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

CHEMICAL COMPOSITION OF ESSENTIAL OILS FROM LEAVES OF VITEX NEGUNDO L.. Verbenaceae were collected from Da Nang City DND41, DND29, DND15 and DND18, the Pu Hoat Nature Reserve, Nghe An p

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, *

1

Center for Advanced Chemistry, Institute of Research and Development, Duy Tan University,

03 Quang Trung, Da Nang, Viet Nam 2

Department of Pharmacy, Duy Tan University, 03 Quang Trung, Da Nang, Viet Nam

3

Graduate University of Science and Technology, Vietnam Academy of Science and Technology,

18 Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam 4

Faculty of Agriculture, Forestry and Fishery, Nghe An College of Economics, 51 Ly Tu Trong,

Vinh City, Nghe An Province, Viet Nam 5

Aromatic Plant Research Center, 230 N 1200 E, Suite 102, Lehi, UT 84043, USA

6

University of Khanh Hoa, 01 Nguyen Chanh, Nha Trang, Khanh Hoa, Viet Nam

7

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

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

1 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

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

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 1

Table 1 Plant collection and hydrodistillation details of Vitex negundo central Viet Nam

Collection site (coordinates; elevation) Collection Date Voucher

number

% (v/w) yield

Bà Nà Hill, Hoa Vang district, Da Nang City

16°02′57ʺ N; 108°09′34ʺ E, elev 8 m June 2018 DND41 0.51

Bà Nà Hill, Hoa Vang district, Da Nang City

16°02′57ʺ N; 108°09′34ʺ E, elev 8 m August 2018 DND29 0.55

Bà Nà Hill, Hoa Vang district, Da Nang City

16°01′39ʺ N; 108°03′42ʺ E, elev 28 m February 2019 DND15 0.57 Hoa Vang district, Da Nang City

16°01′39ʺ N; 108°03′42ʺ E, elev 28 m April 2019 DND18 0.56 Đồng Văn Commune, Quế Phong District, Pu

Hoat Nature Reserve, Nghe An province October 2018 DND712 0.49 Nam Dong district, Thua Thien Hue province

16°13′03ʺ N; 107°43′27ʺ E, elev 109 m July 2018 DND789 0.38

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 2 °C/min to a maximum of 260 °C Aliquots

of each of the V negundo essential oils were diluted to 5 % 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

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

1 % 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

± 2 °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

3 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 2 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 %)

Table 2 Chemical compositions (%) of leaf essential oils of Vitex negundo from central Viet Nam

Da Nang Pu Hoat Nam Dong

RI calc RI db Compound DND41 DND29 DND15 DND18 DND712 DND789

976 974 1-Octen-3-ol - 0.1 - - - -

1006 1006 α-Phellandrene - - - - tr tr

1008 1008 δ-3-Carene - - - - tr -

1016 1017 α-Terpinene 0.1 - tr 0.1 0.2 -

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

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 -

1099 1098 trans-Sabinene hydrate 0.3 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 - - - -

Da Nang Pu Hoat Nam Dong

RI calc RI db Compound DND41 DND29 DND15 DND18 DND712 DND789

1169 1170 δ-Terpineol 0.1 0.3 tr 0.2 - -

1172 1172 cis-α-Necrodol 0.1 - - - - -

1180 1180 Terpinen-4-ol 4.0 2.8 0.7 1.6 2.2 tr

1181 1181 Thuj-3-en-10-al 0.1 tr - - - -

1183 1188 Naphthalene - - 0.1 - - -

1185 1186 p-Cymen-8-ol 0.1 0.1 0.1 - tr -

1189 1190 Methyl salicylate - - - - - tr

1194 1195 α-Terpineol 0.4 0.9 0.1 0.9 0.2 tr

1195 1195 cis-Piperitol 0.1 - - - - -

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

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

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

Da Nang Pu Hoat Nam Dong

RI calc RI db Compound DND41 DND29 DND15 DND18 DND712 DND789

1375 1379 (E)-β-Damascenone - - - tr - -

1376 1378 Geranyl acetate - - - - - tr

1380 1383 cis-β-Elemene - - tr - - 0.6

1382 1382 β-Bourbonene 0.1 0.1 0.1 - 0.1 -

1387 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

Da Nang Pu Hoat Nam Dong

RI calc RI db Compound DND41 DND29 DND15 DND18 DND712 DND789

1501 1503 (E,E)-α-Farnesene - - - - - 0.2

1501 1507 Eremophila-1(10),8,11-triene tr 0.3 0.1 0.1 - -

1505 1508 β-Bisabolene - - - - - 0.7

1511 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

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 - -

1653 1652 α-Eudesmol 2.0 - - - - -

1656 1658 neo-Intermedeol - - - - - 0.4

1659 1664 ar-Turmerone - - - - - 0.1

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 - -

Da Nang Pu Hoat Nam Dong

RI calc RI db Compound 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 - -

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

Total identified 87.4 85.1 87.7 84.3 92.3 99.4

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, terpinene-4-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 2 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 3 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

Figure 1 Dendrogram based on hierarchical cluster analysis (HCA) of the leaf essential oils of Vitex

negundo obtained from different locations in Asia

Table 3 Group centroid concentrations (%) from the hierarchical cluster analysis of Vitex negundo leaf

essential oils

CIMAP, India [40]

Beriparav, India [40]

Pantnagar, India [40]

Bindukhatta, India [40]

Haldwani, India [40]

Kathgodam, India [40]

Bhimtal, India [40]

Dehra Dun, India [40]

Soneshwar, India [40]

Ghingartola, India [40]

Chhati, India [40]

Sirkot, India [40]

Bharadi, India [40]

Tharali, India [40]

Dewalchoura, India [40]

Lohali, India [40]

Kandhar, India [40]

Ashon, India [40]

Gagrigole, India [40]

Lobanj, India [40]

Manan, India [40]

Balighat, India [40]

Tarikhet, India [40] ]

Dungoli, India [40]

Chandigarh, India [39]

DND712 (this work)

DND789 (this work)

Ganeshpur, India [37]

DND29 (this work)

DND18 (this work)

DND15 (this work)

DND41 (this work)

Taichung, Taiwan [38]

Dissimilarity

Group 1

Group 2 Group 3

Group 4

Group 5