BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 11 pps

Potent Mosquito Repellents from the Leaves of Eucalyptus and Vitex Plants Hiroyuki Nishimura and Atsushi Satoh CONTENT 11.1 Mosquito Repellent from Eucalyptus Oils 11.2 Mosquito Repelle

Potent Mosquito Repellents from the Leaves

of Eucalyptus and Vitex Plants

Hiroyuki Nishimura and Atsushi Satoh

CONTENT

11.1 Mosquito Repellent from Eucalyptus Oils

11.2 Mosquito Repellent from E citriodora Oil

11.3 Mosquito Repellent from E camaldulensis Oil

11.4 New Natural Mosquito Repellent from Vitex rotundifolia

References

ABSTRACT N,N-Diethyl-m-toluamide (DEET), which is a commercially available

repel-lent against mosquitoes, has many disadvantages such as an unpleasant odor, skin pene-tration, and carcinogenicity We have explored alternative repellents without such

drawbacks New repellents, p-menthane-3,8-diols (cis and trans), were isolated from the leaves of Eucalyptus citriodora Especially the pentanoyl (C5) and caproyl (C6) esters of the diols exhibited much higher activity than DEET in terms of repellency and repellent

dura-bility against mosquitoes, Aedes albopictus and Culex pipiens In addition, (+)-eucamalol, (1R, 6R)-(+)-3-formyl-6-isopropyl-2-cyclohexene-1-ol, which is a new mosquito repellent, was isolated from E camaldulensis leaves The absolute configuration was determined by synthesizing from (S)-(–)-perillaldehyde While, from information of the traditional usage

of Vitex rotundifolia for repelling mosquitoes at the southern part of Japan, we tried to

iso-late a mosquito repellent from the leaves As a result, a cyclopentene dialdehyde, named rotundial, was identified

11.1 Mosquito Repellent from Eucalyptus 0ils

As a postdoctoral fellow at the University of California, Berkeley (1975 to 1977), I became

interested in research on Eucalyptus oil (Nishimura, 1995) One day, I noticed that mosqui-toes never appeared at the staff barbecues held near the Eucalyptus groves surrounding the campus While enjoying the barbecues, I could smell Eucalyptus volatiles in the air and I observed that the volatile chemicals from Eucalyptus leaves repelled mosquitoes quite a

dis-tance from the groves

N,N-Diethyl-m-toluamide (DEET) has been used as a repellent against blood-sucking

insects such as mosquitoes all over the world However, DEET has many drawbacks such

as an unpleasant odor and its skin penetration (Moody et al., 1986) Furthermore, DEET also reacts with certain plastics and synthetic rubber, resulting in considerable damage to eyeglasses and watchbands, pens, and other plastic items Therefore, a search for new repellents lacking these undesirable properties has been undertaken

A bioassay of repellent activity against mosquitoes was carried out according to the

fol-lowing protocol Pupae of Aedes albopictus (Hatoyama race) were obtained from Laboratory

of Parasitology at Teikyo University and incubated at 25°C for 3 weeks The hatched adults were released into a cage (25 × 25 × 25 cm) made of stainless steel and nylon gauze, and the bioassay was performed in the cage Female Wistar mice (Nippon SLC Ltd.), 6 to 7 weeks old were used Test samples were diluted with acetone at concentrations of 1.0, 5.0, or

10 mg/ml The acetone solution of each test sample was applied to a wire gauze bag (7 cm I.D.× 12 cm) at rate of 50 ml/m2 (50, 250, and 500 mg/m2) and the bag was air-dried at room temperature A mouse was then put into the bag and the bag placed in the cage of mosquitoes Each test was run using 20 female mosquitoes which were 7 days old follow-ing emergence from pupae The total number of mosquitoes landfollow-ing on the mouse was counted immediately The mouse was then taken out of the cage after 10 min, and the mos-quitoes killed in a drying oven at 160°C Each dead mosquito was crushed and the mosqui-toes that had sucked blood were counted Repellency (%) was calculated as

and feed inhibition as

Leaves of several species of Eucalyptus which had been collected in Australia were cut

into small pieces and extracted with acetone in a glass bottle at ambient temperature Ace-tone extracts were steam-distilled to obtain essential oils The repellent activities of the

essential oils against mosquito (Aedes albopictus) are shown in Table 11.1 The larger number indicates the greater repellency From this result, the essential oils from E.citriodora (lemon-scented gum tree) and E.camaldulensis (river red gum tree) had relatively high activities

against mosquitoes

TABLE 11.1

Repellent Activities of Several Eucalyptus Oils Against Mosquito, Aedes albopictus

Repellency (%) Essential oils

2.5 0

0.25 g/m 2

Mouse Skin Area

Total mosquitoes

100%

Total mosquitoes bloodsucking mosquitoes

Total mosquitoes

11.2 Mosquito Repellent from E.citriodora Oil

Silica gel column chromatography of the E.citriodora essential oil was guided by the

bioas-say of repellent activity against mosquitoes Subsequently, two active crystalline com-pounds were isolated

The spectral interpretation of the chemicals isolated from E citriodora oil gave rise to the

identification of p-menthane-3,8-diols (cis type 1; 4.5 mg/g fresh weight leaves and trans

type 2; 2.2 mg/g fresh weight leaves) as shown in Figure 11.1 (Nishimura et al., 1986;

Nish-imura and Mizutani, 1989) The physicochemical data were as follows:

p-Menthane-3.8-cis-diol (1) Mp 81.0-82.5° (crystallized from Et2 O-hexane), [ α] 23

D ± 0° (CHCl 3 ; c = 0.2), IRν KBr

max cm –1 3240, 2930, 2900, 1450, 1420, 1250, 1160, 930 High resolution FIMS m/z(rel.int.):173.1532 [M+H] + (16), 157 [M-CH 3 ] + (37), 154 [M-H 2 O] + (100), 114(9), 96 [M-OH-hydroxyisopropyl] + (83), 77(41), 59(85) EIMS(probe)70eV, (rel.int.): no.M + peak,

157 [M-CH 3 ] + (1), 154 [M-H 2 O] + (2), 139 [M-H 2 O-CH 3 ] + (3), 121(2), 111(2), 96(40), 81 [M-OH-hydroxyisopropyl-CH 3 ] + (100), 68(6), 59(79), 55(21), 54(21), 43(42), 41(34) 1 H NMR(200MHz, CDCl 3, TMS): 0.87(3H,d,J = 6.4Hz, H-7), 1.22(3H,s,H-9), 1.36(3H,s,H-10), 4.41(1H,q,J = 2.4Hz,

H-3) 13 CNMR(50MHz, CDCl 3 , TMS): δ20.4 (t,C-5), 22.3 (q,C-7), 25.7 (d,C-1), 28.8(q,C-9),

29.0(q,C-10), 35.0(t,C-6), 42.6(t,C-2), 48.4(d,C-4), 68.1(d,C-3), 73.3(s,C-8).

p-Menthane-3,8-trans-diol(2) Mp 77.3-78.3° (from Et2 O-hexane), [α] 23

D ± 0° (CHCl 3 ; c = 0.1) IR ν KBr

max cm –1 : 3250, 2960, 2920, 1450, 1420, 1220, 1180, 1000, 910, 870 High resolution FIMS m/z (rel.int.):173.1546 [M+H] + (47), 157 [M-CH 3 ] + (11), 154 [M-H 2 O] + (30), 114(10), 113(15), 96 [M-OH-hydroxyisopropyl] + (54), 77(87), 59(100) EIMS(probe) 70eV, (rel int.):no.M + peak, 157 [M-CH 3 ] + (1),154 [M-H 2 O] + (1), 139[M-H 2 O-CH 3 ] + (3), 121(2), 111(1), 96(38), 81(100), 68(10), 59(90), 55(13), 54(20),43(34), 41(19) 1 H NMR(200MHz,CDCl 3 , TMS):

δ0.92(3H,d,J = 6.4Hz, H-7), 1.22(6H,s,H-9 and H-10), 3.72(1H,dt,J = 10.4,4.3Hz,H-3).13 C NMR(50MHz,CDCl 3 , TMS): δ22.0(q,C-9), 23.8(q,C-10), 27.1(t,C-5), 30.1(q,C-7), 31.4(d,C-1), 34.6(t,C-6), 44.7(t,C-2), 53.5(C-4), 72.9(d,C-3), 75.0(s,C-8).

To explore repellents with higher activity against mosquitoes, several esters of

p-men-thane-3,8-diols were prepared The repellent activities of the esters are shown in Table 11.2 From this result, the pentanoyl (C5) and caproyl (C6) esters of p-menthane-3,8-cis-diol had

FIGURE 11.1

Chemical structures of p-menthane-3,8-diols (cis and trans) from E citriodora leaves.

higher activity than DEET which is a commercially available repellent Interestingly enough, the caproyl ester had higher repellent durability than DEET (unpublished data)

11.3 Mosquito Repellent from E camaldulensis Oil

As shown in Table 11.1, the essential oil from E camaldulensis leaves had significant repel-lent activity against Aedes albopictus (Nishimura et al., 1986) These observations prompted

us to purify the mosquito repellent in the E camaldulensis essential oil.

The leaves of E camaldulensis (1.5 kg) were collected in Matsudo, Chiba prefecture,

Japan They were cut into small pieces and steam-distilled to yield 1.6 g of the essential oil The oil was purified by successive preparative TLC to give two repellents against mosquito Bioassay of repellent activity against mosquitoes was carried out according to the follow-ing methods: A chick whose abdominal feathers were removed with a haircutter was fixed

on a wood board (7× 15 cm) The shaved abdominal skin of the chick (2.5 × 4 cm) was exposed and an ethanol solution of a test compound was applied to the skin (1.5 g/m2)

About 500 adult mosquitoes which were 6 to 8 days old after emergence (Aedes aegypti,

approximately equal numbers of females and males) were released in a cage (21× 21 ×

30 cm) made of stainless steel and nylon gauze Two chicks (one was treated and the other untreated) were put in the cage for 2 min Then, the total number of landed mosquitoes on each chick was counted Repellency (%) was calculated according to following equation

The repellency was evaluated every hour after treatment until the repellency was reduced to less than 80%

TABLE 11.2

Repellent Activities of p-Menthane-3,8-diols and their Derivatives Against Mosquito, Aedes albopictus

cis trans

Repellency (%) R Repellency (%)

Note: Sample concentration: 30 mg/m2 mouse skin area.

Number of landed mosquitoes on untreated chicks

The bioassay-guided chromatography of the essential oil from E camaldulensis leaves

gave rise to the isolation of two active principles From the spectral interpretation, two

repellents were identified as 4-isopropylbenzyl alcohol (3) and a new compound, (+)-euca-malol (4) (3-formyl-6-isopropyl-2-cyclohexen-1-ol) (Watanabe et al., 1993) as shown in

Figure 11.2

The mosquito-repelling activity of 4-isopropylbenzyl alcohol (3) and eucamalol (4) were

examined against A aegypti (Table 11.3) in comparison with that of DEET All tested com-pounds exhibited potent mosquito repelling activities against A aegypti immediately after

the application In 1 h after treatment, the effectiveness of 3 was lost Although the duration

of the effectiveness for DEET was within 2 h after the treatment by our test method,

euca-malol (4) showed 75% repellency even 3 h after the treatment This result indicates that the

repellency of eucamalol against A aegypti is superior to that of DEET.

4-Isopropylbenzyl alcohol(3):MS,m/z 150 [M]+ , 132(M-H 2 O) + (base peak), 107 [M-C 3 H 7 ] + ;

1 H NMRδ TMS

CDC1, 7.05(2H,d,J = 7.0 Hz), 6.95(2H,d,J = 7.0 Hz), 4.00(2H,s,CH2 OH),

2.85(1H,m,CH), 1.00 (6H, d,J = 6.8Hz).

FIGURE 11.2

Chemical structures of 4-isopropylbenzyl alcohol (3) and (+)-eucamalol (4) from E camaldulensis leaves.

TABLE 11.3

Repellent Activities of 4-Isopropylbenzyl

alcohol (3), (+)-Eucamalol (4) and DEET against

the Yellow Fever Mosquito, Aedes aegypti

Repellency (%) Compounds 0 h a 1 h 2 h 3 h

Note: Sample concentration: 1.5 g/m2 chick skin area.

a Immediately after treatment.

D +13.5° (c = 0.80, CH 3 OH); HRMS m/z 168.1151 (C 10 H 16 O 2 , Calcd 168.1150); MS, m/z 168[M] + , 139[M-CHO] + , 125[M-C 3 H 7 ] + , 69(base peak); IRν KBr

max 3400(0H group), 2980, 1680(conjugate aldehyde)cm –1 ; 1 H NMRδ TMS

CDC1a, 9.45(1H,s), 6.63(1H,d,J = 2.2Hz), 4.28(1H,dd,J = 2.2&9.3Hz), 2.37(1H,m), 2.08(1H,m), 2.04(1H,m), 1.78(1H, m), 1.39(1H,m), 1.24(1H,m), 0.98(1H,d,J = 6.8Hz), 0.84(1H,d,J = 6.8Hz).

However, it was difficult to determine the absolute configuration of (+)-eucamalol from

these results Synthesis of (+)-eucamalol (4) and its 1-epimer (5) from (S)-(–)-perillaldehyde

(6) was carried out to determine the absolute configuration and compare their repellent

activities against Aedes albopictus The synthetic scheme is shown in Figure 11.3.

(S)-(–)-Perillaldehyde (6) was converted to 8,9-dihydro-perillaldehyde (7) by

homoge-nous hydrogenation with tris(triphenylphosphine)rhodium chloride as a catalyst in a 73%

yield Conversion of 7 to 3-bromo-8,9-dihydroperillaldehyde (9) was performed by the pro-cedure of Ishihara et al (1990) Enol acetylation of 7 with isopropenyl acetate gave an enol

acetate (8) in a 38% yield This enol acetate (8) was brominated by N-bromo-succinimide.

Since 3-bromo-8, 9-dihydro-perillaldehyde (9) was unstable, nucleophilic substitution of bromide (9) was subsequently carried out by treating with potassium hydroxide to give two alcohols, (+)-eucamalol (4) and (–)-1-epi-eucamalol (5) in yields of 7.7 and 8.4%,

respec-tively (Satoh et al., 1995)

The J1,6value (9.2Hz) of synthetic (+)-eucamalol (4) shows axial–axial coupling, while the

smaller J1,6value (<2.0 Hz) of synthetic (–)-1-epi-eucamalol (5) shows axial-equatorial

cou-pling Thus, the J1,6value of synthetic (+)-eucamalol (4) indicates that the relative

configu-ration at C-1 and C-6 was, like that of natural (+)-eucamalol, of trans-form The optical

rotation of synthetic (+)-eucamalol was +14.1° in methanol, and was very close to the opti-cal rotation of natural eucamalol, [α] = +13.5° (c = 0.80, MeOH) (Watanabe et al., 1993)

Consequently, the absolute configuration of (+)-eucamalol was determined to be

(1R,6R)-(+)-3-formyl-6-isopropyl-2-cyclohexen-1-ol (Satoh et al., 1995)

The repellent activity of synthetic eucamalol and its epimer were evaluated by using

Aedes albopictus as the test mosquito strain (Table 11.4) (+)-Eucamalol and its epimer had

FIGURE 11.3

Synthesis of (+)-eucamalol (4) and its 1-epimer (5) from (S)-(–)-perillaldehyde (6).

repellent and feeding inhibitory activities against A albopictus to the same degree as DEET.

In addition, both the repellent and feeding inhibitory activities of (+)-eucamalol were the same as that of its epimer Structure–activity relationships will be presented elsewhere

11.4 New Natural Mosquito Repellent from Vitex rotundifolia

Vitex rotundifolia has long been used as a medicinal plant for a headache and a cold, and

various compounds such as flavonoid, iridoid glycosides, diterpenoids, and sesquiterpe-noids have been identified in this plant (Kimura et al., 1967; Asaka et al., 1973; Tada and Yasuda, 1984) It also has been reported that the leaves and twigs of this plant can be used for repelling mosquitoes (Okuda, 1967) However, the principle responsible for its activity has not been previously studied Hence, we investigated the mosquito-repelling principle

of V rotundifolia and isolated a new natural cyclopentene dialdehyde named rotundial (10)

as shown in Figure 11.4 In this report, we describe the identification and mosquito repel-ling activity of rotundial

The volatile constituents of fresh leaves (5 kg) from V rotundifolia were subjected to silica

gel column and preparative thin-layer chromatography, eluting with hexane-EtOAc (2:1,

v/v), to afford 250 mg of rotundial (10, 0.005%) as a colorless and odorless oil; C6H12O2 [high resolution MS (HRMS), M+, m/z 152.0780, [calcd as 152.0837], [αl25

D+39.3°] (c = 1.0, CHCl3) The existence of both a simple aldehyde and an α,β-unsaturated aldehyde in 10 was con-firmed by UV (λmax 250nm, e = 11,000), IR (νmax 1720 and 1665 cm–1) and NMR spectral

TABLE 11.4

Repellent and Feeding Inhibition Activities of (+)-Eucamalol

and its (–)-1-Epimer Against Aedes albopictus

Repellent Activity (RA)

Compounds

RA(%)

500 250

50 g/m 2

Mouse Skin Area

Feeding Inhibitory Activity (FIA)

Compounds

FIA(%)

500 250

50 g/m 2

Mouse Skin Area

RA =Total mosquitoes attracted mosquitoes

Total mosquitoes

−

× 100%

FIA =Total mosquitoes b mosquitoes

Total mosquitoes

−

× loodsucking

100%

data [1Hδ: 9.93(1H,s) and 9.70(1H,dd, J = 2.0 and 2.0 Hz); 13Cδ: 201.9(d) and 187.9(d)] The

1H-NMR spectrum of 10 showed a broad singlet at δ2.15(3H,-C = C-CH3), two double dou-ble doudou-blets at δ2.88 (1H,J = 2.0, 4.4 and 17.0Hz) and 2.32 (1H,J = 2.0, 9.1 and 17.0 Hz)

assignable to the methylene protons adjacent to the simple aldehyde (CH2CHO) The 13 C-NMR signals at δ164.2(s) and 139.0(s) indicated full substitution of the double bond in 10.

The1H-NMR signals indicated the existence of a CH2CH2CH moiety which formed a cyclo-pentene ring The long-range correlations from its COLOC spectrum (Kessler et al., 1984)

established the structure of rotundial as 10 as shown in Figure 11.4 (Watanabe, 1995) The absolute stereochemistry of 10 was then determined The LiAlH4 reduction of 10 gave rotundiol (11; [αl25

D-16.6°]) The optical rotation data ([α]D) for its methylated (at C-8)

derivatives, (3R,8S)-isodehydroiridodiol (12) and (3R,8R)-isodehydroiridodiol (13) (Figure 11.5) were –20.7° and –15.3°, respectively, suggesting the 3R configuration of 11 (Sakai et al., 1980) Accordingly, the configuration of 10 at C-3 was deduced to be R This is

FIGURE 11.4

Chemical structure of rotundial (10) and correlations of its COLOC spectrum (→).

FIGURE 11.5

Chemical structures of rotundiol (11), (3R,8S)-dehydroiridodiol (12) and (3R,8R)-isodehydroiridodiol (13).

the first isolation of rotundial (10) from a natural source, compared with enzymatic or acid hydrolysis of the aucubin-terated iridoid giving compound 10 (Bianco et al., 1977).

The mosquito repelling activity of rotundial (10) was examined against Aedes aegypti in

comparison with that of DEET (Table 11.5) Both compounds had potent repellent activity

against A aegypti immediately after the application, while 1 h after treatment, the

effective-ness of 10 was almost equivalent to that of DEET Two h after treatment, the repellency of

10 became higher than that of DEET, indicating that the mosquito repelling activity of

rotundial (10) was superior to that of DEET in respect to its long-lasting effectiveness.

A monoterpene dialdehyde, chrysomedial, has been isolated from larvae of the

chry-somelid beetle, Plagiodra versicolora, as its defensive substance (Meinwald et al., 1977;

Mein-wald and Jones, 1978) The structural similarity between 10 and chrysomedial leads us to speculate that rotundial (10) might serve the plant as a defensive principle against insect

attack which will be the subject of further investigation

References

Asaka, Y., Kamikawa, T., and Kubota, T., Chem Lett., 937-940, 1973.

Bianco, A., Guiso, M., Iavarome, C., Pasacantilli, P., and Trogolo, C., Tetrahedron, 33, 851-854, 1977.

Ishihara, M., Kakiuti, H., Tsuneya, T., and Shiga, M., 34th Symposium on the Chemistry of Terpenes, Essential Oil, and Aromatics, Abstract, 1990, 45-47.

Kessler, H., Griesinger, C., Zarbock, J., and Loosli, H.R., J Mag Res., 57, 331-336, 1986 (The COLOC

spectrum of 1 was recorded with an 8k × 256 matrix and a mixing delay of 45 and 22.5 ms.)

Kimura, Y., Takido, M., and Hiwatashi, Y., Yakugaku Zasshi (in Japanese), 87, 1429-1430, 1967 Meinwald, J., Jones, T.H., Eisner, T., and Hocks, K., Proc Natl Acad Sci., 74, 2189-2193, 1977 Meinwald, J and Jones, T.H., J Am Chem Soc., 100, 1883-1886, 1978.

Moody, R.P., Sidon, E., and Franklin, C.A., 6th Intl Congress of Pesticide Chemistry, Ottawa, Symposium Paper 8A/7E-06, 1986.

Nishimura, H., Mizutani, J., Umino, T., and Kurihara, T., Intl Congress of Pesticide Chemistry, Ottawa, Symposium Paper 2D/E-07, 1986.

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Eucalyptus citriodora plant, the Proc Hokkaido Tokai University, Science and Engineering, no 2, 1989,

57-65.

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Okuda, O., “Kouryou Kagaku Soran” (in Japanese), Hirokawa Shoten, Tokyo, Japan, 1967, 310-311.

Sakai, T., Nakajima, K., Yoshihara, K., and Sakan, T., Tetrahedron, 36, 3115-3119, 1980.

TABLE 11.5

Repellent Activities of Rotundial (10) and DEET

Against the Yellow Fever Mosquito, Aedes aegypti

Repellency (%)

Note: Sample concentration: 1.5 g/m2 chick skin area.

a Immediately after treatment.

Satoh, A., Utamura, H., Nakade, T., and Nishimura, H., Absolute Configuration of a new mosquito

repellent, (+)-eucamalol and the repellent activity of its epimer, Biosci Biotech Biochem., 59,

1139-1141, 1995.

Tada, H and Yasuda, F., Heterocycles, 22, 2203-2205, 1984.

Watanabe, K., Shono, Y., Kakimizu, A., Okada, A., Matsuo, N., Satoh, A., and Nishimura, H., J Agric.

Food Chem., 41, 2164-2166, 1993.

Watanabe, K., Takada, Y., Matsuo, N., and Nishimura, H., Biosci Biotech Biochem., 59, 1979-1980, 1995 Wright, R.H., Sci Am., 233, 104-111, 1975.