	0.7	tr	0.8
1033	1032	1,8-Cineole	tr	1.1	tr	1.7
1035	1039	o-Cymene	tr	-	-	-
1035	1035	(Z)-β-Ocimene	tr	tr	tr	tr
1046	1045	(E)-β-Ocimene	0.1	tr	0.1	0.1
1058	1058	γ-Terpinene	-	0.5	-	0.4
1070	1069	1-Octanol	0.1	-	tr	-
1072	1072	Pinol	0.2	-	tr	-
1081	1082	p-Mentha-2,4(8)-diene	-	tr	-	tr
1084	1086	o-Guaiacol	tr	-	-	-
1085	1086	Terpinolene	-	0.1	-	0.6
1089	1090	Fenchone	-	0.2	-	0.5
1090	1091	p-Cymenene	0.3	-	0.2	0.1
1099	1098	Perillene	tr	-	0.1	tr
1100	1101	Linalool	0.1	0.2	0.1	0.4
1103	1104	Hotrienol	tr	-	tr	-
1105	1105	Nonanal	tr	-	tr	-
1120	1119	endo-Fenchol	tr	1.8	tr	1.3
1127	1127	α-Campholenal	0.7	tr	0.3	0.1
1141	1140	trans-Pinocarveol	1.2	-	0.2	tr
1142	1140	cis-Verbenol	0.5	-	tr	-
1146	1145	trans-Verbenol	2.0	-	0.2	-
1148	1145	Camphor	tr	tr	-	0.1
1156	1156	Camphene hydrate	-	tr	-	0.1
1161	1160	trans-Pinocamphone	0.1	-	0.1	-
1163	1164	Pinocarvone	0.5	-	0.2	0.1
1172	1171	p-Mentha-1,5-dien-8-ol	1.1	-	-	-
1172	1170	Borneol	-	1.2	0.1	1.3
1176	1176	cis-Pinocamphone	-	-	-	tr
1181	1180	Terpinen-4-ol	0.1	0.1	0.1	0.1
1184	1184	p-Methylacetophenone	-	-	tr	-
1187	1186	p-Cymen-8-ol	0.3	-	0.2	-
1195	1195	α-Terpineol	-	0.1	-	0.7
1196	1196	Myrtenal	1.2	-	0.7	-
1207	1205	Verbenone	0.6	-	0.2	-
1219	1218	trans-Carveol	0.4	-	0.1	-
1219	1219	endo-Fenchyl acetate	-	9.4	-	4.2
1224	1226	Nerol	-	-	tr	-
1229	1229	Thymyl methyl ether	-	1.9	-	0.9
1238	1238	Carvacryl methyl ether	-	0.1	-	0.1
1243	1242	Carvone	0.2	-	0.1	-
1272	1275	4-Ethylguaiacol	0.1	-	-	-
1284	1285	Bornyl acetate	0.2	0.3	0.4	0.3
1295	1296	trans-Pinocarvyl acetate	-	-	-	0.1
1308	1309	4-Vinylguaicol	0.1	-	-	-
1322	1322	Myrtenyl acetate	-	-	-	tr
1335	1335	δ-Elemene	-	-	-	tr
1346	1346	α-Terpinyl acetate	-	tr	-	0.1
1347	1348	α-Cubebene	0.3	-	0.2	-
1358	1361	Neryl acetate	-	-	tr	0.1
1369	1367	Cyclosativene	0.7	-	0.4	-
1369	1370	α-Ylangene	-	tr	-	0.1
1376	1375	α-Copaene	8.5	0.1	5.4	0.6
1378	1378	Geranyl acetate	-	-	0.1	-
1387	1387	β-Cubebene	0.1	-	0.1	-
1389	1390	trans-β-Elemene	0.1	0.1	0.2	0.3
1391	1389	Sativene	0.1	-	tr	-
1403	1407	Cyperene	-	-	tr	-
1404	1400	Ylanga-2,4(15)-diene	1.8	tr	0.7	0.1
1412	1411	Thymohydroquinone dimethyl ether	-	0.8	-	0.4
1419	1417	(E)-β-Caryophyllene	1.0	7.0	3.3	4.3
1428	1427	γ-Elemene	-	0.1	0.1	0.4
1435	1436	α-Guaiene	-	tr	0.1	tr
1437	1438	Aromadendrene	-	tr	-	tr
1445	1445	Selina-5,11-diene	-	tr	-	tr
1449	1447	iso-Germacrene D	0.1	tr	0.2	0.1
1452	1452	(E)-β-Farnesene	-	tr	-	tr
1455	1454	α-Humulene	0.3	12.3	1.2	3.3
1459	1458	allo-Aromadendrene	2.1	0.1	1.7	0.2
1472	1473	trans-Cadina-1(6),4-diene	0.2	-	0.1	-
1473	1475	Selina-4,11-diene	-	tr	-	tr
1475	1475	γ-Muurolene	0.9	tr	0.5	0.1
1479	2479	α-Amorphene	-	tr	0.1	0.1
1481	1480	Germacrene D	-	tr	-	0.1
1484	1482	β-Chamigrene	-	1.2	-	1.0
1485	1482	Aristolochene	-	-	0.1	-
1489	1489	β-Selinene	0.8	0.4	0.7	0.7
1492	1490	γ-Amorphene	0.2	0.2	0.2	-
1492	1492	trans-Muurola-4(14),5-diene	-	-	-	0.3
1493	1493	Curzerene	-	0.1	0.1	0.5
1495	1497	epi-Cubebol	0.5	-	0.3	-
1496	1497	α-Selinene	-	0.5	0.4	0.7
1498	1497	α-Muurolene	0.6	-	0.5	0.1
1503	1506	δ-Amorphene	-	-	-	0.1
1505	1509	β-Dihydroagarofuran	-	0.1	-	0.1
1513	1512	γ-Cadinene	3.3	0.5	3.7	1.0
1515	1514	Cubebol	0.4	-	0.2	-
1518	1518	δ-Cadinene	1.8	0.2	2.4	0.5
1519	1520	7-epi-α-Selinene	-	0.4	-	0.4
1521	1519	trans-Calamenene	0.6	tr	0.8	tr
1522	1521	Zonarene	0.1	-	-	-
1530	1533	10-epi-Cubenol	0.1	-	-	-
1532	1533	trans-Cadine-1,4-diene	0.1	-	-	-
1536	1540	Selina-4(15),7(11)-diene	-	0.1	-	0.2
1541	1541	α-Calacorene	1.7	0.1	1.9	0.4
1547	1548	Occidentalol	-	-	-	0.1
1548	1549	α-Elemol	0.5	0.4	0.7	0.3
1551	1551	Isocaryphyllene oxide	1.1	0.2	2.4	0.4
1559	1560	Germacrene B	-	0.2	-	0.4
1561	1560	(E)-Nerolidol	-	0.2	3.6	0.4
1562	1560	β-Calcorene	0.8	-	-	-
1576	1576	Spathulenol	-	tr	0.3	0.1
1579	—	Unidentified^a	1.0	-	-	-
1583	1587	Caryophyllene oxide	15.1	3.3	27.7	4.8
1587	1590	Gleenol	0.8	-	0.4	-
1589	1590	Globulol	-	0.1	-	0.2
1596	1593	Guaiol	-	0.3	0.2	0.4
1598	1593	Humulene epoxide I	-	0.4	0.4	0.3
1604	1605	Ledol	0.5	-	0.6	-
1610	1613	Humulene epoxide II	3.6	3.3	6.4	1.9
1612	1609	Rosifoliol	0.3	-	0.4	-
1615	1616	1,10-di-epi-Cubenol	0.3	0.1	1.0	0.2
1616	1618	α-Corocalene	0.1	-	-	-
1622	1624	10-epi-γ-Eudesmol	-	0.3	-	0.2
1626	1624	Muurola-4,10(14)-dien-1β-ol	0.3	-	0.3	-
1627	1628	1-epi-Cubenol	1.6	0.1	2.1	0.2
1631	1632	γ-Eudesmol	-	1.6	-	1.0
1632	1635	(Z,Z)-Geranyl linalool	-	-	0.8	-
1633	1630	Caryophylla-4(12),8(13)-dien-5α-ol	0.5	-	0.7	-
1637	1636	Caryophylla-4(12),8(13)-dien-5β-ol	0.6	0.5	1.9	0.7
1642	1643	Cubenol	0.5	-	1.2	-
1647	1651	α-Muurolol	-	-	0.5	-
1655	1655	α-Eudesmol	-	1.3	-	2.4
1655	1656	14-Hydroxy-9-epi-(Z)-caryophyllene	0.9	-	4.5	-
1659	1660	Atractylone	-	0.8	-	5.0
1664	1668	Intermedeol	-	1.9	-	1.5
1665	1664	cis-Calamenen-10-ol	-	-	0.4	-
1670	1666	14-Hydroxy-9-epi-(E)-caryophyllene	0.7	0.3	3.2	0.4
1673	1676	Mustakone	3.3	-	1.8	0.1
1673	1677	Cadalene	-	0.1	1.3	0.1
1698	—	Unidentified^b	1.7	-	-	-
1700	1701	10-nor-Calamenen-10-one	0.3	-	0.6	-
1715	1715	Pentadecanal	-	0.1	-	-
1728	1729	Zerumbone	-	0.1	0.2	-
1743	1755	α-Cyperone	0.2	-	0.2	-
1798	—	Ambrial	-	1.1	-	0.5
1836	1836	Neophytadiene (=Phytadiene 1)	0.2	-	-	-
1879	1879	Phytadiene 4	0.1	-	-	-
1885	1886	(Z)-Hexadecatrienal	-	0.1	-	-
1978	—	(E)-15,16-Dinorlabda-8(17),11-dien-13-one	-	0.2	-	-
1990	1989	Manoyl oxide	-	-	-	0.8
2004	—	Atractylenolide I	-	0.2	-	0.4
2020	2022	(E,E)-Geranyl linalool	-	-	1.2	0.2
2107	2106	Phytol	-	-	-	0.4
2118	—	Coronarin E	-	0.7	-	0.2
2312	—	Unidentified^c	-	1.1	-	-
2322	—	Unidentified^d	-	3.9	-	0.5
2342	—	Unidentified^e	-	2.4	-	0.3
2347	—	(E)-Labda-8(17),12-diene-15,16-dial	-	1.7	-	0.3
		Monoterpene hydrocarbons	23.5	29.3	3.7	46.6
		Oxygenated monoterpenoids	9.5	17.3	3.2	12.2
		Sesquiterpene hydrocarbons	26.5	23.7	26.3	15.8
		Oxygenated sesquiterpenoids	31.9	15.3	62.4	21.2
		Diterpenoids	0.0	2.4	1.2	1.9
		Benzenoid aromatics	0.2	0.0	tr	0.0
		Others	0.7	1.5	0.6	0.5
		Total identified	92.4	89.6	97.4	98.3

RIcalc = Retention index calculated with respect to a homologous series of n-alkanes on a ZB-5 ms column. RIdb = Reference retention index from the databases. tr = trace (<0.05%), - = not detected. Major components are highlighted in blue bold font. Unidentified components > 1% are highlighted in red font.

^a MS(EI): 220(6%), 205(14%), 202(11%), 192(14%), 191(70%), 187(20%), 177(42%), 159(91%), 149(42%), 135(84%), 121(100%), 108(66%), 107(68%), 105(60%), 93(91%), 91(60%), 81(63%), 55(45%), 41(40%).

^b MS(EI): 238(27%), 223(11%), 205(13%), 195(20%), 181(31%), 177(20%), 162(58%), 155(25%), 147(17%), 137(28%), 128(47%), 123(30%), 110(40%), 109(54%), 95(99%), 85(27%), 81(32%), 71(54%), 69(53%), 55(51%), 43(100%), 41(41%).

^c MS(EI): 300(32%), 163(23%), 162(29%), 133 (16%), 123(15%), 117(18%), 107(20%), 95(28%), 94(100%), 81(35%), 69(20%), 55(18%), 41(16%).

^d MS(EI): 300(27%), 218(22%), 204(100%), 189(47%), 161(37%), 147(25%), 133(46%), 122(31%), 119(45%), 109(39%), 108(44%), 107(43%), 105(45%), 81(51%), 69(35%), 55(33%), 41(32%).

^e MS(EI): 300(10%), 282(35%), 189(10%), 176(10%), 162(13%), 147(15%), 133(16%), 124(20%), 119(22%), 109(97%), 95(100%), 94(37%), 81(35%), 69(22%), 55(21%), 41(20%).

Larvicidal Activities

Leaf, root, and rhizome essential oils demonstrated larvicidal activities against Aedes mosquitoes with 24h-LC₅₀ values in the range of 14.34-21.79 µg/mL, while the stem essential oil showed with 24-h LC₅₀ values greater than 77.30 µg/mL (Tables 3, 4).

Table 3.

Larvicidal Activity of Essential Oils of Alpinia breviligulata Against Aedes Aegypti (µg/mL).

Material	LC₅₀ (95% limits)	LC₉₀ (95% limits)	χ²	P
		24 h
Leaves	14.34 (12.91-15.94)	29.12 (25.17-35.38)	7.546	.110
Root	21.79 (19.71-24.08)	41.30 (36.03-49.66)	5.464	.243
Stem	77.30 (68.90-87.03)	178.98 (150.43-226.40)	17.120	.004
Rhizome	16.19 (14.48-18.10)	35.76 (30.64-43.80)	10.55	.061
Permethrin	0.00638 (0.00548-0.00744)	0.0232 (0.0182-0.0318)	8.868	.031
		48 h
Leaves	11.11 (9.86-12.49)	26.83 (22.71-33.43)	3.793	.435
Root	17.52 (15.77-19.48)	35.64 (30.81-43.25)	8.047	.090
Stem	75.24 (67.09-84.65)	173.32 (145.92-218.62)	15.542	.008
Rhizome	13.85 (12.42-15.44)	29.45 (25.33-35.93)	7.452	.189

Table 4.

Larvicidal Activity of Essential Oils of Alpinia breviligulata Against Aedes Albopictus (µg/mL).

Material	LC₅₀ (95% limits)	LC₉₀ (95% limits)	χ²	P
		24 h
Leaves	14.34 (12.89-15.95)	29.46 (25.45-35.79)	6.720	.242
Root	21.02 (18.89-23.40)	43.70 (37.72-53.06)	9.953	.077
Stem	>100	>100	nd	nd
Rhizome	nt	nt
Permethrin	0.0024 (0.0021-0.0026)	0.0042 (0.0038-0.0049)	4.64	.031
		48 h
Leaves	12.59 (11.30-14.02)	26.43 (22.77-32.22)	4.942	.423
Root	14.22 (12.79-15.80)	28.91 (25.00-35.08)	8.212	.145
Stem	>100	>100	nd	nd
Rhizome	nt	nt	nd	nd

Nt: not test; Nd: not determined.

Discussion

Comparisons of the chemical composition of A. breviligulata essential oil in this study and previous studies are presented in Figure 1. The leaf essential oils in this study have high similarities with the study of Dũng et al.¹⁶

Figure 1.

Summary and comparison with previous studies on main chemical composition (≥4.0%). (A) This study. (B) Previous studies.^10–13

For the root essential oil, major differences between the present study and the previous study were observed in the components α-pinene (5.5/2.4%), camphene (10.3/3.2%), limonene (4.5/2.0%), 1,8-cineole (1.1/6.0%), borneol (1.2/5.5%), and caryophyllene oxide (3.3/13.0%).¹⁵ Rhizome essential oil in this study showed a greater content of the main component monoterpene and lower content of the main components sesquiterpene compared to the previous study.¹⁵ The chemical composition of stem essential oil showed the dominance of sesquiterpenoids, of which sesquiterpene hydrocarbons accounted for 26.3% and oxygenated sesquiterpenoids accounted for 62.4%.

Our previous study and several other studies, have reported that (E)-β-caryophyllene and caryophyllene oxide exhibits larvicidal activities against Aedes mosquitoes, with 24-h LC₅₀ values greater than 50 µg/mL.^24–26 The α-pinene and β-pinene have demonstrated larvicidal activity against Aedes mosquito species, with 24-h LC₅₀ values ranging from 12.94 to 23.63 µg/mL.^26,27 The α-humulene has shown larvicidal activity, with 24-h LC₅₀ values ranging from 37.89 to 53.05 µg/mL.^26,27 The α-copaene demonstrated weaker larvicidal activity against Bradysia procera compared to α-pinene, caryophyllene oxide, and α-humulene.²⁸

Thus, the additive activity of the main components α-pinene, α-copaene, and caryophyllene oxide in the leaf essential oil may have been responsible for the observed larvicidal activity. Camphene demonstrated larvicidal activity against Ae. aegypti, with 24-h LC₅₀ values ranging from 50 to 100 µg/mL.^29,30

The endo-fenchyl acetate demonstrated larvicidal activity against Ae. albopictus, with 24-h LC₅₀ values greater than 300 µg/mL.³¹ Although limonene showed strong larvicidal activity against Aedes mosquitoes,²⁷ its content in the root essential oil was only 4.5%. Thus, the strong activity of the root essential oil may be due to the synergistic effects between its main components. The observed larvicidal activity of stem essential oil is consistent with the content and larvicidal activity of its main components, caryophyllene oxide (27.7%) and α-copaene (5.4%). The two compounds, α-pinene and β-pinene, may be primarily responsible for the larvicidal activity of rhizome essential oil. Piper arboricola stem essential oil has been characterized by two main components, α-pinene (19.3%) and β-pinene (26.9%), which have shown larvicidal activity against Ae. aegypti with a 24-h LC₅₀ value of 28.45 µg/mL.³² Huong et al showed that β-pinene and α-pinene are the two major components responsible for the larvicidal activity of essential oils of Zingiber species against Ae. aegypti and Ae. albopictus with 24h-LC₅₀ values ranging from 12.72 to 45.58 µg/mL.³³

According to Pavela,⁹ a plant species holds great promise for the development of botanical larvicides if it satisfies the following factors: it can be cultivated and its essential oil has larvicidal activity with a 24-h-LC₅₀ value < 50 µg/mL. In addition, the LC₉₀ value is also an important parameter to consider, because a low LC₅₀ value does not always necessarily mean that a low dose is sufficient to achieve maximum mortality. The essential oils of leaves, roots and rhizomes of A. breviligulata all exhibited LC₅₀ and LC₉₀ values less than 50 µg/mL. Yield is one of the important factors when considering an essential oil as a biopesticide. In this study, the yield of essential oil of A. breviligulata leaves was 0.31% based on the weight of fresh material. Many aromatic plant species have been proposed as sources of essential oils for biopesticide development, with their reported yields equivalent to 0.31% such as Ocimum tenuiflorum L.,³⁴ Coriandrum sativum L.,³⁵ Blumea densiflora DC.³⁶ Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm.³⁷ and Alpinia calcarata (Andrews) Roscoe.³⁸

Conclusion

Essential oils from different parts of A. breviligulata (leaves, roots, stems and rhizome) demonstrated different chemical composition profiles. The concentrations of monoterpenes and sesquiterpenes increased gradually in the order of stem, leaf, root, and rhizome essential oils, respectively. Leaf, root, and rhizome essential oils have shown larvicidal activity against Ae. aegypti and Ae. albopictus, considered very active with 24-h LC₅₀ values ranging from 14.34 to 21.79 µg/mL. These oils can be considered as sources of botanical pesticides for Aedes mosquito control due to their potent larvicidal activities. Further studies should include toxicity assessments on non-target organisms of these essential oils. A limitation of this work is that plat materials were only collected at one point of the year and only from one location. The concentrations of bioactive components can vary due to phenology, geographical location, edaphic or climate conditions so the larvicidal activities of the essential oils are likely to be affected.

Footnotes

Acknowledgements

P.S. and W.N.S. participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, , accessed on March 30, 2024).

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

This research was funded by University of Khanh Hoa, grant number KHTN-23.01.

Ethical Approval

This study was approved by the Duy Tan University Medical–Biological Research Ethics Council, Number DTU/REC2020/NHH02.

Statement of Human and Animal Rights

All of the experimental procedures involving animals were conducted in accordance with the Duy Tan University Medical–Biological Research Ethics Council, Number DTU/REC2020/NHH02.

Statement of Informed Consent

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

ORCID iDs

Prabodh Satyal

Nguyen Huy Hung

William N. Setzer

Dinh Quang Diep

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