Chemical composition of essential oils from leaves of vitex negundo l growing in viet nam and larvicidal activity against aedes aegypti l

Vietnam Journal of Science and Technology 58 (6A) (2020) 142-157 doi:10.15625/2525-2518/58/6A/15499 CHEMICAL COMPOSITION OF ESSENTIAL OILS FROM LEAVES OF VITEX NEGUNDO L GROWING IN VIET NAM AND LARVICIDAL ACTIVITY AGAINST AEDES AEGYPTI L Nguyen Huy Hung1, 2, *, Do Ngoc Dai3, 4, Prabodh Satyal5, Nguyen Thanh Chung3, Bui Van Nguyen6, William N Setzer5, 7, * Center for Advanced Chemistry, Institute of Research and Development, Duy Tan University, 03 Quang Trung, Da Nang, Viet Nam Department of Pharmacy, Duy Tan University, 03 Quang Trung, Da Nang, Viet Nam Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam Faculty of Agriculture, Forestry and Fishery, Nghe An College of Economics, 51 Ly Tu Trong, Vinh City, Nghe An Province, Viet Nam Aromatic Plant Research Center, 230 N 1200 E, Suite 102, Lehi, UT 84043, USA University of Khanh Hoa, 01 Nguyen Chanh, Nha Trang, Khanh Hoa, Viet Nam Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA * Emails: nguyenhuyhung@duytan.edu.vn, wsetzer@chemistry.uah.edu Received: 15 September 2020; Accepted for publication: 24 January 2021 Abstract The leaves of Vitex negundo L (Verbenaceae) were collected from Da Nang City (DND41, DND29, DND15 and DND18), the Pu Hoat Nature Reserve, Nghe An province (DND712), and Nam Dong district, Thua Thien Hue province (DND789), and were hydrodistilled to give the essential oils The leaf oils (six separate samples) were analyzed by gas chromatographic – mass spectrometric methods, and showed very different chemical compositions The major components in the four essential oils from Da Nang were sabinene (6.0 - 19.9 %), 1,8-cineole (1.6 - 13.7 %), α-terpinyl acetate (1.9 - 7.8 %), (E)-caryophyllene (5.7 18.3 %), eremophilene (13.1 - 33.6 %), caryophyllene oxide (4.9 - 18.1 %), and an unidentified diterpenoid (5.2 - 8.3 %); while the major components in the Pu Hoat samples were sabinene (14.7 %), (E)-caryophyllene (57.0 %), and caryophyllene oxide (5.4 %) Meanwhile, the main chemical constituents of the sample in the Nam Dong district, Thua Thien Hue province were trans-β-elemene (11.1 %), (E)-caryophyllene (48.2 %), bicyclogermacrene (7.5 %), phytol (6.3 %) and caryophyllene oxide (3.2 %) One sample of V negundo leaf essential oil from Da Nang and one from Nam Dong were screened for larvicidal activity against Aedes aegypti, a vector of dengue fever, chikungunya, Zika fever, Mayaro and yellow fever viruses The Da Nang essential oil showed only marginal larvicidal activity (24-h LC50 = 82.9 μg/mL; 48-h LC50 = 72.2 μg/mL), but the Nam Dong sample was significantly more active (24-h LC50 = 16.8 μg/mL; 48-h LC50 = 14.2 μg/mL) As a Essential oil composition and larvicidal activity of Vitex negundo test for pesticidal selectivity, V negundo leaf oil from Nam Dong was also screened against the water bug, Diplonychus rusticus, an insect predator of mosquito larvae The essential oil was significantly less toxic to D rusticus (24-h LC50 = 136 μg/mL; 48-h LC50 = 134 μg/mL) Keywords: Verbenaceae, Vitex negundo, Aedes aegypti, arbovirus, dengue fever Classification numbers: 1.2.1, 1.4.6 INTRODUCTION Diseases caused by mosquito vectors have been a continuing threat to mankind The yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culicidae) is an important insect vector of mosquito-borne viral diseases including dengue [1], yellow fever [2], chikungunya [3], and Zika [4], among others All four dengue virus serotypes are present throughout the year in Viet Nam and the country has been classified as hyperendemic [5] In the last few decades, epidemics of dengue fever have increased in occurrence equivalent to a median rate of 232 cases per 100,000 people per year [5] Additionally, both Zika and chikungunya infections have been recently recorded in Viet Nam [6] Exacerbating this problem, Zika virus infection has been recently shown to enhance the future risk and severity of dengue disease [7] Vector control has been one of the principal methods to limit the spread of arboviral infections Unfortunately, however, current procedures for controlling Aedes mosquitoes have been largely unsuccessful [8] Insecticide resistance in Aedes mosquitoes has been increasing worldwide potentially leading to a re-emergence of mosquito-borne diseases [9 - 11] In addition to insecticide resistance, detrimental environmental effects of synthetic insecticides have been an enduring problem for several decades [12, 13] Broad application of insecticides has had serious consequences on non-target organisms such as imidacloprid on honey bee (Apis mellifera) [14], damselfly (Ischnura senegalensis) [15], fathead minnow (Pimephales promelas), or the amphipod (Hyalella azteca) [16] There is an obvious need for complementary vector control approaches and essential oils may provide renewable and environmentally-benign alternatives to synthetic insecticides for mosquito control [17 - 20] Vitex negundo L., syn Vitex paniculata Lam (Verbenaceae), known as Ngũ trảo, Quan âm, or Hoàng kinh in Vietnamese, is a shrub or small tree that is native to East Africa and Asia [21] In Viet Nam, the plant has been recorded in many provinces from the northern mountainous regions to the Mekong Delta provinces [22, 23] The phytochemistry, ethnobotanical uses, and biological activities of V negundo have been extensively reviewed [24 - 30] In Viet Nam, leaves of the plant are used to treat aching tendons, polio, and enteritis, while the roots are used for cough and malaria [31] As part of our continuing efforts to identify readily-available essential oils for control of mosquitoes, we have assessed the essential oil from leaves of Vitex negundo (Table 1) growing in the wild in central Viet Nam for larvicidal action against Ae aegypti MATERIALS AND METHODS 2.1 Plant materials Leaves of V negundo were selected from several individual plants (Table 1) Identification of the plants was carried out by Dr Do Ngoc Dai, who deposited voucher specimens (Table 1) in the School of Natural Science Education, Vinh University Fresh leaves of V negundo (2.0 kg 143 Nguyen Huy Hung, et al per sample) were shredded and subjected to hydrodistillation using a Clevenger type apparatus (Witeg Labortechnik, Wertheim, Germany) over a 4-h period The essential oil yields are presented in Table Table Plant collection and hydrodistillation details of Vitex negundo central Viet Nam Collection site (coordinates; elevation) Collection Date Voucher number % (v/w) yield June 2018 DND41 0.51 August 2018 DND29 0.55 February 2019 DND15 0.57 April 2019 DND18 0.56 October 2018 DND712 0.49 July 2018 DND789 0.38 Bà Nà Hill, Hoa Vang district, Da Nang City 16°02′57ʺ N; 108°09′34ʺ E, elev m Bà Nà Hill, Hoa Vang district, Da Nang City 16°02′57ʺ N; 108°09′34ʺ E, elev m Bà Nà Hill, Hoa Vang district, Da Nang City 16°01′39ʺ N; 108°03′42ʺ E, elev 28 m Hoa Vang district, Da Nang City 16°01′39ʺ N; 108°03′42ʺ E, elev 28 m Đồng Văn Commune, Quế Phong District, Pu Hoat Nature Reserve, Nghe An province Nam Dong district, Thua Thien Hue province 16°13′03ʺ N; 107°43′27ʺ E, elev 109 m 2.2 Gas chromatography – Mass spectrometry The V negundo essential oil samples were analyzed by gas chromatography – mass spectrometry (GC-MS) The instrument was a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) carried out in electron impact (EI) mode with electron energy of 70 eV, a scan rate of 3.0 scans/s, a scan range of 40 - 400 atomic mass units (AMU), using the GC-MS solution software A ZB-5ms fused silica capillary column (Phenomenex, Torrance, CA, USA) with dimensions of 30 m length × 0.25 mm internal diameter and a stationary phase of (5% phenyl)-polymethylsiloxane with a film thickness of 0.25 μm was used Helium was the carrier gas with a flow rate of 1.37 mL/min and a column head pressure of 552 kPa The temperature of the injector was 250 °C and the temperature of the ion source was 200 °C The GC oven temperature was programmed for an initial temperature of 50 °C, and the temperature was increased at the rate of °C/min to a maximum of 260 °C Aliquots of each of the V negundo essential oils were diluted to % w/v solutions in dichloromethane and a volume of 0.1 μL was injected using the split (30:1) mode The essential oil components were identified using both their retention indices, which were calculated in reference to a homologous series of normal alkanes (C8-C40), and by comparison of their mass spectral fragmentations with those recorded in available databases [32 - 35] The concentrations of each component in the essential oils were calculated based on total ion current without standardization and normalized to 100 % total essential oil composition 2.3 Mosquito larvicidal assay Ae aegypti eggs were obtained from the Institute of Biotechnology, Vietnam Academy of Science and Technology, and mosquito larvae raised in the Laboratory of Department of 144 Essential oil composition and larvicidal activity of Vitex negundo Pharmacy of Duy Tan University, Da Nang, Viet Nam Larvicidal activity of essential oils were evaluated according to the protocol of Hoi and co-workers [36] For each test, an aliquot of a % stock solution of the essential oil of V negundo in DMSO was placed in a 250-mL beaker and added to water that contained 20 third and early fourth instar mosquito larvae For each assay, DMSO was used as a negative control and permethrin was used as the positive control Acute larvicidal activity was recorded after 24 h and again after 48 h of exposure during which no nutritional supplement was added The experiments were conducted at room temperature (25 ± °C) Each assay was carried out in quadruplicate with several concentrations of essential oil (100, 50, 25, 12.5, 6.0, 3.0, 1.5, 1.0, and 0.5 μg/mL) 2.4 Water bug lethality assay Water bug lethality assay was performed according to the protocol of Hoi and co-workers [36] Adults of Diplonychus rusticus were collected in the field and maintained in glass aquaria (60 cm long  50 cm wide) containing water at 25 °C and a water depth of 20 cm Each insecticidal assay was carried out in quadruplicate using several concentrations of essential oil (200, 150, 100, 75, 50, and 25 μg/mL) Twenty D rusticus adults were introduced into each solution and acute mortality was assessed after 24 h and 48 h exposure The negative control was DMSO 2.5 Statistical analysis The insect lethality data obtained were subjected to log-probit analysis [37] to obtain LC50 values, LC90 values, 95 % confidence limits, and chi square values using Minitab® 18 (Minitab Inc., State College, PA, USA) Agglomerative hierarchical cluster (AHC) analysis was carried out based on the essential oil compositions from this work and from the published literature The chemical compositions were treated as operational taxonomic units (OTUs) The percentages of the major essential oil components were used to determine the chemical similarities between the various essential oil samples using the XLSTAT software, version 2018.1.1.62926 (Addinsoft™, Paris, France) Euclidean distance was used to measure dissimilarity, and Ward’s method was used for cluster definition RESULTS AND DISCUSSION 3.1 Essential oil composition The essential oils from the leaves of V negundo were obtained by hydrodistillation in yields ranging from 0.38 % to 0.57 % yield The chemical compositions of the V negundo leaf oils, determined using gas chromatography – mass spectrometry, are summarized in Table The major components in the four essential oils from Da Nang were sabinene (6.0 - 19.9 %), 1,8-cineole (1.6 - 13.7 %), α-terpinyl acetate (1.9 - 7.8 %), (E)-caryophyllene (5.7 - 18.3 %), eremophilene (13.1 - 33.6 %), caryophyllene oxide (4.9 - 18.1 %), and an unidentified diterpenoid (5.2 - 8.3 %) In contrast, the major components in the Pu Hoat sample were sabinene (14.7 %), (E)-caryophyllene (57.0 %), and caryophyllene oxide (5.4 %) Meanwhile, the main chemical constituents of the sample in the Nam Dong district, Thua Thien Hue province were trans-β-elemene (11.1 %), (E)-caryophyllene (48.2 %), bicyclogermacrene (7.5 %), phytol (6.3 %) and caryophyllene oxide (3.2 %) 145 Nguyen Huy Hung, et al Table Chemical compositions (%) of leaf essential oils of Vitex negundo from central Viet Nam Da Nang Pu Hoat Nam Dong RIcalc RIdb Compound DND41 DND29 DND15 DND18 DND712 DND789 923 923 α-Thujene 0.4 0.1 0.2 0.2 0.3 - 948 950 Camphene tr tr - - tr tr 972 972 Sabinene 19.9 7.1 8.5 6.0 14.7 tr 976 974 1-Octen-3-ol - 0.1 - - - - 977 978 β-Pinene 1.7 0.6 0.9 2.4 1.2 0.1 983 984 3-Octanone - - 0.1 0.2 tr tr 988 989 Myrcene 0.4 0.2 0.3 0.5 0.4 tr 995 996 3-Octanol tr - tr 0.3 tr - 1006 1006 α-Phellandrene - - - - tr tr 1008 1008 δ-3-Carene - - - - tr - 1016 1017 α-Terpinene 0.1 - tr 0.1 0.2 - 1023 1024 p-Cymene 1.5 1.2 1.6 1.1 1.0 0.1 1028 1028 Limonene 0.3 0.3 0.3 1.0 0.4 0.1 1029 1029 β-Phellandrene 0.3 0.1 0.3 1.4 0.4 tr 1031 1032 1,8-cineole 1.6 11.2 3.9 13.7 0.6 0.1 1033 1034 (Z)-β-Ocimene tr - tr tr tr tr 1044 1045 (E)-β-Ocimene 0.1 tr 0.1 0.2 0.1 tr 1056 1057 γ-Terpinene 0.3 tr 0.1 0.2 0.5 - 1069 1069 cis-Sabinene hydrate 0.5 0.1 tr 0.1 tr - 1084 1086 Terpinolene 0.1 - 0.1 0.1 0.2 - 1098 1099 Linalool 0.5 0.3 0.2 0.7 0.8 0.2 1099 1098 trans-Sabinene hydrate 0.3 tr - tr - - 1101 1104 Hotrienol - - - tr - - 1103 1104 Nonanal tr tr - tr - tr 1103 1109 Isopentyl isovalerate - - - tr tr - 1118 1118 3-Octyl acetate 0.1 - tr 0.1 tr - 1123 1124 cis-p-Menth-2-en-1-ol 0.3 0.1 tr 0.1 0.1 - 1134 1137 Benzeneacetonitrile - - - - tr - 1137 1137 trans-Sabinol tr tr - - - - 1139 1141 cis-Verbenol - - tr - - - 1140 1141 trans-Pinocarveol 0.1 0.1 - - - - 1141 1142 trans-p-Menth-2-en-1-ol 0.2 0.1 tr 0.1 tr - 1156 1157 Sabina ketone 0.1 0.1 - - - - 1158 1160 (Z)-Isocitral tr tr - - - - 1160 1160 Pinocarvone - tr - - - - 1162 1165 Lavandulol 0.9 - - 0.3 - - 1168 1168 trans-Phellandrene epoxide 0.1 tr - - - - 146 Essential oil composition and larvicidal activity of Vitex negundo Da Nang RIcalc RIdb Compound DND41 DND29 1169 1170 δ-Terpineol 0.1 1172 1172 cis-α-Necrodol 0.1 1180 1180 Terpinen-4-ol 1181 1181 Thuj-3-en-10-al 1183 1188 Naphthalene 1185 1186 p-Cymen-8-ol 1186 1187 Cryptone 1189 1190 1193 1194 Pu Hoat Nam Dong DND15 DND18 DND712 DND789 0.3 tr 0.2 - - - - - - - 4.0 2.8 0.7 1.6 2.2 tr 0.1 tr - - - - - - 0.1 - - - 0.1 0.1 0.1 - tr - 0.1 0.1 - 0.2 - - Methyl salicylate - - - - - tr 1194 Myrtenol 0.1 tr - - tr - 1195 α-Terpineol 0.4 0.9 0.1 0.9 0.2 tr 1195 1195 cis-Piperitol 0.1 - - - - - 1196 1198 Safranal - - - - - tr 1204 1206 Decanal - - - - - tr 1205 1202 (5Z)-Octenyl tiglate - - - - 0.1 - 1207 1207 trans-Piperitol 0.1 tr - - - - 1216 1219 β-Cyclocitral - - - - - tr 1222 1222 cis-iso-Ascaridole 0.1 0.1 tr 0.1 - - 1238 1240 Ascaridole - 0.1 tr 0.1 - - 1241 1238 Cumin aldehyde - tr - - - - 1272 1275 trans-Ascaridol glycol 0.1 0.1 tr 0.1 - - 1281 1284 Lavandulyl acetate 2.6 - - 1.1 - - 1282 1282 Bornyl acetate 0.1 0.1 tr tr tr tr 1287 1287 Dihydroedulan IA - - - - 0.1 1.0 1289 1291 cis-Ascaridol glycol 0.2 - - 0.1 - - 1292 1294 Dihydroedulan IIA - - - - tr tr 1296 1302 cis-α-Necrodol acetate 0.2 - - - - - 1297 1298 cis-Theaspirane - - tr 0.1 tr tr 1300 1300 Tridecane - - - - - 0.1 1302 1306 trans-iso-Ascaridole - - tr 0.1 - - 1310 1313 δ-Terpinyl acetate - 0.2 0.1 0.1 - - 1313 1314 trans-Theaspirane - - tr 0.1 tr tr 1330 1332 Bicycloelemene 0.1 - 0.1 - - 0.4 1334 1335 δ-Elemene 0.1 0.1 0.8 - tr 0.1 1345 1346 α-Terpinyl acetate 1.9 7.3 3.8 7.8 0.5 - 1346 1348 α-Cubebene - - - - - tr 1351 1352 Dehydro-ar-ionene - - - - - tr 1368 1371 α-Ylangene - - - - - tr 1369 1370 iso-Ledene - - - - - tr 147 Nguyen Huy Hung, et al Da Nang RIcalc RIdb Compound DND41 DND29 1375 1375 α-Copaene - 1375 1379 (E)-β-Damascenone - 1376 1378 Geranyl acetate 1380 1383 1382 1387 Pu Hoat Nam Dong DND15 DND18 DND712 DND789 - tr tr 0.1 0.1 - - tr - - - - - - - tr cis-β-Elemene - - tr - - 0.6 1382 β-Bourbonene 0.1 0.1 0.1 - 0.1 - 1387 β-Cubebene - - - - tr - 1389 1390 trans-β-Elemene 0.2 0.1 0.4 0.2 0.2 11.1 1404 1405 (Z)-Caryophyllene 0.1 0.1 0.1 tr 0.1 0.2 1406 1406 α-Gurjunene - - - - - 0.1 1410 1411 Thymohydroquinone dimethyl ether - - 0.1 - - - 1418 1417 (E)-Caryophyllene 12.8 5.7 18.3 16.4 57.0 48.2 1427 1427 γ-Elemene - - 0.1 - 0.1 - 1429 1430 β-Copaene - - - - 0.1 0.2 1432 1432 trans-α-Bergamotene - - - - - 1.0 1434 1436 α-Guaiene - - - - - 0.6 1437 1438 Aromadendrene - - - - - 0.5 1440 1442 6,9-Guaiadiene 0.1 - 0.1 - - 1.2 1444 1445 Selina-5,11-diene - - - - - 0.1 1451 1451 (E)-β-Farnesene - - - - - 0.1 1454 1454 α-Humulene 0.6 0.4 0.8 0.8 2.9 3.1 1458 1458 allo-Aromadendrene - 0.1 - 0.1 tr 0.1 1468 1473 4,5-di-epi-Aristolochene 0.1 0.2 0.1 0.1 - 0.1 1469 1471 β-Acoradiene - - - - - 0.1 1471 1475 Selina-4,11-diene - - - - - 0.4 1473 1474 α-Neocallitropsene - - - - - 0.2 1474 1475 γ-Gurjunene - - tr - - - 1475 1481 (E)-β-Ionone - - - - - 0.1 1477 1479 α-Amorphene - - 0.1 - - - 1480 1480 Germacrene D - - 0.2 - 0.9 0.9 1482 1483 trans-β-Bergamotene - - - - - 0.3 1487 1491 Eremophilene 19.5 19.4 33.6 13.1 - - 1487 1487 β-Selinene - - - - - 2.0 1489 1491 Viridiflorene - - - 0.1 - 0.2 1490 1492 trans-Muurola-4(14),5-diene - - tr - - - 1493 1492 Valencene - - 0.1 - - - 1493 1497 α-Selinene - - 0.1 - - - 1495 1497 Bicyclogermacrene - - - 0.4 - 7.5 1500 1505 α-Bulnesene - - - - - 0.2 148 Essential oil composition and larvicidal activity of Vitex negundo Da Nang RIcalc RIdb Compound 1501 1503 (E,E)-α-Farnesene 1501 1507 1505 1508 1511 Pu Hoat Nam Dong DND41 DND29 DND15 DND18 DND712 DND789 - - - - - 0.2 Eremophila-1(10),8,11-triene tr 0.3 0.1 0.1 - - β-Bisabolene - - - - - 0.7 1512 γ-Cadinene - - - - - 0.1 1516 1518 δ-Cadinene - - 0.1 0.1 0.4 0.2 1517 1520 7-epi-α-Selinene - - - - - 0.1 1519 1519 trans-Calamenene - - - - - 0.1 1521 1522 cis-Dihydroagarofuran - - - - - 0.1 1538 1540 (E)-α-Bisabolene - - - - - 0.3 1550 1551 (Z)-Caryophyllene oxide 0.6 1.3 0.5 0.5 0.5 - 1557 1557 Germacrene B - - 0.2 - 0.1 0.1 1559 1560 (E)-Nerolidol 0.1 - - - tr 0.4 1569 1571 (3Z)-Hexenyl benzoate 0.2 - - 0.2 0.1 - 1575 1576 Spathulenol - - tr 1.2 - 3.5 1579 1577 Caryophyllene oxide 6.9 18.1 5.9 4.9 5.4 3.2 1584 1590 Globulol - - - - - 0.4 1592 1594 Viridiflorol - - - - - 0.2 1594 1596 Cubeban-11-ol - - - - - 0.1 1596 1600 Curzerenone - - - - - 0.1 1608 1613 Humulene epoxide II 0.3 0.9 0.3 0.2 0.2 0.2 1628 1629 iso-Spathulenol 0.3 0.1 0.6 0.2 - 0.3 1631 1630 Caryophylla-4(12),8(13)-dien-5α-ol tr - 0.1 - - - 1635 1636 Caryophylla-4(12),8(13)-dien-5β-ol 0.3 0.3 0.2 0.2 - - 1637 1640 Hinesol - - 0.1 0.1 - - 1639 1640 τ-Cadinol - - tr - - - 1653 1652 α-Eudesmol 2.0 - - - - - 1653 1655 α-Cadinol - 1.5 1.8 0.8 - 0.1 1656 1658 neo-Intermedeol - - - - - 0.4 1659 1664 ar-Turmerone - - - - - 0.1 1669 1672 Apiole - - - - - 0.7 1680 1683 Germacra-4(15),5,10(14)-trien-1α-ol - - 0.5 0.5 - - 1727 1729 Zerumbone - - - - 0.1 - 1796 1797 Solavetivone 0.4 2.0 0.3 0.2 - - 1822 1813 Nootkatone 0.1 0.4 - - - - 1834 1836 Neophytadiene - - - - - 0.1 1904 1907 Isopimara-9(11),15-diene - - - - 0.2 - 1938 - Unidentified diterpenoid 5.2 5.4 6.4 8.3 - - 1976 1978 Manool 0.3 0.1 - 0.4 0.1 - 149 Nguyen Huy Hung, et al Da Nang RIcalc RIdb Compound Pu Hoat Nam Dong DND41 DND29 DND15 DND18 DND712 DND789 1977 - Unidentified diterpenoid 1.4 1.0 1.0 1.5 1.2 - 1988 1989 Manoyl oxide 0.1 - - 0.1 - - 1993 1998 (Z,E)-Geranyl linalool 0.1 - - - - - 2048 2049 Abietatriene 2.0 0.3 0.3 2.0 - - 2105 2106 Phytol - - 0.2 0.3 - 6.3 Monoterpene hydrocarbons 25.1 9.6 12.5 13.3 19.3 0.4 Oxygenated monoterpenoids 14.7 23.9 8.9 27.2 4.3 0.3 Sesquiterpene hydrocarbons 33.6 26.4 55.3 31.3 61.9 81.4 Oxygenated sesquiterpenoids 11.1 24.7 10.2 8.8 6.2 9.0 Diterpenoids 2.5 0.5 0.5 2.7 0.3 6.4 Others 0.3 0.0 0.2 1.0 0.3 1.9 Total identified 87.4 85.1 87.7 84.3 92.3 99.4 RIcalc = Retention Indices determined with respect to a homologous series of n-alkanes on an ZB-5ms column; RIdb = Retention Indices from the databases [32 - 35]; tr = trace (< 0.05 %) Each compound was identified based on both RI and MS comparison There is wide variation in the leaf essential oil compositions of V negundo between the samples collected in Viet Nam and compared to leaf oil compositions from other geographical locations [38 - 41] In order to provide some insight into the chemotypes based on leaf essential oil composition and to place the Vietnamese samples into chemical context with samples from other geographical locations, a hierarchical cluster analysis was carried out based on the major essential oil components (i.e., sabinene, (E)-caryophyllene, viridiflorol, eremophilene, terpinene4-ol, caryophyllene oxide, 1,8-cineole, α-terpinyl acetate, trans-β-elemene, α-pinene, α-copaene, p-cymene, camphene, α-thujene, α-humulene, β-pinene, bicyclogermacrene, phytol, and two unidentified diterpenoids) The cluster analysis (Figure 1) reveals five groups based on chemical composition Group 1, composed of the four V negundo essential oils from Da Nang and an essential oil sample from Taiwan, is composed largely of (E)-caryophyllene and sabinene Group is a single sample from Ganeshpur, India, and is dominated by α-copaene, camphene, and trans-β-elemene Group 3, dominated by (E)-caryophyllene, is comprised of the Pu Hoat samples from Viet Nam and a sample from Chandigarh, India Group 4, made up entirely of samples from India, can best be described as a sabinene/viridiflorol/(E)-caryophyllene group, and group 5, also made up of samples from India, has large concentrations of viridiflorol, (E)caryophyllene, sabinene, and an unidentified diterpenoid Centroid concentrations for each of the groups are listed in Table The chemical groupings of V negundo essential oils from India have already been described by Padalia and co-workers [41], and are in agreement with the present work Thus, in general, the chemical compositions of V negundo from Viet Nam are markedly different from the compositions of V negundo from India 150 Essential oil composition and larvicidal activity of Vitex negundo Taichung, Taiwan [38] DND41 (this work) DND15 (this work) DND18 (this work) DND29 (this work) Ganeshpur, India [37] DND789 (this work) DND712 (this work) Chandigarh, India [39] Dungoli, India [40] Group Group Group Tarikhet, India [40] Balighat, India [40] Manan, India [40] Lobanj, India [40] Gagrigole, India [40] Ashon, India [40] Kandhar, India [40] Lohali, India [40] Dewalchoura, India [40] Tharali, India [40] Bharadi, India [40] Sirkot, India [40] Chhati, India [40] Ghingartola, India [40] Soneshwar, India [40] Dehra Dun, India [40] Bhimtal, India [40] Kathgodam, India [40] Haldwani, India [40] Bindukhatta, India [40] Pantnagar, India [40] Beriparav, India [40] CIMAP, India [40] ] Group Group 1000 2000 3000 4000 5000 6000 Dissimilarity Figure Dendrogram based on hierarchical cluster analysis (HCA) of the leaf essential oils of Vitex negundo obtained from different locations in Asia Table Group centroid concentrations (%) from the hierarchical cluster analysis of Vitex negundo leaf essential oils Compound Group Group Group Group Group α-Thujene 0.2 9.9 0.3 0.2 0.0 α-Pinene 0.0 7.7 1.7 0.5 1.1 Camphene 0.0 21.1 0.0 0.0 0.0 Sabinene 12.1 3.1 7.3 29.1 9.0 β-Pinene 1.1 0.0 1.1 0.0 0.0 151 Nguyen Huy Hung, et al p-Cymene 2.3 0.0 0.8 0.5 0.2 1,8-Cineole 6.1 0.0 0.6 0.6 1.5 Terpinen-4-ol 2.4 0.0 3.6 3.9 2.2 α-Terpinyl acetate 6.0 0.0 0.4 0.5 0.1 α-Copaene 0.0 25.3 0.1 0.0 0.0 trans-β-Elemene 0.2 19.2 3.8 0.1 0.2 (E)-Caryophyllene 14.3 2.7 44.4 11.9 10.6 α-Humulene 0.6 0.0 2.5 0.1 0.1 Eremophilene 19.7 0.0 0.0 0.0 0.0 Bicyclogermacrene 0.1 0.0 2.5 0.0 0.0 Caryophyllene oxide 9.4 0.0 3.3 0.8 0.8 Viridiflorol 0.0 0.0 0.1 14.5 20.3 Unidentified diterpenoid (1871) 0.4 0.0 0.5 8.3 10.3 Unidentified diterpenoid (1938) 5.4 0.0 0.3 0.0 0.0 Phytol 0.1 0.0 2.1 0.0 0.0 3.2 Aedes aegypti mosquito larvicidal activity One of the V negundo leaf essential oil samples from Da Nang (DND41) and sample from Nam Dong district, Thua Thien Hue province (DND789) were screened for larvicidal activity against Aedes aegypti In addition, essential oil sample from Nam Dong was screened against the water bug, Diplonychus rusticus, an insect predator of mosquito larvae The 24-h and 48-h mosquito larvicidal activities of the V negundo leaf essential oils are assembled in Table Table Aedes aegypti larvicidal activities (μg/mL) and Diplonychus rusticus insecticidal activity (μg/mL) for Vitex negundo leaf essential oils Activity Sample Aedes aegypti DND41 DND789 Permethrin 24-h LC50 82.90 (77.72-87.90) 16.78 (15.50-18.16) 0.0094 (0.0082-0.0107) 24-h LC90 106.3 (100.3-114.1) 24.29 (22.45-26.79) 0.0211 (0.0185-0.0249) 48-h LC50 72.21 (67.14-77.88) 14.23 (12.87-15.64) 0.0087 (0.0074-0.0102) 48-h LC90 101.6 (94.3-110.8) 23.01 (20.99-25.84) 0.0204 (0.0181-0.0236) 24-h LC50 n.t 136.3 (125.2-149.5) n.t 24-h LC90 n.t 245.2 (221.2-279.6) n.t 48-h LC50 n.t 134.2 (123.5-146.7) n.t 48-h LC90 n.t 239.3 (216.6-271.5) n.t Diplonychus rusticus LC50 = median lethal concentration, μg/mL (95 % confidence limits in parentheses), LC90 = 90 % lethal concentration, n.t = not tested 152 Essential oil composition and larvicidal activity of Vitex negundo The V negundo leaf oil from Da Nang showed only marginal larvicidal activity on Ae aegypti with 24-h LC50 of 82.9 μg/mL In contrast, the essential oil from Nam Dong was much more active with 24-h LC50 of 16.8 μg/mL The Nam Dong sample was rich in (E)caryophyllene (48.2 %), trans-β-elemene (11.1 %), and phytol (6.0 %), which may account for the observed larvicidal activity Although (E)-caryophyllene has shown only marginal larvicidal activity against Ae aegypti (LC50 = 38.6 μg/mL) [42], β-elemene has shown excellent larvicidal activity on Ae albopictus (LC50 = 11.2 μg/mL) [43] As far as we are aware, phytol has not been screened for mosquito larvicidal activity, but the compound has shown insecticidal activity against the aphid, Metopolophium dirhodum [44] Essential oils containing (E)-caryophyllene, trans-β-elemene, and phytol have shown larvicidal activities For example, the major components in the leaf essential oil of Premna latifolia included (E)-caryophyllene (8.4 %), trans-β-elemene (8.3 %), and phytol (27.2 %), and had a moderate larvicidal activity against Ae albopictus of IC50 = 38.9 μg/mL [45] Atalantia racemosa leaf essential oil had (E)-caryophyllene (10.1 %), trans-β-elemene (5.4 %), and phytol (2.2 %), but showed relatively weak larvicidal activity against Ae aegypti with an IC50 of 97.1 μg/mL [46] The leaf essential oil of Bauhinia cheilantha was composed of (E)-caryophyllene (21.7 %), trans-β-elemene (4.9 %), and phytol (0.8 %), and showed larvicidal activity on Ae aegypti with an IC50 of 40.8 μg/mL [47] In order to compare the mosquito larvicidal activity of V negundo leaf essential oil with potential insecticidal activity on a beneficial aquatic species, the activity was determined against the water bug, Diplonychus rusticus, an insect predator of mosquito larvae [48] The leaf essential oil of V negundo was substantially less toxic to D rusticus than it was to the Ae aegypti mosquito larvae CONCLUSIONS Vitex negundo leaf essential oil, obtained from Nam Dong district, Thua Thien Hue province (DND789), was found to be rich in (E)-caryophyllene, trans-β-elemene, and phytol, and showed remarkable larvicidal activity against Ae aegypti The essential oil from Da Nang had significantly lower concentrations of these three compounds and showed reduced larvicidal activity Furthermore, V negundo leaf oil was significantly less toxic to the beneficial insect species, D rusticus Thus, depending on the chemical composition, V negundo essential oil may serve as an alternative, environmentally-friendly mosquito control agent Acknowledgements This research was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED), grant number 106.03-2019.25 P.S and W.N.S participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/) CRediT authorship contribution statement NHH: Methodology, Investigation, Funding acquisition DND: Formal analysis Prabodh Satyal: Methodology, Investigation NTC: Formal analysis 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