plants Article Premna Species in Vietnam: Essential Oil Compositions and Mosquito Larvicidal Activities Nguyen Huy Hung 1,2, * , Le Thi Huong 3 , Nguyen Thanh Chung 4 , Nguyen Cong Truong 5 , Do Ngoc Dai 4,5 , Prabodh Satyal 6 , Thieu Anh Tai 2 , Vu Thi Hien 7 and William N Setzer 6,8, * 1 2 3 4 5 6 7 8 * Center for Advanced Chemistry, Institute of Research and Development, Duy Tan University, 03 Quang Trung, Da Nang 550000, Vietnam Department of Pharmacy, Duy Tan University, 03 Quang Trung, Da Nang 550000, Vietnam; anhtai0808qn@gmail.com School of Natural Science Education, Vinh University, 182 Le Duan, Vinh City 43000, Nghe An Province, Vietnam; lehuong223@gmail.com Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi 10000, Vietnam; chungpuhoat@gmail.com (N.T.C.); daidn23@gmail.com (D.N.D.) Faculty of Agriculture, Forestry and Fishery, Nghe An College of Economics, 51-Ly Tu Trong, Vinh City 43000, Nghe An Province, Vietnam; congtruong777@gmail.com Aromatic Plant Research Center, 230 N 1200 E, Suite 102, Lehi, UT 84043, USA; psatyal@aromaticplant.org Faculty of Hydrometerology, Ho Chi Minh City University of Natural Resources and Environment, Ho Chi Minh City 70000, Vietnam; hiensphoa@gmail.com Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA Correspondence: nguyenhuyhung@duytan.edu.vn (N.H.H.); wsetzer@chemistry.uah.edu (W.N.S.) Received: 13 August 2020; Accepted: 31 August 2020; Published: 31 August 2020



 Abstract: Essential oils have emerged as viable alternatives to synthetic insecticides for control of mosquito-borne pathogens. The leaf essential oils of eight species of Premna (Lamiaceae) growing in central Vietnam have been obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry. Sesquiterpene hydrocarbons dominated most of the Premna essential oils, with the notable exception of Premna mekongensis from Ngoc Linh Nature Reserve, which had α-pinene as the major component. Larvicidal activities against Aedes aegypti have been determined and all of the Premna essential oils showed larvicidal activity with 24-h LC50 < 65 µg/mL. The leaf essential oils of Premna cambodiana from Chu Mom Ray National Park and Premna mekongensis from Ngoc Linh Nature Reserve showed the best larvicidal activities with 24-h LC50 of 16.8 and 18.0 µg/mL, respectively. The essential oil compositions and larvicidal activities of P. cambodiana, Premna flavescens, Premna maclurei, P. mekongensis, and Premna puberula are reported for the first time. Although the larvicidal activities of Premna leaf essential oils are promising, the essential oil yields are relatively low (0.10–0.25%). Keywords: Lamiaceae; Aedes aegypti; sesquiterpene hydrocarbons 1. Introduction Mosquito-borne infectious diseases have been a persistent problem in Vietnam. Dengue fever and dengue hemorrhagic fever are especially problematic and chikungunya fever is an emerging threat in the country [1,2]. Aedes aegypti (L.) (Diptera: Culicidae), the yellow fever mosquito, and Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito, are important vectors of several viral pathogens, including dengue fever virus [3], yellow fever virus [4], chikungunya fever virus [5], and possibly Zika virus [6]. Culex quinquefasciatus Say (Diptera: Culicidae), the southern house mosquito, is a vector of Plants 2020, 9, 1130; doi:10.3390/plants9091130 www.mdpi.com/journal/plants Plants 2020, 9, 1130 2 of 16 lymphatic filariasis [7] as well as several arboviruses such as West Nile virus and St. Louis encephalitis virus [8], and possibly Zika virus [9]. Insecticide resistance has been emerging in many insect disease vectors, including mosquitoes [10–14]. Furthermore, the environmental impacts of synthetic insecticides have been felt for many years [15,16]. It has been reported that insecticide use has detrimental effects on non-target organisms, for example imidacloprid on honey bee (Apis mellifera) [17], damselfly (Ischnura senegalensis) [18], fathead minnow (Pimephales promelas), or the amphipod (Hyalella azteca) [19]. Thus, there is a need for new and complementary methods for controlling insect vectors, and essential oils have shown promise as renewable and environmentally-safe alternatives to the use of synthetic insecticides [20–25]. The Lamiaceae has been an important family in terms of biologically active essential oils. Essential oils from members of this family have demonstrated potential as natural insect pest control agents [24,26–32]. The genus Premna L. was formerly included in the family Verbenaceae, but has been reassigned to the Lamiaceae [33]. The genus is distributed in tropical regions of the Old World, from Africa, eastward through China, Southeast Asia and Malesia, to Australia and islands in the Pacific [34]. The number of species has been estimated to be as few as 50, or as many as 200 [34]. The ethnopharmacology, pharmacognosy, and phytochemistry of the genus have been reviewed [33,35–37]. As part of our ongoing efforts in identifying readily-available essential oils for mosquito control, we have examined the leaf essential oils of eight species of Premna (Table 1) found growing wild in central Vietnam for larvicidal activity against Aedes aegyptae, Aedes albopictus, and Culex quinquefasciatus. Several of these Premna species have been used traditionally in Vietnam (Table 1). Table 1. Premna species examined in this study. Premna Species Native Range Ethnobotanical Use in Vietnam Premna cambodiana Dop (Vietnamese name Cách cam bốt) Laos, Cambodia and Vietnam (Kon Tum, Gia Lai, and Ðắk Nông provinces) [38–40]. Used to treat spermatorrhea and gynecological diseases [40]. Premna chevalieri Dop (syn. Premna acuminatissima Merr.) (Vietnamese name Cách vàng) Thailand, Laos, Vietnam, China (Hainan, Yunnan) [41]. In Vietnam, the plant has been recorded in Thái Nguyên, Phú Thọ, Bắc Giang, Hà Nội, Hòa Bình, Ninh Bình, Nghệ An, Hà Tĩnh, and Quảng Nam provinces [39,40]. The plant is used to treat polio, jaundice [40]. Premna corymbosa Rottler & Willd. (syn. Premna serratifolia L., Cornutia corymbosa Burm. f., Premna integrifolia L., Gumira corymbosa (Rottler & Willd.) Kuntze) (Vietnamese name Vọng cách, Cách biển) Ranges from Madagascar, through tropical and subtropical Asia, to Australia and Pacific islands [38]. In Vietnam, P. corymbosa has been found in Quảng Ninh, Hà Nội, Hải Phòng, Hà Nam, Ninh Bình, Thanh Hóa, Thừa Thiên Huế, Ðà Nẵng, Quảng Nam, Khánh Hòa, Kon Tum, Ðắk Nông, Ðồng Nai, Hồ Chí Minh, Bà Rịa-Vũng Tàu, Long An, and Kiên Giang provinces [39,40]. The plant used to treat skin diseases. Additionally, the leaves are used as culinary additives [40]. Premna flavescens Buch.-Ham. ex C.B. Clarke (syn. Premna lucidula Miq.) Southern China (Guangdong, Guangxi, and southern Yunnan), India, Indonesia, Malaysia, and Vietnam [41]. In Vietnam, P. flavescens has been recorded in Vĩnh Phúc, Nghệ An, Quảng Nam, Kon Tum, Gia Lai, Ðắk Nông, and Ðồng Nai provinces [39,40]. A species commonly grown in Vietnam; a decoction of the leaves is taken daily as a tonic [40]. Premna maclurei Merr. (Vietnamese name Cách maclura) China (Hainan) [41] as well as the provinces of Nghệ An and Quảng Nam, Vietnam [39,40]. Premna mekongensis W.W. Sm. (Vietnamese name Cách mê công) China (northwestern and western Yunnan province) [41] and in Vietnam (Hà Giang and Quảng Nam Provinces) [39,40]. Premna puberula Pamp. (syn. Premna martini H.Lév.) (Vietnamese name Cách lún phún) China (Fujian, Gansu, Guangdong, Guangxi, Guizhou, Hubei, Hunan, southern Shanxi, Sichuan, and Yunnan) [41] as well as Vietnam (Hà Giang, Bắc Giang, and Nghệ An) [39,40]. Used in traditional medicine [40]. Premna tomentosa Willd. (syn. Premna cordata Blanco) (Vietnamese name Cách lông tơ) Ranges from China (Guangdong), through tropical Asia, to North Queensland, Australia [38]. In Vietnam, the plant has been recorded in Nghệ An province and South Vietnam [39,40]. Leaves, roots as medicine [40]. A perusal of the literature has revealed no previous phytochemical reports on P. cambodiana, P. flavescens, P. maclurei, P. mekongensis, or P. puberula. 2. Results and Discussion 2.1. Plant Collection and Essential Oils The leaves of eight species of Premna were collected from several sites in Vietnam. The collection sites, voucher numbers, and essential oil yields are summarized in Table 2. Plants 2020, 9, 1130 3 of 16 Table 2. Collection details and yields for Premna leaf essential oils from central Vietnam. Premna Species Voucher Numbers Essential Oil Yield (% v/w) Premna cambodiana Chu Mom Ray National Park 14◦ 25′ 33.5” N, 107◦ 43′ 15.6” E, 672 m elevation DND 88 0.14 Premna chevalieri Tay Giang District, Quang Nam Province 15◦ 49′ 59” N 107◦ 21′ 10” E, 962 m elevation DND 101 0.10 Nậm Giải Commune, Quế Phong district, Pu Hoat Nature Reserve, Nghe An province 19◦ 41′ 40” N, 104◦ 49′ 29” E, 670 m elevation DND 788 0.22 Son Tra Peninsula, Da Nang province 16◦ 05′ 57” N 108◦ 15′ 59” E, 6 m elevation DND 49 0.25 Nậm Giải Commune, Quế Phong district, Pu Hoat Nature Reserve, Nghe An province 19◦ 41′ 40” N, 104◦ 49′ 29” E, 670 m elevation DND 787 0.11 Ðồng Văn Commune, Quế Phong District, Pu Hoat Nature Reserve, Nghe An province 19◦ 50′ 45” N, 105◦ 06′ 09” E, 511 m elevation DND 711 0.12 Nậm Giải Commune, Quế Phong district, Pu Hoat Nature Reserve, Nghe An province 19◦ 41′ 40” N, 104◦ 49′ 29” E, 670 m elevation DND 747 0.12 Ngoc Linh Nature Reserve, Quang Nam Province 15◦ 50′ 16.0” N, 107◦ 22′ 54.7” E, 1341 m elevation DND 102 0.19 Chu Mom Ray National Park 14◦ 25′ 33.5” N, 107◦ 43′ 15.6” E, 672 elevation DND 84 0.21 Premna puberula Ðồng Văn Commune, Quế Phong District, Pu Hoat Nature Reserve, Nghe An province 19◦ 50′ 45” N, 105◦ 06′ 09” E, 511 m elevation DND 710 0.11 Premna tomentosa Nghia Dan District, Nghe An province 19◦ 20′ 23” N 105◦ 25′ 18” E, 49 elevation DND 23 0.12 Premna corymbosa Premna flavescens Premna maclurei Premna mekongensis Collection Site 2.2. Essential Oil Compositions The Premna leaf essential oils were analyzed by gas chromatography–mass spectrometry and the chemical compositions are summarized in Table 3. 2.2.1. Premna cambodiana A total of 72 compounds were tentatively identified in the leaf essential oil of P. cambodiana, accounting for 97.4% of the total composition (Table 3). Sesquiterpene hydrocarbons dominated P. cambodiana leaf essential oil with α-copaene (23.3%), α-gurjunene (11.3%), (E)-caryophyllene (12.8%), and δ-cadinene (5.5%) as the major sesquiterpene components. There have been no previous phytochemical investigations on P. cambodiana reported in the literature; this is the first report on its essential oil composition. 2.2.2. Premna chevalieri Eighty-five components (99.8% of the composition) were tentatively identified in P. chevalieri essential oil. The major components in the leaf essential oil of P. chevalieri were the sesquiterpenes (E)-caryophyllene (31.5%) and α-humulene (7.5%) and the monoterpenes α-pinene (12.2%) and β-pinene (16.8%) (Table 3). There have been no previous phytochemical investigations on P. chevalieri reported in the literature; this is the first report on the leaf essential oil composition of this plant. 2.2.3. Premna corymbosa (syn. P. integrifolia, P. serratifolia) Leaves of P. corymbosa were collected from two different sites (i.e., Nậm Giải Commune, Quế Phong district, Pu Hoat Nature Reserve, Nghe An province, and Son Tra Peninsula, Da Nang province). Although the two essential oil compositions are qualitatively similar, there are notable quantitative differences (Table 3). The sample from Nghe An province was rich in oxygenated sesquiterpenoids, e.g., spathulenol (17.3%) and caryophyllene oxide (16.8%), while the sample from Da Nang was dominated by sesquiterpene hydrocarbons, including allo-aromadendrene (39.7%), (E)-caryophyllene (13.3%), and α-copaene (8.1%). Plants 2020, 9, 1130 4 of 16 Table 3. Chemical compositions of leaf essential oils of Premna species from central Vietnam. RIcalc 922 925 931 947 949 952 971 976 978 978 983 987 997 1001 1006 1008 1017 1023 1027 1029 1031 1034 1045 1057 1085 1098 1102 1103 1107 1111 1125 1136 1139 1143 1146 1161 1170 1179 1182 1182 1184 1185 1189 1189 1192 1193 1200 1204 1205 1206 1227 1247 1248 1251 a RIdb b 923 927 933 948 953 953 971 978 978 986 986 991 999 1004 1007 1009 1018 1025 1030 1031 1032 1034 1045 1057 1086 1101 1104 1104 1108 1113 1126 1138 1141 1145 1153 1164 1173 1180 1187 1188 1187 1188 1193 1192 1196 1195 1202 1208 1208 1207 1229 1257 1250 1252 Compound c P. cambodiana P. chevalier P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Nậm Giải) P. flavescens (Ðồng Văn) P. maclurei P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula P. tomentosa Tricyclene α-Thujene α-Pinene α-Fenchene Camphene Thuja-2,4(10)-diene Sabinene β-Pinene 1-Octen-3-ol 6-Methylhept-5-en-2-one 3-Octanone Myrcene 3-Octanol p-Mentha-1(7),8-diene α-Phellandrene δ-3-Carene α-Terpinene p-Cymene Limonene β-Phellandrene 1,8-Cineole (Z)-β-Ocimene (E)-β-Ocimene γ-Terpinene Terpinolene Linalool 2-Methylbutyl 2-methylbutanoate Nonanal p-Mentha-2,8-dien-1-ol (3E)-4,8-Dimethyl-1,3,7-nonatriene α-Campholenal Benzeneacetonitrile trans-Pinocarveol trans-Verbenol p-Vinylanisole Pinocarvone Borneol Terpinen-4-ol (3Z)-Hexenyl butyrate Naphthalene Cryptone p-Cymen-8-ol Butyl hexanoate Methyl salicylate Myrtenal α-Terpineol cis-Sabinol Decanal Verbenone (3E)-Octenyl acetate Thymol methyl ether p-Anisaldehyde Linalyl acetate Isopentyl hexanoate — — 1.9 — — — — 1.7 — — — — — — — — — tr 0.2 — — — — — — tr — tr — — — — tr — — — — — — — — — — — tr — — tr — — — — — — tr d 0.1 12.2 tr 0.1 tr tr 16.8 — — 0.1 0.2 0.1 — — — tr 0.1 1.0 tr tr 0.1 1.3 tr tr 0.8 — 0.1 — 1.1 — — 0.1 — — tr — — — — — — — 0.1 — 0.1 — — — 0.1 — — — — — 0.1 0.5 — — — 0.8 0.6 — — — — — — — — — 0.5 0.2 — 0.1 — — — — 0.8 — — — — — — — — — — — 0.1 — — — — — — — — — — — — — — — — — — 0.5 — — — tr 0.1 — — — 0.4 — tr 0.2 — — 1.9 1.7 0.6 — — — — — 0.2 — tr — — — — — — — — — — tr — tr — — tr — tr 0.1 — — — — — — — — — 0.2 — tr — — 0.1 — — — — — — — — — 0.1 0.1 tr 0.1 — — — — 0.1 — tr — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.1 — tr — tr 0.1 0.3 — — 0.3 — — — tr — tr 0.1 — tr — tr — — 0.4 — — — — — tr — — — — — — 0.1 0.1 — — — — — — — — — — — — — — — — 0.4 — — — tr 0.2 — tr — tr — — — — — tr 0.1 — — — — — — 0.2 — tr — tr — — — — 0.1 — — tr — — — — — tr tr tr — — — — — tr — — — — 66.9 — 0.2 0.1 0.9 0.5 0.2 — — 1.6 — — 0.3 — — 0.3 1.1 0.4 0.9 0.1 0.1 0.1 tr 1.4 — 0.1 — — — — — 0.1 — — — — — — — — — — — 0.1 — — — — — — — — tr tr 1.5 — 1.5 — 0.1 0.7 — — — 3.1 — — 1.6 — tr 0.6 1.7 1.3 tr tr 0.2 tr 0.1 0.3 tr — — — — — — — — — tr tr — — tr — 0.6 — — tr tr — — — 0.1 — tr 0.3 — — 1.3 — — — — 0.1 — — — — — — — — — 0.1 0.3 — — — — — — 0.1 — — 0.4 — 0.1 0.1 0.2 0.3 — — — — — — — 0.1 — — 0.1 — — — 0.1 — — — — — — 0.1 3.0 — tr — 2.0 0.7 tr — — 0.1 tr — tr — 0.1 0.4 0.2 tr tr tr 0.1 0.2 0.1 0.2 — — — tr — — — — 0.2 — — 0.3 — — — — — tr — tr — — — — — — — — Plants 2020, 9, 1130 5 of 16 Table 3. Cont. RIcalc 1282 1286 1291 1297 1300 1307 1312 1313 1317 1329 1332 1345 1345 1356 1362 1365 1367 1368 1374 1374 1376 1378 1379 1381 1384 1386 1387 1402 1402 1402 1405 1412 1419 1427 1427 1428 1429 1430 1432 1433 1437 1443 1444 1445 1448 1448 1450 1450 1451 1454 1458 1458 1461 1462 a RIdb b 1282 1287 1294 1299 1300 1306 1315 1318 1328 1335 1349 1349 1361 1367 1372 1371 1367 1378 1375 1379 1382 1383 1382 1390 1392 1390 1405 1403 1406 1406 1415 1417 1427 1430 1433 1430 1437 1436 1438 1438 1444 1445 1446 1453 1445 1447 1455 1452 1454 1457 1463 1459 1466 Compound c Bornyl acetate Dihydroedulan IA Dihydroedulan IIA Theaspirane A Tridecane Isoascaridole Unidentified e Theaspirane B 3-Hydroxycineole Bicycloelemene δ-Elemene α-Cubebene α-Terpinyl acetate Neryl acetate Decanoic acid Isoledene α-Ylangene Cyclosativene Geranyl acetate α-Copaene (E)-β-Damascenone (3Z)-Hexenyl hexanoate cis-β-Elemene β-Bourbonene Hexyl hexanoate β-Cubebene trans-β-Elemene (Z)-Caryophyllene 9,10-Dehydroisolongifolene Cyperene α-Gurjunene β-Maaliene (E)-Caryophyllene γ-Elemene γ-Maaliene β-Copaene trans-α-Bergamotene β-Gurjunene (= Calarene) α-Guaiene α-Maaliene Aromadendrene Guaia-6,9-diene Selina-5,11-diene Neryl acetone trans-Muurola-3,5-diene β-Barbatene iso-Germacrene D Valerena-4,7(11)-diene (E)-β-Farnesene α-Humulene allo-Aromadendrene cis-Cadina-1(6),4-diene Rotundene cis-Muurola-4(14),5-diene P. cambodiana P. chevalier P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Nậm Giải) P. flavescens (Ðồng Văn) P. maclurei P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula P. tomentosa — — — — — — — — — — 0.2 0.7 — — — — 0.3 tr 0.1 23.3 — — 0.2 tr — 1.1 3.5 — — — 11.3 0.2 12.8 0.7 — — — — 0.1 0.1 0.1 0.1 — — — — — 0.1 — 3.7 1.6 0.1 — — tr tr 0.1 tr — — — tr — — — 0.1 — — tr — — — — 0.9 0.1 tr tr tr — 0.2 0.3 0.1 — — — — 31.5 tr — tr tr — 0.1 — tr — 0.1 tr — tr — — 0.1 7.5 0.1 — — — — — — — — — — — — — — 0.5 — — — — 0.6 — — 6.8 — — — — — 1.8 1.5 — — — 0.6 — 6.9 — — 0.2 — — — — 0.5 — — — — — — — — 2.6 7.7 — — — — — — — — — — — — 0.1 — 0.3 — — — — 0.1 — — 8.1 — — tr tr — 0.9 1.0 — — — 0.1 — 13.3 — — 0.2 0.1 — 4.6 — 0.1 — — — — — — 0.4 — 3.7 39.7 — — — tr 0.8 — — tr — — — — 0.6 0.1 — — — — — — tr — 0.1 — — 0.5 — — — 9.9 0.2 — — 0.1 — 41.0 — — — 0.7 — 0.5 — 0.4 1.1 — — — — — — 0.1 2.4 0.1 — — — — — tr — — — 1.2 — — 0.1 tr 0.2 — — — — — 0.1 — 2.9 — — 0.5 0.1 — 0.6 8.7 — — 1.1 19.6 0.4 11.8 — — 0.1 — 0.1 6.1 — 0.1 tr — — — — — 0.3 — 3.1 3.0 — 0.1 — — tr 0.4 — — — — — — — 0.2 0.5 tr — — tr 0.1 — — 2.6 0.1 — 0.1 0.1 — 1.6 1.8 — — — — — 30.7 — — 0.4 — — 0.4 — 0.3 — — — — — — — — 5.3 0.2 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.5 — — — — — 14.7 — — — — — 0.5 — — — — — — — — — — 2.5 — — — — 3.9 0.2 — — — — 3.4 — tr 1.1 0.6 0.4 1.5 tr — — 0.3 0.1 — 1.6 — — 0.1 — 2.1 0.3 1.3 — — 0.1 0.3 — 3.9 — 0.1 1.2 — — — 0.1 1.0 — 0.1 — 0.1 — — 0.1 — 2.0 1.0 — — 0.2 — — — — — — — — — — — 0.5 — — — — — — — 5.3 — — — — — 1.5 1.9 — 0.8 — 1.3 — 0.6 — — — — — 0.4 — — — — — — — — — — 0.4 4.1 — — — — tr 0.1 — — tr — — — 0.2 0.2 0.2 — — — — 0.1 — — 3.1 tr — 0.3 tr — 0.7 5.0 — — — 5.2 2.8 22.0 2.4 0.3 — — — 0.3 — 3.4 0.1 — — — — tr 0.1 — 3.9 1.0 — — 0.1 Plants 2020, 9, 1130 6 of 16 Table 3. Cont. RIcalc 1469 1471 1472 1473 1474 1475 1476 1476 1479 1479 1480 1484 1486 1487 1491 1491 1491 1491 1493 1493 1495 1495 1496 1497 1499 1502 1503 1508 1511 1513 1516 1518 1518 1519 1522 1532 1534 1535 1536 1538 1539 1539 1546 1547 1550 1557 1559 1560 1563 1565 1568 1569 1572 1575 a RIdb b 1473 1472 1478 1476 1475 1477 1481 1479 1480 1482 1483 1491 1488 1487 1492 1492 1491 1490 1493 1498 1497 1497 1497 1502 1505 1503 1508 1511 1512 1515 1518 1518 1521 1519 1521 1538 1536 1529 1540 1540 1546 1544 1546 1551 1549 1557 1560 1560 1558 1566 1568 1571 1568 1578 Compound c 4,5-di-epi-Aristolochene trans-Cadina-1(6),4-diene γ-Muurolene γ-Gurjunene Selina-4,11-diene β-Chamigrene (E)-β-Ionone α-Amorphene Germacrene D γ-Himachalene trans-β-Bergamotene Eremophilene δ-Selinene β-Selinene trans-Muurola-4(14),5-diene Valencene Viridiflorene (= Ledene) γ-Amorphene Curzerene epi-Cubebol α-Muurolene α-Selinene Bicyclogermacrene ε-Amorphene α-Bulnesene (E,E)-α-Farnesene β-Bisabolene Germacrene A γ-Cadinene Cubebol δ-Cadinene 7-epi-α-Selinene α-Panasinsen trans-Calamenene Zonarene α-Cadinene trans-Cadina-1,4-diene cis-Calamenene (E)-α-Bisabolene Selina-4(15),7(11)-diene cis-Sesquisabinene hydrate α-Calacorene α-Elemol (Z)-Caryophyllene oxide cis-Muurol-5-en-4β-ol Germacrene B (E)-Nerolidol β-Calacorene 1-Tetradecanol 1,5-Epoxysalvial-4(14)-ene Palustrol Cedroxyde Dendrolasin Furopelargone B P. cambodiana P. chevalier P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Nậm Giải) P. flavescens (Ðồng Văn) P. maclurei P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula P. tomentosa — — 0.6 0.8 — — — 0.3 2.2 0.1 — — — 1.4 — — 0.1 0.2 — — 0.5 1.0 — — — — — — 0.4 0.1 5.5 — — 0.5 — 0.2 — — — — — 0.9 — 0.4 — 0.6 — 0.4 — — 0.3 — — — 0.1 — — — 2.0 — tr — 0.3 — — — — 1.2 — — — — — — — 1.5 — 0.1 0.1 3.4 — — tr — 0.3 — — 0.1 — — — — — — — — 0.1 — — — 0.2 — 0.3 — — — 2.0 — 1.0 — 0.4 — — — — — 0.4 — — — — 4.0 — 0.2 — — — — 0.2 2.7 — — — — — — 0.2 0.6 0.4 0.2 — 0.4 — — — — — — — 0.6 — — — — — — — — — — — — — — 0.4 — — — — 0.2 3.0 — — — — 0.2 — — 0.3 — — — 0.3 — 1.1 — 4.1 — 0.4 — 0.3 — 2.0 — — 0.3 — 0.1 — — — — — 0.1 — — — — — — — — — 0.1 — — — — 0.1 — 0.2 — — — 0.7 — 0.2 0.1 — 1.3 — — 0.1 — — — 0.2 — 7.8 — 0.2 0.3 0.5 — 0.1 — 0.1 — — — — — — — 0.3 — — — — — — 0.1 0.3 — — — — — — 0.2 0.1 — — 0.4 0.1 1.3 — — 3.8 — — — — 9.7 — — 1.8 — — — — 8.7 — — 5.4 — — 0.2 0.2 — 0.9 — 0.5 — — — — — — — — — — 0.1 — tr — — — — 0.6 — — — — — 1.2 — — 0.1 — — 3.0 — — — — 0.3 — 0.2 — — — — 0.4 — 1.9 — 0.3 — — — 0.4 0.2 8.4 — — 0.3 — 0.1 — 0.2 — — — 0.6 — 0.6 — — — 0.7 — — 6.8 — — — — — — — 0.5 — — — 0.1 — — — — 0.4 — — — — — — — 0.4 — — 0.1 0.1 — — — — — — — — — — — — — — — — — — — — 0.1 — — — — — — — — 0.4 1.7 0.3 — — — 0.6 5.6 — — — 0.2 0.7 1.7 — — — 1.2 — — — 11.9 — — — — — 1.1 0.3 3.2 — — 1.0 0.2 0.3 0.2 — — — — 0.2 0.2 — 0.1 0.5 7.5 — — — 1.7 — — — — — — — — — — — — — — — — 0.7 — — — — — 0.9 — — — — 0.6 — — — — 0.6 — — — — — — — — — — — 3.4 — 1.5 — — — 0.8 — 1.1 1.9 0.9 — — — — 0.4 0.3 — — — — 11.4 — — — — 4.3 — — — 0.3 — — 0.2 5.5 — — 0.1 1.0 — 0.1 0.2 tr 1.8 — — 0.1 — — 0.1 — — 0.1 0.1 — — — — 3.5 — — — — — — — — Plants 2020, 9, 1130 7 of 16 Table 3. Cont. RIcalc 1575 1580 1584 1590 1593 1595 1598 1602 1607 1612 1614 1622 1624 1629 1633 1633 1637 1641 1642 1642 1643 1643 1647 1653 1654 1654 1657 1659 1662 1663 1664 1668 1669 1671 1674 1682 1685 1692 1699 1700 1727 1744 1746 1748 1749 1758 1759 1761 1806 1830 1837 1859 1873 1886 a RIdb b 1576 1587 1590 1596 1594 1593 1600 1605 1613 1615 1616 1632 1627 1629 1635 1634 1641 1643 1643 1643 1643 1645 1649 1652 1663 1653 1658 1664 1670 1664 1662 1677 1668 1681 1677 1683 1688 1701 1704 1708 1729 1746 1748 1763 1757 1768 1813 1836 1841 1860 1879 1884 Compound c P. cambodiana P. chevalier P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Nậm Giải) P. flavescens (Ðồng Văn) P. maclurei P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula P. tomentosa Spathulenol (E)-Caryophyllene oxide Globulol Cubeban-11-ol Viridiflorol Guaiol Curzerenone Ledol Humulene epoxide II Rosifoliol 1,10-di-epi-Cubenol Muurola-4,10(14)-dien-1β-ol 1-epi-Cubenol iso-Spathulenol Caryophylla-4(12),8(13)-dien-5β-ol cis-Cadin-4-en-7-ol allo-Aromadendrene epoxide τ-Cadinol Cubenol Hedycariol α-Muurolol (= δ-Cadinol) τ-Murrolol β-Eudesmol α-Cadinol cis-Calamenen-10-ol Pogostol Selin-11-en-4α-ol ar-Turmerone trans-Calamenen-10-ol Bulnesol 9-Methoxycalamenene Cadalene 14-Hydroxy-9-epi-(E)-caryophyllene Mustakone Apiole Germacra-4(15),5,10(14)-trien-1α-ol α-Bisabolol 10-nor-Calamenen-10-one cis-Thujopsenol δ-Dodecalactone Zerumbone α-Cyperone Geranyl hexanoate β-Costol Cyclocolorenone Squamulosone Unidentified f Unidentified g Nootkatone Neophytadiene Phytone Platambin 4-Phytadiene Corymbolone 0.8 4.3 0.5 — — — — 0.2 0.8 — — 0.1 0.4 — 0.1 0.2 0.6 — — — 0.3 0.2 — 0.6 0.4 — 0.3 — 0.6 — — 0.3 — — — — — — — — — — — — 1.1 — — — — — 0.2 — — — 0.6 5.3 0.1 — — 1.2 — — 1.0 1.1 — — — — — — — — — 0.1 — — — 0.3 — 0.3 — — — 0.4 — — — — — — 0.1 — — — — — — — — — — — — — — — — — 17.3 16.8 1.7 — 1.8 — — 0.3 3.4 — — — 0.4 1.0 — — — 0.4 — — 0.3 0.4 — 1.9 — 0.3 0.9 — — — 0.7 — 0.5 — 1.6 1.0 — — — — — 0.6 — — — — — — 0.5 — — — — — 1.4 3.0 0.3 0.1 0.3 — — 0.3 0.4 — — — 0.2 0.1 — — 0.3 0.2 — — 0.1 0.2 — 0.3 — — — — — — tr — — — — — — — 0.1 — — — — 0.4 — — — — 0.3 — — — — — 1.8 1.7 0.3 — 0.2 — — — 0.1 — — — — 0.4 0.2 — — — — — 0.1 — — 0.3 — — 0.4 — — — — — — — 0.6 — — — — — — — — — — — 14.7 2.0 — 0.1 0.1 — 0.1 — 1.0 1.3 0.2 — 0.1 — — 0.3 0.2 — — — — — — — 0.1 — — — — — — 0.1 — — 0.2 — — — — — — — — — — — — 0.1 0.4 — — — 0.1 — — — — — — — — — — 12.3 0.3 — — — — — 1.0 — — 0.2 0.1 0.5 0.6 — — — — — — — 0.1 0.3 0.9 0.8 — — 0.7 — — — — 3.6 — — — 0.4 — — — — — — — — — — — — 0.3 0.8 — — — 0.8 — — — — — — 0.1 — — — — — 0.1 — — — — — — — — — — 1.4 — 0.1 — — — — — — — — — — — — — — — — — — — — — — — — — — 2.8 0.3 1.2 — 5.6 — 0.3 1.2 — 0.1 0.2 — 1.0 — — 0.9 0.1 — 1.5 — 0.4 — — 1.6 — — 0.3 — — — — — — — — — — — — — — — 0.2 — — — — — — — — — — — 7.7 21.2 2.0 — 0.7 — — 0.6 4.7 — — — — — — — — — — — — — — 0.6 0.8 1.2 — — 1.0 — — — — 2.6 — — — — 1.8 — 1.1 — — — 2.0 0.6 — — 1.4 — 1.9 — — 1.1 0.8 1.1 0.7 — — — — 6.1 0.1 — — — — 0.1 — — 0.2 — — — 0.1 0.1 — 0.3 — 0.4 — — — — — — — — — — — — — — — — — — — — — — — — — 0.1 — — Plants 2020, 9, 1130 8 of 16 Table 3. Cont. RIcalc 1939 1958 1983 2019 2102 2131 a a RIdb b 1947 1958 1995 2022 2102 2138 P. cambodiana P. chevalier P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Nậm Giải) P. flavescens (Ðồng Văn) P. maclurei P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula P. tomentosa iso-Phytol (Z,Z)-Geranyl linalool Manool oxide (E,E)-Geranyl linalool (E)-Phytol Palmitaldehyde, diallyl acetal — — — — 4.3 0.6 — 0.1 — 0.1 2.2 — — — — — — — — — — — 0.3 — 0.1 — — — 4.9 — — — — — — — — — 0.4 — 2.4 0.4 — — — — 2.2 — — — — — — — — — — — — — — — — — 0.7 — Monoterpene hydrocarbons Oxygenated monoterpenoids Sesquiterpene hydrocarbons Oxygenated sesquiterpenoids Diterpenoids Others Total Identified 3.8 0.1 76.1 12.3 4.5 0.6 97.4 31.8 1.0 49.9 12.6 2.4 2.0 99.8 2.7 1.0 40.2 50.6 0.0 1.6 96.1 5.3 0.2 85.4 8.2 0.3 tr 99.6 0.5 0.2 69.9 6.0 5.2 1.4 83.3 0.6 0.4 92.2 4.8 0.0 0.6 98.6 0.7 0.2 62.5 30.1 3.1 1.1 97.7 72.5 2.5 19.6 2.5 2.2 0.3 99.6 12.5 6.0 46.9 27.4 0.0 3.2 96.0 1.8 1.4 22.4 58.2 1.9 0.1 85.7 7.0 0.5 81.0 10.2 0.7 0.3 99.8 Compound c RIcalc = Retention indices determined with respect to a homologous series of n-alkanes on a ZB-5ms column. b RIdb = Retention indices from the databases [42–45]. c Tentative identification based on RI and MS fragmentation agreement. d tr = Trace (<0.05%). e MS: 162(42%), 147(54%), 133(24%), 120(24%), 119(36%), 105(100%), 91(79%), 79(37%), 77(26%), 65(14%), 55(23%), and 41(21%). f MS: 204(28%), 147(5%), 134(12%), 133(100%), 120(45%), 107(41%), 105(16%), 91(8%), 77(11%), 55(7%), and 41(6%). g MS: 206(10%), 107(100%), 77(6%), and 41(3%). Concentrations of major components are highlighted in bold. Plants 2020, 9, 1130 9 of 16 The major components of the leaf essential oil of P. corymbosa (reported as P. integrifolia) from Bangladesh were phytol (27.3%), α-humulene (14.2%), spathulenol (12.1%), 1-octen-3-ol (8.2%), eugenol (6.7%), and phenylethyl alcohol (5.8%) [46]. Neither 1-octen-3-ol, phenylethyl alcohol, nor eugenol were detected in the samples from Vietnam. Likewise, neither α-copaene nor allo-aromadendrene were reported from the Bangladeshi sample. In contrast, P. corymbosa leaf essential oil (reported as P. serratifolia) displayed a very simple composition of eugenol (47.9%), eugenyl acetate (9.1%), massoialactone (32.9%), and a compound identified as cis-2-oxabicyclo[4.4.0]decane (12.4%) (likely incorrect based on relative retention times) [47]. Thus, there is wide variation in the essential oil compositions of this plant, which suggests different chemotypes are possible or these three plants represent different species. 2.2.4. Premna flavescens Leaves of P. flavescens were collected from two different sites (i.e., Nậm Giải Commune, Quế Phong district, Pu Hoat Nature Reserve, Nghe An province, and Ðồng Văn Commune, Quế Phong District, Pu Hoat Nature Reserve, Nghe An province). The leaf essential oils from the two sites showed notable differences in compositions (Table 3). (E)-Caryophyllene was abundant in both samples (41.0% and 11.8% in the Nậm Giải and Ðồng Văn samples, respectively), as was trans-β-elemene (9.9% and 8.7%, respectively). The sample from Ðồng Văn was rich in α-gurjunene (19.6%), but only a minor component (0.1%) in the sample from Nậm Giải. Likewise, α-guaiene and α-bulnesene were relatively abundant in the Ðồng Văn sample (6.1% and 5.4%), but minor in the sample from Nậm Giải (0.5% and 0.2%, respectively). Interestingly, bicyclogermacrene (7.8%) and an unidentified component (RI 1759, 14.7%) in the sample from Nậm Giải, were not detected in the sample from Ðồng Văn. Conversely, α-selinene, 8.7% in the sample from Ðồng Văn, was not detected in the sample from Nậm Giải. As far as we are aware, there have been no previous reports on the essential oil chemistry of P. flavescens. 2.2.5. Premna maclurei The leaf essential oil composition of P. maclurei is shown in Table 3. The essential oil was dominated by sesquiterpene hydrocarbons (62.5%) and oxygenated sesquiterpenoids (30.1%) with (E)-caryophyllene (30.7%), α-humulene (5.3%), δ-cadinene (8.4%), spathulenol (6.8%), and caryophyllene oxide (12.3%) as the major components. To our knowledge, there have been no previous reports on the essential oil composition of P. maclurei. 2.2.6. Premna mekongensis Essential oils were obtained from leaves of P. mekongensis from two different locations, Ngoc Linh Nature Reserve in Quang Nam Province, and Chu Mom Ray National Park. The leaf essential oil compositions are listed in Table 3. The two samples showed very different compositions. The Ngoc Linh sample was dominated by α-pinene (66.9%) and (E)-caryophyllene (14.7%). The leaf essential oil from Chu Mom Ray, on the other hand, had relatively low concentrations of α-pinene (1.5%) and (E)-caryophyllene (3.9%). In addition, the Chu Mom Ray essential oil was much more complex with 95 identified components compared to only 37 in the Ngoc Linh sample. The high concentration of α-pinene in P. mekongensis leaf essential oil from Ngoc Linh was unexpected and uncharacteristic of Premna leaf essential oils, which are generally low in monoterpene hydrocarbon concentrations (see below). To our knowledge, there have been no previous studies on the essential oil composition of P. mekongensis. 2.2.7. Premna puberula The chemical composition of the leaf essential oil of P. puberula is shown in Table 3. The major chemical classes present in the essential oil were sesquiterpene hydrocarbons (22.4%), with α-copaene (5.3%) and allo-aromadendrene (4.1%) as major components, and oxygenated sesquiterpenoids (58.2%), Plants 2020, 9, 1130 10 of 16 dominated by (E)-caryophyllene oxide (21.2%) along with spathulenol (7.7%) and humulene epoxide II (4.7%). There have been no previous reports on the essential oil of P. puberula. 2.2.8. Premna tomentosa The leaf essential oil composition of P. tomentosa is shown in Table 3. A total of 82 compounds were tentatively identified in the essential oil accounting for 99.8% of the composition, which was dominated by sesquiterpene hydrocarbons, especially (E)-caryophyllene (22.0%) and germacrene D (11.4%). The only previous examination of the essential oil of P. tomentosa is a relatively old work by Narayan and Muthana in 1953 [48]. These workers identified limonene (57.8%), (E)-caryophyllene (17.2%), an unidentified cadinane sesquiterpene (7.8%), an unidentified sesquiterpene alcohol (5.6%), and an unidentified diterpene hydrocarbon (5.5%) in the leaf essential oil from southern India. 2.2.9. Species Composition Comparison Analogous to most of the Premna essential oil compositions observed in this study, leaf essential oils of other Premna species have shown compositions dominated by sesquiterpene hydrocarbons, e.g., Premna coriacea (55.2%) [49], Premna latifolia (76.4%) [50], Premna quadrifolia (65.5%) [51], and Premna odorata (62.3%) [52]. On the other hand, other Premna species are particularly rich in low molecular weight alcohols such as 1-octen-3-ol in Premna barbata (37.3%) [53], P. latifolia (35.7%) [54], and Premna angolensis (28.0%) [51]. In contrast, the essential oil of Premna microphylla was dominated by the sesquiterpenoid derivative blumenol C (49.7%) [55]. Compounds common to all eight of the Premna leaf essential oils in this study were α-pinene, β-pinene, p-cymene, limonene, linalool, trans-β-elemene, (E)-caryophyllene, α-humulene, β-selinene, and caryophyllene oxide. These are all relatively common essential oil constituents, and therefore cannot be considered as key compounds defining the genus. Furthermore, leaf essential oils of other Premna species were missing several of these components. The leaf essential oil of P. coriacea from Karnataka, India, was devoid of α-pinene, β-pinene, linalool, and β-selinene [49]. Likewise, the leaf oil from P. microphylla from Zhejiang Province, China, contained no α-pinene, β-pinene, linalool, (E)-caryophyllene, or α-humulene [55]. Premna integrifolia leaf essential oil from Bangladesh did not show p-cymene, limonene, linalool, β-elemene, or β-selinene [46]; P. odorata leaf oil from Giza, Egypt, showed no α-pinene, β-pinene, p-cymene, limonene, β-elemene, or β-selinene [52]; P. angolensis leaf oil from Comé, Benin, contained no β-pinene or caryophyllene oxide, and P. quadrifolia from Comé, Benin, contained no α-pinene, limonene, or linalool [51]. 2.3. Mosquito Larvicidal Activity The Premna leaf essential oils have been screened for mosquito larvicidal activity against Aedes aegypti and, if sufficient mosquito larvae were available, also against Ae. albopictus and Culex quinquefasciatus. The 24-h and 48-h larvicidal activities are shown in Tables 4 and 5, respectively. Considering larvicidal activities against Ae. aegypti, the most active Premna leaf essential oils were P. cambodiana (24-h LC50 = 16.8 µg/mL) and P. mekongensis from Nghe An (24-h LC50 = 16.8 µg/mL). The pronounced larvicidal activity of P. mekongensis (Ngoc Linh) against Ae. aegypti can be attributed to the high concentration of α-pinene. This monoterpene has shown larvicidal activity against Ae. aegypti with LC50 values ranging from 15.4 µg/mL to 79.1 µg/mL [56]. Interestingly, the larvicidal activity of P. mekongensis (Ngoc Linh) against Cx. quinquefasciatus was less (24-h LC50 = 42.7 µg/mL), consistent with the reduced activity of α-pinene against this mosquito larva (LC50 = 95 µg/mL) [57]. Plants 2020, 9, 1130 11 of 16 Table 4. Twenty-four-hour mosquito larvicidal activities of Premna leaf essential oils. Premna Species (Collection Site) LC50 (95% Limits), µg/mL P. cambodiana (Chu Mom Ray) P. chevalieri (Quang Nam) P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Ðồng Văn) P. maclurei (Nghe An) P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula (Nghe An) P. tomentosa (Nghe An) Permethrin (control) 16.79 (14.66–18.68) 31.72 (29.20–34.48) 37.96 (33.16–43.18) 61.78 (57.16–67.71) 64.67 (58.99–71.10) 43.66 (40.67–47.07) 17.98 (14.79–20.71) 41.63 (38.79–44.49) 50.88 (46.25–56.36) 34.21 (31.02–37.67) 0.0094 (0.0082–0.0107) LC90 (95% Limits), µg/mL χ2 p 0.00624 2.54 4.53 5.75 13.23 1.211 4.14 35.0 12.7 0.225 57.6 0.997 0.281 0.104 0.056 0.001 0.546 0.126 0.000 0.002 0.893 0.000 1.55 4.51 12.2 4.64 0.460 0.105 0.007 0.031 6.94 1.68 11.8 12.4 24.1 0.031 0.431 0.003 0.002 0.000 Aedes aegypti 28.02 (25.18–32.82) 46.88 (43.14–52.00) 75.43 (66.72–88.43) 83.01 (75.71–93.93) 106.1 (95.9–120.2) 60.72 (56.03–67.58) 35.81 (31.76–42.26) 55.94 (52.45–60.49) 80.60 (72.74–91.86) 54.36 (49.42–61.35) 0.0211 (0.0185–0.0249) Aedes albopictus P. corymbosa (Da Nang) P. flavescens (Ðồng Văn) P. puberula (Nghe An) Permethrin (control) 45.89 (42.61–49.88) 90.02 (80.92–99.87) 115.9 (108.2–124.1) 0.0024 (0.0021–0.0026) P. chevalieri (Quang Nam) P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula (Nghe An) Permethrin (control) 75.68 (68.51–84.52) 42.66 (38.71–47.43) 33.16 (30.30–36.25) 60.59 (55.77–66.33) 0.0188 (0.0173–0.0206) 64.70 (59.15–73.12) 165.4 (148.9–189.2) 176.7 (165.0–191.8) 0.0042 (0.0038–0.0049) Culex quinquefasciatus 129.8 (115.9–150.0) 69.35 (62.21–79.95) 52.01 (47.55–58.29) 87.68 (80.11–98.09) 0.0294 (0.0270–0.0326) Table 5. Forty-eight-hour mosquito larvicidal activities of Premna leaf essential oils. Premna Species (Collection Site) LC50 (95% Limits), µg/mL LC90 (95% Limits), µg/mL χ2 p 0.00399 4.59 2.98 8.07 2.33 0.922 0.0364 0.130 3.40 0.0878 39.6 0.998 0.101 0.225 0.018 0.312 0.631 0.982 0.937 0.182 0.957 0.000 0.148 9.87 37.2 0.929 0.007 0.000 6.65 0.584 3.55 2.20 0.036 0.747 0.169 0.333 Aedes aegypti P. cambodiana (Chu Mom Ray) P. chevalieri (Quang Nam) P. corymbosa (Nghe An) P. corymbosa (Da Nang) P. flavescens (Ðồng Văn) P. maclurei (Nghe An) P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula (Nghe An) P. tomentosa (Nghe An) Permethrin (control) 13.68 (10.72–15.77) 30.23 (27.75–32.92) 33.59 (28.68–38.65) 60.43 (55.81–66.17) 62.42 (56.58–69.12) 41.63 (38.85–44.63) 17.62 (15.37–19.67) 38.70 (36.18–41.21) 45.71 (41.21–50.97) 31.4 (28.32–34.69) 0.0087 (0.0074–0.0102) 25.62 (22.82–30.59) 45.11 (41.41–50.23) 71.64 (62.98–84.86) 83.54 (76.24–94.13) 105.9 (96.5–119.0) 57.07 (53.07–62.68) 30.00 (26.76–35.65) 49.94 (47.01–53.73) 76.15 (68.30–87.56) 50.80 (46.13–57.36) 0.0204 (0.0181–0.0236) Aedes albopictus P. corymbosa (Da Nang) P. flavescens (Ðồng Văn) P. puberula (Nghe An) 35.13 (31.93–38.74) 74.14 (66.55–81.95) 98.1 (91.0–105.7) P. chevalieri (Quang Nam) P. mekongensis (Ngoc Linh) P. mekongensis (Chu Mom Ray) P. puberula (Nghe An) 52.10 (44.16–60.92) 38.72 (34.62–43.45) 27.02 (23.51–30.51) 41.31 (37.07–46.27) 56.97 (51.54–64.86) 133.2 (121.1–149.9) 151.1 (140.3–165.0) Culex quinquefasciatus 121.1 (104.4–147.9) 68.87 (61.30–80.21) 51.55 (45.96–60.01) 72.04 (64.28–83.52) The major components in P. cambodiana leaf essential oil were the sesquiterpene hydrocarbons α-copaene (23.3%), (E)-caryophyllene (12.8%), and α-gurjunene (11.3%). (E)-Caryophyllene has shown larvicidal activity against Ae. aegypti with reported LC50 values of 38.6 µg/mL [58] and 88.3 µg/mL [59]. As far as we are aware, there have been no reports on the larvicidal activities of either α-copaene or α-gurjunene. However, essential oils rich in α-copaene have shown notable larvicidal activity. For example, the essential oil from the inflorescences of Piper marginatum Jacq. (Piperaceae) (9.4% α-copaene and 13.1% (E)-caryophyllene) showed larvicidal activity against Ae. aegypti with LC50 of 19.9 µg/mL [60]; the ripe peel essential oil of Hymenaea courbaril L. (Fabaceae) (11.1% α-copaene) had an LC50 of 14.8 µg/mL on Ae. aegypti [61]. Note, however, that the leaf essential oil of P. corymbosa from Da Nang was also rich in α-copaene (8.1%) and (E)-caryophyllene (13.3%), but the larvicidal activity against Ae. aegypti was weaker (LC50 = 61.8 µg/mL). Similarly, the leaf essential oil of P. flavescens from Plants 2020, 9, 1130 12 of 16 Ðồng Văn had α-gurjunene (19.6%) and (E)-caryophyllene (11.8%) as major components, but also showed weak larvicidal activity against Ae. aegypti (LC50 = 64.7 µg/mL). The mere presence of the sesquiterpene hydrocarbons α-copaene, α-gurjunene, and (E)-caryophyllene is not sufficient to impart good larvicidal activity; there are likely synergistic effects of these compounds with minor components that account for the activities. 3. Materials and Methods 3.1. Plant Material Leaves of Premna species were collected from several different locations in central Vietnam (Table 1). The plants were identified by Dr. Do Ngoc Dai, and voucher specimens (see Table 2) have been deposited in the plant specimen room, Faculty Agriculture, Forestry, and Fishery, Nghe An, College of Economics. The fresh leaves (2.0 kg each), immediately after collection, were shredded and hydrodistilled for 4 h using a Clevenger type apparatus (Witeg Labortechnik, Wertheim, Germany). Essential oil yields are summarized in Table 2. Essential oils were dried over anhydrous Na2 SO4 and stored in sealed glass vials at 4 ◦ C until analyzed. 3.2. Gas Chromatography–Mass Spectrometry The Premna leaf essential oils were analyzed by gas chromatography–mass spectrometry (GC-MS) as described previously [56]: Shimadzu GCMS-QP2010 Ultra, electron impact (EI) mode (electron energy = 70 eV), scan range = 40–400 atomic mass units, scan rate = 3.0 scans/s; ZB-5ms column (30 m length × 0.25 mm inner diameter × 0.25 µm film thickness); He carrier gas, head pressure of 552 kPa, flow rate of 1.37 mL/min; injector temperature was 250 ◦ C, ion source temperature was 200 ◦ C; GC oven temperature program: 50 ◦ C initial temperature, increased 2 ◦ C/min to 260 ◦ C; 5% solution of essential oil in CH2 Cl2 , 0.1 µL injection, splitting ratio 30:1. Putative identification of the essential oil components was based on their calculated retention indices (RI), based on a homologous series of n-alkanes (C8 -C40 ), and their mass spectral fragmentation patterns compared with those reported in the databases [42–45], with RI values within ±10 units and with matching factors >80%. The concentrations of the essential oil components were calculated from raw peak areas, normalized to 100%, without standardization. 3.3. Mosquito Larvicidal Assay Eggs of Aedes aegypti were purchased from Institute of Biotechnology, Vietnam Academy of Science and Technology and maintained at the Laboratory of Department of Pharmacy of Duy Tan University, Da Nang, Vietnam. Adults of Culex quinquefasciatus and Aedes albopictus collected in Hoa Khanh Nam ward, Lien Chieu district, Da Nang city (16◦ 03′ 14.9” N, 108◦ 09′ 31.2” E) and were identified by National institute of Malariology, Parasitology, and Entomology, Ho Chi Minh City. Adult mosquitoes were maintained in entomological cages (40 × 40 × 40 cm) and fed a 10% sucrose solution and were allowed to blood feed on 1-week-old chicks and mice, respectively. Egg hatchings were induced with tap water. Larvae were reared in plastic trays (24 × 35 × 5 cm). The larvae were fed on Koi fish food. All developmental stages were maintained at 25 ± 2 ◦ C, 65–75% relative humidity and a 12:12 h light:dark cycle at the Laboratory of the Faculty of Environmental and Chemical Engineering of Duy Tan University, Da Nang, Vietnam. Larvicidal activities of the Premna essential oils were determined following the protocol previously reported [62]: 250-mL beakers, 150 mL of water, and 20 larvae (fourth instar), aliquots of the Premna essential oils dissolved in EtOH (1% stock solution) were added to give final concentrations of 100, 50, 25, 12.5, and 6 µg/mL; EtOH only (negative control) and permethrin (positive control), mortality recorded after 24 h and 48 h of exposure, experiments were carried out at 25 ± 2 ◦ C, each test was conducted with four replicates. The data obtained were subjected to log-probit analysis [63] to obtain Plants 2020, 9, 1130 13 of 16 LC50 values, LC90 values and 95% confidence limits using Minitab® 19 (Minitab, LLC, State College, PA, USA). All experimental procedures that involved animals (mice, mosquitoes, chicks, and non-target organisms) were conducted in accordance with the “Guideline for the Care and Use of Laboratory Animals” which was approved by the Medical-Biological Research Ethics Committee of Duy Tan University (DTU/REC2020/NHH01), Vietnam. 4. Conclusions The leaf essential oils of eight species of Premna have been obtained in yields ranging from 0.10% to 0.25%. The mosquito larvicidal activities of these species have been determined for the first time and this is the first report of the essential oil compositions of P. cambodiana, P. flavescens, P. maclurei, P. mekongensis, and P. puberula. The essential oil compositions were largely dominated by sesquiterpene hydrocarbons and oxygenated sesquiterpenoids. The larvicidal activities against Aedes aegypti (LC50 < 65 µg/mL) are promising and can probably be attributed to these components. The essential oil yields, however, are low and likely preclude their consideration as viable alternatives to other essential oils for control of mosquito vectors. However, potential utility of these essential oils will necessitate exploration of cultivation, including plant breeding aimed at increasing oil yield and/or larvicidal activity, potential detrimental effects of the essential oils on the environment, as well as field experiments on application of the essential oils, effects of environmental conditions and potential formulations on essential oil evaporation rates. Author Contributions: Conceptualization, N.H.H.; methodology, N.H.H., P.S. and W.N.S.; software, P.S.; validation, N.H.H., L.T.H., P.S. and W.N.S.; formal analysis, P.S. and W.N.S.; investigation, L.T.H., N.T.C., N.C.T., D.N.D., T.A.T. and V.T.H.; resources, N.H.H.; data curation, W.N.S.; writing—original draft preparation, W.N.S.; writing—review and editing, N.H.H., L.T.H., P.S. and W.N.S.; supervision, N.H.H.; project administration, N.H.H.; and funding acquisition, N.H.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NAFOSTED (Vietnam), grant number 106.03-2019.25. Acknowledgments: 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/). Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 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