Fumigant toxicity of the essential oils of some African plants against Anopheles gambiae sensu stricto

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Phytomedicine 12 (2005) 241–246 www.elsevier.de/phymed

Fumigant toxicity of the essential oils of some African plants against Anopheles gambiae sensu stricto M.O. Omoloa,b, D. Okinyoa,b, I.O. Ndiegea,b,, W. Lwandeb, A. Hassanalib a

Chemistry Department, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, Nairobi, Kenya Behavioural and Chemical Ecology Department (BCED), International Centre for Insect Physiology and Ecology, Nairobi, Kenya

b

Received 14 October 2003; accepted 29 October 2003

Abstract The essential oils from 15 species of African plants selected by ethnobotanical considerations and field inspection (odour and presence of insects) were screened for fumigant toxicity to Anopheles gambiae s.s. in the laboratory. Essential oils from 6 species showed varying levels of toxicity, with Conyza newii (Compositae) and Plectranthus marruboides (Labiateae) being the most potent. Fifty compounds representing
74% of the essential oil of C. newii were identified by GC-MS and GC-coinjection (for available standards). The major and some of the minor constituents of the two oils were assayed at different doses. Two compounds, from C. newii, perillaldehyde and perillyl alcohol, exhibited higher fumigant toxicity (LD50=1.05  104 and 2.52  104 mg cm3, respectively) than the parent oil (2.0  103 mg cm3). GC-MS analysis of the essential oil of P. marruboides gave results similar to that previously reported. Interestingly, none of its components were active, suggesting that the insecticidal activity of the oil results from either some of the minor components or as a blend effect of some of the major constituents. r 2004 Elsevier GmbH. All rights reserved. Keywords: Conyza newii; Plenctranthus marruboides; Compositae; Labiateae; Anopheles gambiae; Fumigant toxicity; Essential oil composition; Perillaldehyde; Perillyl alcohol

Introduction Essential oils of a large number of plant species have been found to have toxic and/or repellent effects against different insects (Curtis et al., 1991; Regnault-Roger, 1997). Although the oils generally consist of complex mixtures of monoterpenes and sesquiterpenes, the antiinsect activities are associated with a smaller group of the constituents, acting additively or synergistically

Corresponding author. Chemistry Department, School of Pure and Applied Sciences, Kenyatta University, P.O. Box 43844, Nairobi, Kenya. Tel.: +254 20 810901 20; fax: +254 20 811575. E-mail address: [email protected] (I.O. Ndiege).

0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2003.10.004

(Singh and Agarwal, 1988; Regnault-Roger et al., 1993; Bekele and Hassanali, 2001). As part of our bioprospecting programme to identify readily cultivable African plants with potential use in protection against malaria vectors, we have been screening the essential oils of different African plant species against Anopheles gambiae s.s. for repellency and fumigant toxicity. The plants were selected primarily on the basis of ethnobotanical information (Kokwaro, 1993), supplemented, in some case, by inspection of the plants growing naturally in the field for evidence of emission of specific odour and avoidance by insects. In this communication, we report the results of fumigant toxicity assays on the essential oils of 15 African plant species, and the analysis and identification of the active constituents of the oil of Conyza newii.

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Materials and methods Plant materials Plant materials (leaves and flowers, or whole aerial parts) were collected from different parts of Nyanza, Western, Rift Valley and Central provinces of Kenya in September 1999, February and June 2000. The collection comprised of C. newii, Tarchonanthus camphoratus, Bidens pilosa, Psaidia punctulata and Helichrysum spp. (Compositae); Plectranthus marruboides, Tetradenia riparia, Ocimum lamiifolia and Hyptis pectinata (Labiatae); Lippia ukambensis, Lippia javanica and Lantana camara (Verbenaceae); Croton dichogamus (Euphorbiaceae) [from two different localities, Ngong’ and Got Ramogi]; and Teclea simplisifolia (Rutacea) and Schinus molle (anacardiaceae) all identified by a plant taxonomist from the University of Nairobi (UoN), Botany Department. Voucher specimens were deposited at the UoN Herbarium. For plants with active essential oils, the voucher numbers LUK/0361/2000 (Lippia ukambensis, Verbenaceae); PMA/0362/2000 (Plectranthus marruboides, Labiateae); TCA/0364/2000 (Tarchonanthus camphoratus, Compositae); LJA/0373/2000 (Lippia javanica, Verbenaceae); CNE/0376/2000 (C. newii, Compositae); and TRI/0385/2000 (Tetradenia riparia, Labiateae), respectively, were assigned. The materials were dried under shade for one week before extraction.

Isolation of essential oils The oils were isolated by steam-distillation of aerial parts, or (leaves with flowers) using Clavenger apparatus, collected, dried using anhydrous sodium sulphate and stored in amber-coloured vials at 0 1C until use.

Analysis of essential oils Gas chromatographic (GC) analysis was performed on a Hewlett Packard (HP) 5890 Series II equipped with a split-less capillary injector system, a cross-linked methyl silicone capillary column (50 m  0.2 mm i.d. and 0.33 mm film thickness) and a flame ionization detector (FID) coupled to a HP 3393A Series II integrator. Carrier gas (N2) was set at a flow rate of 0.7 ml/min. The GC oven program comprised of an initial temperature of 50 1C (5 min) to 280 1C at 5 1C/min and held at the final temperature for 10 min. Identification of the constituents of C. newii oil was performed on a HP 8060 Series II GC linked to a VG Platform II mass spectrometer (MS), operated in the EI mode at 70 eV, with the temperature of the source held at 180 1C and multiplier voltage at 300 V. Instrument calibration was performed using heptacosafluorotributylamine [CF3(CF2)3]3N (Apollo Scientific Ltd., UK). The col-

umn used for GC-MS was of the same specification as the one for GC analysis except for the film thickness (0.5 mm). The GC temperature programme comprised of an initial temperature of 50 1C (5 min) to 90 1C at 5 1C/ min to 200 1C at 20 1C/min to 280 1C at 20 1C/min and held at the final temperature for 20 min. Identification of the components was made by comparison of mass spectra with published data (NIST, Wiley) and confirmed, where possible, by GC co-injections with authentic samples.

Fumigant assays These were carried out according to WHO (1996) protocol in small cages (20  20  35.5 cm). Test materials in acetone applied to Whatman filter papers (7 cm diameter) in a Petri dish (8 cm) acted as sources of fumigants. The Petri dishes were covered with wire gauzes to prevent the mosquitoes from making direct contact with the filter papers. Rolled filter papers (5  10 cm) dipping into glucose solution (6%) served as sources of food for the insects. Control cages were similarly set, with the solvent (acetone) replacing the test solution. In each assay, 25 or 30 females An. gambiae (4–6 days old) were introduced into the test and control cages and monitored every 20 or 30 min for 6 h. Each test was replicated 10 times. In the preliminary screening of essential oils from the 15 plant species, single doses of the oils (1 ml of 0.1 g/ml in acetone) were used, and the time for 50% (T i50 ) and 100% (T i100 ) mortality computed. Six of the most potent oils were assayed over a range of doses (1 ml of 0.02, 0.04, 0.06, 0.08 and 0.1 g/ml). The major available constituents of C. newii (perillaldehyde, limonene, 1,8-cineole, perillyl alcohol, geraniol) and P. marruboides (camphor, 1, 8-cineole, p-cymene, a-terpenene, isocaryophyllene, camphene) and some minor constituents of the two (a-pinene, bpinene, neral, myrtenol, d-4-carene, linalool, citral, gterpenene, a-terpeneol, borneol, a-fenchyl alcohol and geranyl acetate) were assayed at lower dose range (1 ml of 0.002, 0.004, 0.006, 0.008 and 0.01 g/ml).

Computation and statistical analyses of results Percent fumigant toxicity of the oils and individual compounds, PI, were calculated according to the formula PI ¼ ðN T 2N C Þ=N I  100; where N T and N C represent the number of dead mosquitoes in the test and control cages, respectively, and N I represents the initial number of mosquitoes introduced into each cage. T i50 and T i100 means were analysed using Statistical Analysis Systems (SASs, 2000) and means ranked according to Student Newman–Kuels (SNK) test. Dose–response data was subjected to probit analysis and LC50 values

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obtained from the derived regression equations (Finney, 1971; Busvine, 1971). LC50 values (95% CI) were converted to LD50 by applying the formula LD50=LC50/V, where V (14,200 cm3) is the volume of the experimental cage.

Results Table 1 gives mean duration of exposure of the mosquitoes to fumigants of different essential oils for 50% (T i50 ) and 100% (T i100 ) mortality. Oils of 6 plant species (Tarchonanthus camphoratus, Lippia javanica, Plectranthus marruboides, Tetradenia riparia, Lippia ukambensis and C. newii were found to be relatively toxic, with C. newii and P. marruboides showing the highest potency. LD50 values obtained for the essential oils (Table 1) also confirmed the high fumigant toxicity of the two oils (Fig. 1). GC-MS analysis (Fig. 2) of the essential oil of C. newii showed a complex blend of constituents of which 50 (74.3% of the oil) were identified by comparison with NIST Registry of Mass Spectral data. Of these, the identity of 43 were confirmed by GC co-injections with standard samples. The major constituents were perillaldehyde (29.3%), limonene (10.1%), 2-methyl-5-(methylethyl)-2-cyclohexene-1-ol (7.3%), 1,8-cineole (6.8%), perillyl alcohol (4.3%), germacrene B (1.45%), trans-bocimene (1.35%), geraniol (1.17%), b-myrecene (1.16%) and a-amorphene (1.11%) (Table 2). GC-MS analyses of the essential oils of T. camphoratus, P. marruboides, T. riparia, L. ukambensis and L. javanica gave results similar to those previously reported (Mwangi et al., 1986, 1991a, b, 1994; Campbell et al., 1997; Chagonda et al., 2000). Of the 23 available constituents of the essential oils, only perillaldehyde and perillyl alcohol showed substantial fumigant toxicity at the dose range tested (Fig. 1). LD50 values of the two compounds (Fig. 3)

Table 1.

243

were calculated as 1.05  104 and 2.2  104 mg cm3, respectively.

Discussion Of the essential oils from the 15 plants screened for fumigant toxicity against An. gambiae in the present study, six showed relatively high activity, two were moderately active and the rest were inactive. The plants were selected primarily on the basis of ethnobotanical information, such as their uses in traditional medicine and protection against biting insects (Kokwaro, 1993). This was augmented by field inspection of the plants for evidence of emission of aromatic volatiles and the absence of insects or insect attack. For example, C. newii was found to have a strong pungent odour in the field and there was no evidence of plant feeding insects in the proximity. The procedure may be useful in selecting candidate plants with potential anti-insect botanicals. Bioassays of major individual components of the essential oils of C. newii and P. marruboides, the two plants with potent fumigant toxicity against An. gambiae in our study, showed that only perillaldehyde and perillyl alcohol had intrinsic activities. Both compounds were identified in the essential oil from C. newii. None of the major constituents of P. marruboides [camphor (45%), 1,8-cineole (9.0%), a-terpene (2.6%), p-cymene (3.1%), isocaryophyllene (1.7%) and camphene (1.58%)], which make up
68% of the essential oil, were active at the dose range tested, suggesting that the activity of the oil may be due to additive or synergistic blend effect of some of the constituents. Such an effect has been previously demonstrated with the essential oils of some Ocimum spp. against post-harvest pests (Bekele and Hassanali, 2001). In this study, lethal toxicities of the oils of O. kilimandscharicum and O. kenyense against Sitophilus zeamais and Rhyzopertha dominica, respectively, were due to the combination of the major constituents none of which was found to exhibit

Fumigant toxicity of essential oils from insecticidal plants against An. gambiae

Plant Oil

T i50 (h)7SE

T i100 (h)7SE

LD50 (mg cm3 95% CI)

Tarchonanthus camphoratus Lippia javanica Plectranthus marruboides Tetradenia riparia Lippia ukambensis Conyza newii Ocimum lamiifolia Croton dichogamus (N) Croton dichogamus (GR)

1.3370.045b 1.5070.067c 1.3370.042b 1.5070.058c 0.6770.006a 1.3370.057b 1.5070.078c 24.070.008d 24.070.004d

2.3370.037b 2.3370.048b 1.6770.065a 2.1770.238b 2.6770.043c 1.6770.075a 24.070.082d — —

3.8  103 4.3  103 2.8  103 4.4  103 4.7  103 2.0  103 + —

Bidens pilosa, Schinus molle, Lantana camara, Teclea simplisifolia, Helichrysum spp, Hyptis pectinata, and Psaidia punctulata were inactive. Means with the same letters in a column are not significantly different at P ¼ 0:05 (SNK Test)+Not determined; N=Ngong’ GR=Got Ramogi.

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Probit

3 2 1 0 -1 0

2

1

3

4

-2 -3 -4 -5 -6 -7 Log (dose + 5) C. newii

L. javanica

P. marruboides

L. ukambensis

T. riparia

T. camphoratus

Perillyl alcohol

Perillaldehyde

Fig. 1. Log dose–probit relationships for insecticidal essential oils and two constituents of C. newii. Regression equation y ¼ mx þ c; where y=probit, x ¼ logðdose þ 5Þ; m=slope, c=intercept; C. newii, m ¼ 2:427; c ¼ 3:363; LC50=27.9 mg/ml; L. javanica, m ¼ 2:664; c ¼ 5:096; LC50=61.6 mg/ml; P. marruboides, m ¼ 1:823; c ¼ 1:564; LC50=39.9 mg/ml; L. ukambensis, m ¼ 2:809; c ¼ 5:731; LC50=66.1 mg/ml; T. riparia, m ¼ 2:823; c ¼ 5:724; LC50=62.9 mg/ml; T. camphoratus, m ¼ 2:885; c ¼ 5:763; LC50=27.9 mg/ml; perillyl alcohol, m ¼ 3:422; c ¼ 3:738; LC50=3.576 mg/ml; perillaldehyde, m ¼ 3:93; c ¼ 3:534; LC50=1.484 mg/ml.

31

Temperature Program

100 10

@20 C/m

280 C (20.0min)

200 C @2 C/m (0.0min) 90 C

@5 C/m

50 C (5.0min)

(0.0min)

42 22

41

39

9

% 47 46 45

5

48

19 18 17

40

30

49

38 34

14

1 4 2

0 20.00

25.00

3

7

8

6

30.00

15

11 12

20 21

13 16

35.00

40.00

23 24 25

32

26 27

44

29 28

45.00

43 35

50.00

50

37

33 36

55.00

60.00

65.00

70.00

75.00

Time 80.00

Fig. 2. Gas chromatogram of the essential oil of Conyza newii. HP cross-linked methyl silicone capillary column, 50 m  0.22 mm (id)  0.33 mm (thickness).

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Table 2.

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Chemical composition of the essential oil from C. newii leaves

Compound

%

GC coinjection

Compound

%

GC coinjection

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

0.35 0.13 t 0.18 1.16 t t t 10.06 6.84 1.35 t t t 0.11 t t 0.21 t 0.12 0.17 1.17 t t t

| | | | | * | | | | | | | | | | | | |

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

t t t t t 29.28 0.69 0.89 4.27 0.12 t t t t 0.37 0.88 7.34 0.27 0.56 1.45 0.78 0.58 0.65 1.11 0.19

| | | | | | | * | | | * | * | | * | | | | | * | |

a-Pinene Camphene b-Phellandrene b-Pinene b-Myrecene p-Mentha-1,3,8-triene d-2-Carene d-4-Carene Limonene 1,8-Cineole Trans-b-ocimene g-Terpinene Trans-sabinene hydrate a-Terpinene Linalool Phenylethyl alcohol p-Cymene a-Fenchyl alcohol Cis-2-pinanol Artemisia ketone Camphor Geraniol Borneol cis-Sabinene hydrate a-Terpineol

*

| | | | |

Myrtenol a-Terpinolene Neral Carvone a-Fenchene Perillaldehyde Geranyl acetate Limonenyl-10-acetate Perillyl alcohol Myrtenyl acetate a-Cadinol g-Curcumene b-Eudesmol Ylangene Methyleugenol p-Mentha-1(7),8(10)-dien-9-ol 2-Methyl-5-(methylethyl)-2-cyclohexen-1-ol. a-Copaene a-Caryophyllene Germacrene B 4-Isopropylbenzaldehyde Isocaryophyllene Germacrene D a-Amorphene Spathulenol

|Identification done by both GC-MS and GC coinjection. * Identification done by GC-MS only; t=trace amounts (o0.1%).

CHO

H3C

CH2

Perillaldehyde

CH2OH

H3C

CH2

Perillylalcohol

Fig. 3. Formulae of the major constituents of P. marruboides.

significant activity individually. A similar study with different blends of the major constituents of P. marruboides may help to identify those that contribute to the fumigant toxicity of the essential oil. The LD50 values of perillaldehyde (1.05  104 mg cm3) and the corresponding alcohol (2.52  104 mg cm3) are 19- and 8-fold, respectively, higher than that of the essential oil of C. newii

(2.0  103 mg cm3). Thus, the activity of the essential oil is at least 3-fold lower than the value expected from proportionate contribution of the aldehyde, which constitutes about 29.3% of the oil. This suggests that other components of the essential oil of C. newii may actually inhibit the fumigant toxicity of the active compounds. Further studies with the aldehyde and the alcohol blended with other constituents of the oil may help throw further light on the paradox. No studies have previously been reported on the biological activities and chemical constituents of C. newii. In traditional medicine, the leaves of the plant are chewed to treat chest and similar ailments (Kokwaro, 1993). Related species (C. floribunda and C. aegyptiaca) have been shown to have anti-inflamatory, anti-viral and anti-microbial activities (De las Heras et al., 1998; Abad et al., 1999; Anani et al., 2000). Phytochemical investigations of other members of the genus Conyza have revealed the presence of sesquiterpenes (C. hypoleuca) (Zdero et al., 1991), diterpenes (C. hypoleuca, C. steadii and C. welwitschii) (Zdero et al., 1990a, b; Ahmed, 1991) and triterpenes (C. stricta) (Ahmed and Ahmed, 1990). A more detailed investigation on the medicinal, pesticidal and phytochemistry of C. newii is clearly warranted.

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Acknowledgement This work was supported by funds from UNDP/ World Bank/WHO/TDR/MIM (Grant No. 990056) and NIH (Grant No. U19A14511-01). We also acknowledge Wanyama Kaye (ICIPE), for running GC-MS of the essential oil; Basilio Njiru, Milka Gitau and Jeremiah Ojude (ICIPE), for rearing the insects; Simon Mathenge (University of Nairobi), for plant identification.

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