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Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of Malaria, Filariasis and Dengue Fever in India
Phytomedicine, Vo!. 5(4), pp. 324-329 © Gustav Fischer Verlag 1998
_
Phytomedicine
Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of Malaria, Filariasis and Dengue Fever in India B. K. Tyagi ', A. K. Shahi- and B. L. KauF lDesert Medicine Research Centre, Jodhpur, India 2Regional Research Laboratory, CSIR, Jammu Tawi, India
Summary Essential oils of four species and two hybrid varieties of Cymbopogon grasses were evaluated for their repellent properties against the major vector mosquitoes, namely, Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti, both in laboratory and field. The magnitude of repellency in the Cymbopogon essential oils was found to be of moderate to high order. All grass species protected completely from mosquito bites for 4 hrs, whereas C. nardus provided protection for as much as 8-10 hrs overnight. Key words: Cymbopogon spp., essential oil, repellent activity, mosquitos, malaria, filariasis, Dengue fever
Introduction Mosquitoes belonging to genera Anopheles, Culex and Aedes are serious vectors of several important diseases in India, such as malaria, filariasis, Japanese encephalitis and dengue (including dengue haemorrhagic fever and dengue shock syndrome) (Tyagi, 1994). So far vector control has been largely carried out by employing residual insecticides, but due to development of resistance by these vectors against most of the conventional insecticides, both the abatement of vector population and cessation of disease transmission has become very difficult (Tyagi, 1992; Sharma and Mehrotra, 1986). At present, one of the most effective methodologies to prevent transmission of mosquito-borne infections is to use repellents which over-sensitivitize the chemoreceptive organs of the vectors and disorient them from seeking blood meal from the human host (Bowen, 1991). Among various mechanisms involved in this process the antifeedent effect of repellents is the most important as it works at a distance and keeps the vectors away from feeding (Sukumar et al., 1991). Although, certain chemically synthesized repellents like N,N-diethyl-rn-toluamide (DEET) and Dimethylphthalate
(DMP) have already been used in dispelling bloodsucking insects, mites, ticks and leeches etc., development of partial tolerance of A. aegypti against DEET (Rutledge et al., 1994) has warranted an urgent need to look for some repellent materials from natural resources. The genus Cymbopogon (Poaceae), comprises 27 species found all over India (Shahi and Sen, 1990). Many a species of this genus are well known for their repellent characteristics against different kinds of haematophagous insects, besides being used in medicine and perfumery. The present paper describes the repellent behaviour of some of these species widely growing in north-western parts of India.
Materials & Methods Test Material
Four plant species, viz. Cymbopogon commutatuslwinterianus Jowitt., C. martinii (Roxb.) Wats., C. pendulus (Nees ex Steud.) Wats., C. nardus (L.) Rendie and
B. K. Tyagi et al.
325
cages from the field to the laboratory where they were allowed to lay eggs. The freshly emerged female mosquitoes were starved for 2-3 days before use in the experiments. Hands of the volunteer human test subjects (# 3; males only) were first washed with liquid soap and subsequently cleaned with 70 % ethanol. After air drying, the left hand of the test person was smeared up to the wrist with 0.25 ml of the essential oil. Then the hand was introduced into the mosquito cage harboring 20 females, at regular intervals of 1, 4, 6 and 8 hours for 15 minutes each time. Controls were carried separately with another male volunteer (# 1) without any essential oil application. Each test and control was replicated in triplicate. • Test procedure: Th e antifeedant effect wa s exp ressed as 100 (1-T/C)%, where T is the mean of sucking mosquitoes after 4, 6 an d 8 hrs ., and C is the corresponding number of blood sucking mosquitoes in the control. The number of mosquitoes landing without sucking was noted and all landing mosquitoes (LT), were expressed in two ways, (i) as a percentage of all mosquitoes, landing (LT), and feeding (FT), i. e., 100 (LT/[LT + FT]), and (ii) landing in the test (LT) compared with the total landings in .the control (LC) expressed as (LT/LC) x100. All landings were recorded with the help of a counter.
two hybrids named Jamrosa and Lemongrass (coded as CKP2s-RRL Jammu), collected from different parts of the north-eastern and north-western parts of the country, were assayed for their repellent activities against A. stephensi, C. quinquefasciatus and A . aegypti. All the mosquito species were collected from the residential settlements near the DMRC campus. Extraction of esse ntial oils
Various Cymbopogon species were collected from the field, while the hybrid varieties were developed and grown in the experimental farms of the Regional Research Laboratory, Jamnu. The essential oils fro m all plant materials were obtained by hydrodistillation of the foliage parts usin g a Clevenger apparatus. To obtain pure forms of th e volatile oils, triplicate dist illation was carried out in succession of each lot of the fresh material weighing 500 gm. The combined oils were eventually dried over anhydrous sodium sulfate before being subjected to experimentation. Assessment of repellent activities
• Laboratory assay: All tests were conducted at room temperature 28°C ± 1 and relative humidity 50-60 %. Feral female mosquitoes were brought in Barraud's
Tab le 1. Percentage landings of vector mosquitoes against essential oils of different Cymbopogon species (figures in parenthesis is the denominator refering to total landings). Plants Species/variety
C. commutatus C. martinii C. pendulus C. nardus CKP25 RRL jarnrosa
Pooled
Vector species
Pooled (aver.)
A. stephensi
C. fatigans
A. aegypti
55.5 (9) 36.0 (25) 44.4 (36) 60 .0 (10) 44.4 (9) 40 .0 (20)
43.7 (16) 38.0 (19) 42 .8 (21) 36.6 (11) 38 .0 (19) 47.3 (38)
41.6 (12) 42.2 (42 ) 50. 0 (58) 40 .0 (20) 37.9 (29) 46.1 (52 )
45.9 (37) 41.9 (86) 47.0 (115) 43.9 (4 1) 40.4 (57) 45.5 (110)
44.0 (109)
43 .5 (124)
45 .1 (213)
44.4 (446 )
Table 2. ANOVA for landing of major vector species against the repellent effect of Cymbopogon species. Source of Variation Between vector spp . Between grass spp. Interaction Error ** p < 0.01; *p < 0.05
Degree of freedom
2 5 10 36
Sum of aqua res
M. S. S.
Variance ratio
351.15 651.94 241.09 248.20
175.57 130.39 24 .11 6.89
25.48 ** 10.92 ** 3.50*
326
Evaluation of repellent activities of Cymbopogon
... landing
20 ...---- ---=- - - - - - - - - - - - - - - - - - - - --,
15
10
5
o
commutatua
mart in i;
pendu lua
nardus
Jamroaa
CKP-215
Fig.1. Percentage landings of the three major vector mosquitoes against the six Cym bopogon essential oils in the laboratory evaluation.
Cymbopogon spec ies •
A.stephensl
~ C.qulnquefasc latus
D
Ae. aegyptl
• Field assay: Repellent activity of the vario us essential oils was tested against C. quinquefasciatus only. T he volati le oil (0.5 m!) was applied on the bare feet, ha nds and for ehead of a volun teer human subject w ho was made to sleep on a cot. Overnight sampling of females was carried out with the help of an aspi rator and a flash light. Co ntrol without any application of th e volatile oil was kep t sepa ra tely. Another experime nt to assess the distance ~f effectiveness of repellency by each essential oil was also carried ou t betwee n 8 p . m. and 9 p. m. against C. quinquefasciatus feeding on h uman subjects sitti ng or lying
on a cot at fixed distances of 2, 4, 6, 8 an d 10 ft., respectively, fro m the source of essen tial oil. • Chemical analysis: The chemical analysis of th e oils were ca rried out by using GC and GC/MS. T he apparatus was a DELCI 121 C gas chromatography (F. I. D .) WCOT capillary column wit h fused silica (25 m x 0.3 mm) and CP WAX 51 sta tionary phase. The temperature was programmed fro m 50 °C with 5 min initial hold and then to 210 °Clmin, using N 2 as the carrier gas . T he identification of th e oil components was carried out by comparing the retention indices of standard substances wi th those of substances present in
Table 3. Antifeedent activity (AFA) of different Cym bopogo n species against three major vector mosquitoes in the laboratory. Grass species C. commutatus C. martinii C. pendulus C. nardus
CKP25 Jamrosa
AFA(%)
Vector species A. stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A . stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A. step hensi C. quinquefasciatus A. aegypti
Average
1 hr
4 hr
6 hr
8 hr
AFA(%)
100.0 100.0 100.0 100.0 100.0 97.1 99.0 100.0 94.1 100.0 100.0 100.0 100.0 100.0 100.0 100.0 98.7 98.8
100.0 100.0 100.0 97.1 98.6 91.8 99.1 100.0 89.6 100.0 98.7 93.7 100.0 94.2 98.1 97.3 90.5 87.2
98.9 96.1 83.3 94.7 98.1 90.4 90.2 92.6 87.1 96.3 98.5 90.9 97.9 97.7 91.2 93.3 95.0 91.5
97.7 95.3 91.5 99.9 90.9 85.0 92.1 92.6 74.4 96.3 92.8 94.1 94.1 92.7 73.8 94.3 87.6 71.9
99.0 97.0 94.8 96.0 96.5 90.9 94.1 96.2 85.4 98.5 97.4 95.1 98.2 99.4 90.7 95.8 93.3 88.1
B. K. Tyagi et al.
327
% antifeeding 100 - r - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
95
90
85
80 +-------,.------r-----,------,-------1
Fig. 2. Percentage antifeeding behaviour of the three vector mosquito species against the six Cymbopogon essential oils in the laboratory evaluation.
commutatus
martini!
-
A. stephensi
the mixture. The identification was confirmed for GC/MS were: ionization energy 70 eV, fused silica WCOT capillary column (50 mx 0.3 mm), temperature programmed from 50 DC to 230 DC at 3 DC/min using He as the carrier gas.
Results and Discussion When the landing in the test alone was considered, maximum protection was recorded against the group of CKP 25 (40.4%), C. martinii (41.9%) and C. nardus (43.9%), followed by another group of Jamrosa (45.5%), C. commutatus (45.9%) and C. pendulus (47%) (Table 1). This was amply substantiated in another evaluation when landings of vector species were compared with those in the control (Fig. 1). By and large A. stephensi and C. quinquefasciatus were easily
Table 4. ANOVA for feeding activity of major vector species against different Cymbopogon species. Source of variation Sum of squares Variance ratio
Degree of freedom M. S. S.
Between vector spp. 393.86 23.45*" Between grass spp, 666.78 15.88':'" Interaction 133.56 1.59 Error 302.32
2 196.93
** p
< 0.01
5 133.36 10 13.36 36 8.398
pendulus
nardus
CKP-25
Jamrosa
Cymbopogon species -+- C. quinquefasciatus
--*- Ae. aegypti
repelled, particularly by C. nardus, CKP25 and C. commutatus. The vector species A. aegypti appeared most refractory among all the species and only C. commutatus and C. nardus could disorient its landing behaviour to an appreciable level. The analysis of variance determined for the vector landings against different Cymbopogon species elucidates that behaviour of the vector species differ from one species of plant to another quite appreciably (Table 2). In the laboratory evaluation all the essential oils invariably offered more than 85% repellent activity against all the vector mosquitoes (Table 3). Although none of the volatile oils seemed to deter mosquitoes from feeding on the inserted hand for a full 8 hours, nevertheless, the antifeedant activity of most of the essential oils was high. While the C. commutatus essential oil offered full protection for the initial four hours against all three vector species, both C. nardus and CKP2S- RRL Jammu hybrid variety of Lemongrass prevented feeding by A. stephensi for the initial four hours. Essential oils from the latter two plant materials protected feeding by all vectors for the initial 1 hour. The analysis of variance (ANOVA) determined for feeding activity of major vector species against different Cymbopogon material (Table 4) supports the view that the feeding behavior of various mosquito species differs, not only against different plant material, but also among themselves. Consequently, the repellent activity of different plants can be arranged as follows: C. nardus (97%), C. commutatus (96.93%), CKP25 RRL Jammu (96.1 %), C. martinii (94.46%), Jamrosa (92.4%) and C. pendulus (91.7%) (Fig. 2). Field experiments with human bait and C. quinquefasciatus as the vector species suggests a maximum protection of 9 hrs by C. nardus, followed by Jamrosa for 8 hrs and C. commutatus and C. martinii for 6-7 hrs (Fig. 3). The Lemongrass variety offered the least pro-
328
Evaluation of repellent activitie s of Cymbop ogon
% protection from feeding 120 ...--- - - - - - - - - - = . - - - - - - - - - - - - - --.
100 +-----+---+----= ~:----=----::~----.,::--.
80
--
60 -
------ -·· -- -.----.---.-- - --....----..... - - -
- - - -- .....-- -- -- -.-..----....- --.----.-..-
40 .-..-.-- - -.- --.- - -
- ---
-\--+ .<-
-..---.- -.-.- - -- -
20
O+--. - -..-----.----r----,---.,--,--.,...---.----.-----1 2
1
3
4
5
6
7
9
8
10
11
Hours -
commutatu8
-I- marti nI!
--*- pendulus
-fr-
nllrdus
--- CKP 26
- - Jamrosa
12
Fig. 3. Percentage protection from biting of C. quinquefasciatus against various Cym bopogon essential oils during overnight peri od in the field evaluation.
Table 5. Percentage composition of various major chemical compounds analyzed in the Cymb opogon species.
Cymbop ogon species
Compound a-pinene camphene ~ -pin cne
sabinene myrccne limoncne 1,8-cineole (Z ) -~-ocimene (E)-~-ocimene
p-cyrnene terpinolene camphor linalool trans-caryphyllene tra ns-bergamotene gymnomitrene o-rerpineol borneol/terpineol ~-sclinene
Zingiberene cuparene p-cymen-8-o1 trans oxide of car yoph yllene methyl eugenol meth yl isoeugenol neo-i nterrnedeol v trans cad inol elemicin asarone isoasarone 3,4,5 tr irnethoxy benzaldeh yde neral geranial geranyl acetate geraniol
1
2
8.03 11.36 0.16 0.27 0.65 1.08 0.29 0.91 0.56 0.06 0.15 0.31 1.70 2.55 1.13 0.34 0.88 2.65 5.44 1.40 0.39 1.14 0.29 0.11 0.17 0.96 0.21 7.94 0.13 9.15 0.28
2.50 2.94 0.05 0.12 18.67 5.62 0.21 2.80 2.14 0.04 0.24 0.45 0.28 3.10 0.07 0.03 0.6 5 8.37 0.90 4.67 0.54 0.19 0.35 1.15 0.1 7 0.30 0.05 29.42 0.02 2.46 0.02
3
5
4
6
6.28
8.12 0.12 0.25 12.74 5.96 0.3 5 1.11 0.81 0.08 0.39 0.35 0.76 2.14 0.16 0.14 1.10 7.23 1.64 2.82 1.04 7.47 0.26 3.76 1.46 0.20 tr 22.9 5 0.1 7 3.42 0.04
1.14 0.66 0.02 0.19 0.12 0.02 0.06 0.56 0.30 2.64 0.90 0.28 0.72 2.20 1.17 1.34 1.69 0.20 1.64 1.56 0.25 0.25 36.05 0.16 1.92 1.24 3.90 4.60 5.70 74.20
0.04 0.10 0.02 0.02
0.90 1.42 0.07 0.13 0.23 2.97 0.7 3 0.17 1.56 0.26 0.51 2.58 1.62 0.12 0.15 56.44 0.31 3.54 5.53 0.06 1.20 9.80 65.60
4.36 5.68 0.10 0.18 4.57 2.28 0.23 0.47 0.04 0.05 0.20 0.34 0.42 1.87 0.12 0.36 1.00 4.78 2.00 2.24 0.79 0.02 0.69 1.15 0.77 0.08 tr 33.90 0.42 8.33 0.4 7
1 = C. martinii; 2 = C. pendulus; 3 = Jamrosa; 4 = C. comm utatus; 5 = C. nardus; 6 = CKP25; tr = trace « 0.01 %)
B. K. Tyagi et al. tection of 5 hrs. When compared with controls, the protection from bites in tests has always been more than 89%. Cymbopogon nardus (95.7%) proved to be the most repellent essential oil, followed by Jamrosa (94.84%), C. commutatus (94%), C. martinii (93.4%), C. pendulus (89.56%) and CPK2S-RRL Jammu (89.18%). Of all the essential oils C. martinii prevented all mosquito biting within 6 ft. distance, followed by C. commutatus, C. nardus, C. pendulus and the hybrid species all being effective up to four feet distance. Some essential oils such as those of C. pendulus, C. commutatus and the Lemongrass variety of CKP2SRRL Jammu gave a feeling of slight itching on the sites of mosquito feeding on the hands in the laboratory evaluation. Mosquito repellent activities of Cymbopogon species are comparable to those of Ocimum, Betula, Geranium and Pinus (Chokechaijaroenporn et al., 1994). Although, the essential oils of Cymbopogon are of high volatility when compared with DEET and DMP, nonetheless, some of the species examined here adequately protect from mosquito bites for a good period of time. In fact the protection rate from mosquito feeding and landing in at least four species, viz., C. nardus, Jamrosa, C. commutatus and C. martinii can be paralleled with those of Achillea millefolium and DEET or DEPA (Tunon et al., 1994; Rao and Rao, 1991). Gas chromatographic and GC-MS analyses manifested the occurrence of several active compounds possibly known for their potential, repellent activities against a variety of blood sucking insects (e. g. camphene, myrcene, limonene, trans-caryophyllene, methyl eugenol and 1,8-cineole) (Masui and Kochi, 1974; Hubert and Weimer, 1985; Nutting et al., 1974). It is, however, interesting to note from our investigations that the two species, viz. C. nardus and C. commutatus which are found to greatly repel the vector mosquitoes contain geraniol (65.6% to 74.2%) as a major chemical constituent. Certain other less known repellent compounds linke (Z)-~-ocimine and (E)-~-ocimine are also present in small proportions. Camphene, limonene and myrcene containing species were found to have less repellency. Elemicin containing Jamrosa proved a good repellent to vector mosquitoes. It is, therefore, of further interest to ponder upon the role of such compounds which have earlier got little attention from repellency point of view (Table 5).
329
Prof. M. L. Sharma, Punjab University, Chandigarh for identifying the Cymbopogon species. For his cogent suggestions on statistical matters, authors are grateful to Mr. T. Ramnath, Assistant Director, DMRC. Thanks are also due to Dr. ]. C. Chalchat, Laboratory of Chemistry of Essential Oils, University Blaise Pascal, Clermont, France for his help in chemically analyzing some of the plant species studied in this paper.
References Bowen, M. E: The sensory physiology of host seeking behaviour in mosquitoes. Ann. Rev. Entomol. 36: 139-158, 1991. Chokechaijaroenporn, 0., Bunyapraphatsara, N. and Kongchuensin, S.: Mosquito repellent activities of Ocimum volatile oils. Phytomedicine 1: 135-139, 1994. Hubert, T. D., Weimer, O. E: Anti-repellent terpenoids from Melampodium divaricatum. Phytochemistry 24: 11971198, 1985. Masui, K., Kochi, H.: Camphor and tricyclodecane in deodorants and insect repelling compositions. Japan Kokai 74: 100239. 1974. Through Chem. Abstr. 48: 165913 F (1976). Nutting, W.L., Blum, M. S., Fales, H. M.: Behaviour of the north American termite, Tenuirostritermes tenuirostris, with special reference to the soldier frontal gland secretion, its chemical composition, and its use in defence. Psyche 81: 16, 1974. Through Chem. Abstr. 82: 1995 H (1975). Rao, S. S., Rao, K. M.: Insect repellent N,N-diethylphenylacetamide: an update. J. Med. Entomol. 28: 303-306, 1991. Rutledge, L. c., Gupta, R. K., Piper, G. N., Lowe, C. A.: Studies on the inheritance of repellent tolerances in Aedes aegypti. J. Amer. Mosquito Control Assoc. 10: 93-100,1994. Shahi, A. K., Sen, D. N.: The grasslands: their distribution, existence and utilization, especially under arid environment. Indian Rev. Life Sci. 10: 3-26, 1990. Sharma, V. P., Mehrotra, K. N.: Malaria resurgence in India: a critical study. Soc. Sci. Med. 22: 835-845, 1986. Sukumar, K., Perich, M.]., Boobar, L. R.: Botanical derivatives in mosquito control: a review. J. Amer. Mosquito Control Assoc. 7: 210-237, 1991. Tunon, H., Thorsell, W., Bohlin, L.: Mosquito repelling activity of compounds occuring in Achillea millefolium L. (Asteraceae). Econ. Botany 48: 111-120, 1994. Tyagi, B. K.: Alternate control strategies for mosquito vectors of human diseases in India. [In: Perspectives in Entomological Researches. Ed. O. P. Aggarwal], Scientific Publishers, Jodhpur: 359-372, 1994. Tyagi, B. K.: Control of malaria vectors in India. Indian Rev. Life Sci. 12: 211-238, 1992.
Address Acknowledgement
Authors are thankful to Dr. R. C. Chaudhary, Deputy Director & Officer-In-Charge, Desert Medicine Research Centre, Jodhpur for critically going through the manuscript. Thanks are also due to Dr. S. N. Sharma, RRL, Jammu-Tawi for his help in extracting and providing essential oil samples, and to
B. K. Tyagi, Deputy Director, Desert Medicine Research Centre, Post Box 122, New Pali Road, Jodhpur342005, India Tel.: 0291-741022; Fax India-0291-741022; e-mail: [email protected]
_
Phytomedicine
Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of Malaria, Filariasis and Dengue Fever in India B. K. Tyagi ', A. K. Shahi- and B. L. KauF lDesert Medicine Research Centre, Jodhpur, India 2Regional Research Laboratory, CSIR, Jammu Tawi, India
Summary Essential oils of four species and two hybrid varieties of Cymbopogon grasses were evaluated for their repellent properties against the major vector mosquitoes, namely, Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti, both in laboratory and field. The magnitude of repellency in the Cymbopogon essential oils was found to be of moderate to high order. All grass species protected completely from mosquito bites for 4 hrs, whereas C. nardus provided protection for as much as 8-10 hrs overnight. Key words: Cymbopogon spp., essential oil, repellent activity, mosquitos, malaria, filariasis, Dengue fever
Introduction Mosquitoes belonging to genera Anopheles, Culex and Aedes are serious vectors of several important diseases in India, such as malaria, filariasis, Japanese encephalitis and dengue (including dengue haemorrhagic fever and dengue shock syndrome) (Tyagi, 1994). So far vector control has been largely carried out by employing residual insecticides, but due to development of resistance by these vectors against most of the conventional insecticides, both the abatement of vector population and cessation of disease transmission has become very difficult (Tyagi, 1992; Sharma and Mehrotra, 1986). At present, one of the most effective methodologies to prevent transmission of mosquito-borne infections is to use repellents which over-sensitivitize the chemoreceptive organs of the vectors and disorient them from seeking blood meal from the human host (Bowen, 1991). Among various mechanisms involved in this process the antifeedent effect of repellents is the most important as it works at a distance and keeps the vectors away from feeding (Sukumar et al., 1991). Although, certain chemically synthesized repellents like N,N-diethyl-rn-toluamide (DEET) and Dimethylphthalate
(DMP) have already been used in dispelling bloodsucking insects, mites, ticks and leeches etc., development of partial tolerance of A. aegypti against DEET (Rutledge et al., 1994) has warranted an urgent need to look for some repellent materials from natural resources. The genus Cymbopogon (Poaceae), comprises 27 species found all over India (Shahi and Sen, 1990). Many a species of this genus are well known for their repellent characteristics against different kinds of haematophagous insects, besides being used in medicine and perfumery. The present paper describes the repellent behaviour of some of these species widely growing in north-western parts of India.
Materials & Methods Test Material
Four plant species, viz. Cymbopogon commutatuslwinterianus Jowitt., C. martinii (Roxb.) Wats., C. pendulus (Nees ex Steud.) Wats., C. nardus (L.) Rendie and
B. K. Tyagi et al.
325
cages from the field to the laboratory where they were allowed to lay eggs. The freshly emerged female mosquitoes were starved for 2-3 days before use in the experiments. Hands of the volunteer human test subjects (# 3; males only) were first washed with liquid soap and subsequently cleaned with 70 % ethanol. After air drying, the left hand of the test person was smeared up to the wrist with 0.25 ml of the essential oil. Then the hand was introduced into the mosquito cage harboring 20 females, at regular intervals of 1, 4, 6 and 8 hours for 15 minutes each time. Controls were carried separately with another male volunteer (# 1) without any essential oil application. Each test and control was replicated in triplicate. • Test procedure: Th e antifeedant effect wa s exp ressed as 100 (1-T/C)%, where T is the mean of sucking mosquitoes after 4, 6 an d 8 hrs ., and C is the corresponding number of blood sucking mosquitoes in the control. The number of mosquitoes landing without sucking was noted and all landing mosquitoes (LT), were expressed in two ways, (i) as a percentage of all mosquitoes, landing (LT), and feeding (FT), i. e., 100 (LT/[LT + FT]), and (ii) landing in the test (LT) compared with the total landings in .the control (LC) expressed as (LT/LC) x100. All landings were recorded with the help of a counter.
two hybrids named Jamrosa and Lemongrass (coded as CKP2s-RRL Jammu), collected from different parts of the north-eastern and north-western parts of the country, were assayed for their repellent activities against A. stephensi, C. quinquefasciatus and A . aegypti. All the mosquito species were collected from the residential settlements near the DMRC campus. Extraction of esse ntial oils
Various Cymbopogon species were collected from the field, while the hybrid varieties were developed and grown in the experimental farms of the Regional Research Laboratory, Jamnu. The essential oils fro m all plant materials were obtained by hydrodistillation of the foliage parts usin g a Clevenger apparatus. To obtain pure forms of th e volatile oils, triplicate dist illation was carried out in succession of each lot of the fresh material weighing 500 gm. The combined oils were eventually dried over anhydrous sodium sulfate before being subjected to experimentation. Assessment of repellent activities
• Laboratory assay: All tests were conducted at room temperature 28°C ± 1 and relative humidity 50-60 %. Feral female mosquitoes were brought in Barraud's
Tab le 1. Percentage landings of vector mosquitoes against essential oils of different Cymbopogon species (figures in parenthesis is the denominator refering to total landings). Plants Species/variety
C. commutatus C. martinii C. pendulus C. nardus CKP25 RRL jarnrosa
Pooled
Vector species
Pooled (aver.)
A. stephensi
C. fatigans
A. aegypti
55.5 (9) 36.0 (25) 44.4 (36) 60 .0 (10) 44.4 (9) 40 .0 (20)
43.7 (16) 38.0 (19) 42 .8 (21) 36.6 (11) 38 .0 (19) 47.3 (38)
41.6 (12) 42.2 (42 ) 50. 0 (58) 40 .0 (20) 37.9 (29) 46.1 (52 )
45.9 (37) 41.9 (86) 47.0 (115) 43.9 (4 1) 40.4 (57) 45.5 (110)
44.0 (109)
43 .5 (124)
45 .1 (213)
44.4 (446 )
Table 2. ANOVA for landing of major vector species against the repellent effect of Cymbopogon species. Source of Variation Between vector spp . Between grass spp. Interaction Error ** p < 0.01; *p < 0.05
Degree of freedom
2 5 10 36
Sum of aqua res
M. S. S.
Variance ratio
351.15 651.94 241.09 248.20
175.57 130.39 24 .11 6.89
25.48 ** 10.92 ** 3.50*
326
Evaluation of repellent activities of Cymbopogon
... landing
20 ...---- ---=- - - - - - - - - - - - - - - - - - - - --,
15
10
5
o
commutatua
mart in i;
pendu lua
nardus
Jamroaa
CKP-215
Fig.1. Percentage landings of the three major vector mosquitoes against the six Cym bopogon essential oils in the laboratory evaluation.
Cymbopogon spec ies •
A.stephensl
~ C.qulnquefasc latus
D
Ae. aegyptl
• Field assay: Repellent activity of the vario us essential oils was tested against C. quinquefasciatus only. T he volati le oil (0.5 m!) was applied on the bare feet, ha nds and for ehead of a volun teer human subject w ho was made to sleep on a cot. Overnight sampling of females was carried out with the help of an aspi rator and a flash light. Co ntrol without any application of th e volatile oil was kep t sepa ra tely. Another experime nt to assess the distance ~f effectiveness of repellency by each essential oil was also carried ou t betwee n 8 p . m. and 9 p. m. against C. quinquefasciatus feeding on h uman subjects sitti ng or lying
on a cot at fixed distances of 2, 4, 6, 8 an d 10 ft., respectively, fro m the source of essen tial oil. • Chemical analysis: The chemical analysis of th e oils were ca rried out by using GC and GC/MS. T he apparatus was a DELCI 121 C gas chromatography (F. I. D .) WCOT capillary column wit h fused silica (25 m x 0.3 mm) and CP WAX 51 sta tionary phase. The temperature was programmed fro m 50 °C with 5 min initial hold and then to 210 °Clmin, using N 2 as the carrier gas . T he identification of th e oil components was carried out by comparing the retention indices of standard substances wi th those of substances present in
Table 3. Antifeedent activity (AFA) of different Cym bopogo n species against three major vector mosquitoes in the laboratory. Grass species C. commutatus C. martinii C. pendulus C. nardus
CKP25 Jamrosa
AFA(%)
Vector species A. stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A . stephensi C. quinquefasciatus A. aegypti A. stephensi C. quinquefasciatus A. aegypti A. step hensi C. quinquefasciatus A. aegypti
Average
1 hr
4 hr
6 hr
8 hr
AFA(%)
100.0 100.0 100.0 100.0 100.0 97.1 99.0 100.0 94.1 100.0 100.0 100.0 100.0 100.0 100.0 100.0 98.7 98.8
100.0 100.0 100.0 97.1 98.6 91.8 99.1 100.0 89.6 100.0 98.7 93.7 100.0 94.2 98.1 97.3 90.5 87.2
98.9 96.1 83.3 94.7 98.1 90.4 90.2 92.6 87.1 96.3 98.5 90.9 97.9 97.7 91.2 93.3 95.0 91.5
97.7 95.3 91.5 99.9 90.9 85.0 92.1 92.6 74.4 96.3 92.8 94.1 94.1 92.7 73.8 94.3 87.6 71.9
99.0 97.0 94.8 96.0 96.5 90.9 94.1 96.2 85.4 98.5 97.4 95.1 98.2 99.4 90.7 95.8 93.3 88.1
B. K. Tyagi et al.
327
% antifeeding 100 - r - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
95
90
85
80 +-------,.------r-----,------,-------1
Fig. 2. Percentage antifeeding behaviour of the three vector mosquito species against the six Cymbopogon essential oils in the laboratory evaluation.
commutatus
martini!
-
A. stephensi
the mixture. The identification was confirmed for GC/MS were: ionization energy 70 eV, fused silica WCOT capillary column (50 mx 0.3 mm), temperature programmed from 50 DC to 230 DC at 3 DC/min using He as the carrier gas.
Results and Discussion When the landing in the test alone was considered, maximum protection was recorded against the group of CKP 25 (40.4%), C. martinii (41.9%) and C. nardus (43.9%), followed by another group of Jamrosa (45.5%), C. commutatus (45.9%) and C. pendulus (47%) (Table 1). This was amply substantiated in another evaluation when landings of vector species were compared with those in the control (Fig. 1). By and large A. stephensi and C. quinquefasciatus were easily
Table 4. ANOVA for feeding activity of major vector species against different Cymbopogon species. Source of variation Sum of squares Variance ratio
Degree of freedom M. S. S.
Between vector spp. 393.86 23.45*" Between grass spp, 666.78 15.88':'" Interaction 133.56 1.59 Error 302.32
2 196.93
** p
< 0.01
5 133.36 10 13.36 36 8.398
pendulus
nardus
CKP-25
Jamrosa
Cymbopogon species -+- C. quinquefasciatus
--*- Ae. aegypti
repelled, particularly by C. nardus, CKP25 and C. commutatus. The vector species A. aegypti appeared most refractory among all the species and only C. commutatus and C. nardus could disorient its landing behaviour to an appreciable level. The analysis of variance determined for the vector landings against different Cymbopogon species elucidates that behaviour of the vector species differ from one species of plant to another quite appreciably (Table 2). In the laboratory evaluation all the essential oils invariably offered more than 85% repellent activity against all the vector mosquitoes (Table 3). Although none of the volatile oils seemed to deter mosquitoes from feeding on the inserted hand for a full 8 hours, nevertheless, the antifeedant activity of most of the essential oils was high. While the C. commutatus essential oil offered full protection for the initial four hours against all three vector species, both C. nardus and CKP2S- RRL Jammu hybrid variety of Lemongrass prevented feeding by A. stephensi for the initial four hours. Essential oils from the latter two plant materials protected feeding by all vectors for the initial 1 hour. The analysis of variance (ANOVA) determined for feeding activity of major vector species against different Cymbopogon material (Table 4) supports the view that the feeding behavior of various mosquito species differs, not only against different plant material, but also among themselves. Consequently, the repellent activity of different plants can be arranged as follows: C. nardus (97%), C. commutatus (96.93%), CKP25 RRL Jammu (96.1 %), C. martinii (94.46%), Jamrosa (92.4%) and C. pendulus (91.7%) (Fig. 2). Field experiments with human bait and C. quinquefasciatus as the vector species suggests a maximum protection of 9 hrs by C. nardus, followed by Jamrosa for 8 hrs and C. commutatus and C. martinii for 6-7 hrs (Fig. 3). The Lemongrass variety offered the least pro-
328
Evaluation of repellent activitie s of Cymbop ogon
% protection from feeding 120 ...--- - - - - - - - - - = . - - - - - - - - - - - - - --.
100 +-----+---+----= ~:----=----::~----.,::--.
80
--
60 -
------ -·· -- -.----.---.-- - --....----..... - - -
- - - -- .....-- -- -- -.-..----....- --.----.-..-
40 .-..-.-- - -.- --.- - -
- ---
-\--+ .<-
-..---.- -.-.- - -- -
20
O+--. - -..-----.----r----,---.,--,--.,...---.----.-----1 2
1
3
4
5
6
7
9
8
10
11
Hours -
commutatu8
-I- marti nI!
--*- pendulus
-fr-
nllrdus
--- CKP 26
- - Jamrosa
12
Fig. 3. Percentage protection from biting of C. quinquefasciatus against various Cym bopogon essential oils during overnight peri od in the field evaluation.
Table 5. Percentage composition of various major chemical compounds analyzed in the Cymb opogon species.
Cymbop ogon species
Compound a-pinene camphene ~ -pin cne
sabinene myrccne limoncne 1,8-cineole (Z ) -~-ocimene (E)-~-ocimene
p-cyrnene terpinolene camphor linalool trans-caryphyllene tra ns-bergamotene gymnomitrene o-rerpineol borneol/terpineol ~-sclinene
Zingiberene cuparene p-cymen-8-o1 trans oxide of car yoph yllene methyl eugenol meth yl isoeugenol neo-i nterrnedeol v trans cad inol elemicin asarone isoasarone 3,4,5 tr irnethoxy benzaldeh yde neral geranial geranyl acetate geraniol
1
2
8.03 11.36 0.16 0.27 0.65 1.08 0.29 0.91 0.56 0.06 0.15 0.31 1.70 2.55 1.13 0.34 0.88 2.65 5.44 1.40 0.39 1.14 0.29 0.11 0.17 0.96 0.21 7.94 0.13 9.15 0.28
2.50 2.94 0.05 0.12 18.67 5.62 0.21 2.80 2.14 0.04 0.24 0.45 0.28 3.10 0.07 0.03 0.6 5 8.37 0.90 4.67 0.54 0.19 0.35 1.15 0.1 7 0.30 0.05 29.42 0.02 2.46 0.02
3
5
4
6
6.28
8.12 0.12 0.25 12.74 5.96 0.3 5 1.11 0.81 0.08 0.39 0.35 0.76 2.14 0.16 0.14 1.10 7.23 1.64 2.82 1.04 7.47 0.26 3.76 1.46 0.20 tr 22.9 5 0.1 7 3.42 0.04
1.14 0.66 0.02 0.19 0.12 0.02 0.06 0.56 0.30 2.64 0.90 0.28 0.72 2.20 1.17 1.34 1.69 0.20 1.64 1.56 0.25 0.25 36.05 0.16 1.92 1.24 3.90 4.60 5.70 74.20
0.04 0.10 0.02 0.02
0.90 1.42 0.07 0.13 0.23 2.97 0.7 3 0.17 1.56 0.26 0.51 2.58 1.62 0.12 0.15 56.44 0.31 3.54 5.53 0.06 1.20 9.80 65.60
4.36 5.68 0.10 0.18 4.57 2.28 0.23 0.47 0.04 0.05 0.20 0.34 0.42 1.87 0.12 0.36 1.00 4.78 2.00 2.24 0.79 0.02 0.69 1.15 0.77 0.08 tr 33.90 0.42 8.33 0.4 7
1 = C. martinii; 2 = C. pendulus; 3 = Jamrosa; 4 = C. comm utatus; 5 = C. nardus; 6 = CKP25; tr = trace « 0.01 %)
B. K. Tyagi et al. tection of 5 hrs. When compared with controls, the protection from bites in tests has always been more than 89%. Cymbopogon nardus (95.7%) proved to be the most repellent essential oil, followed by Jamrosa (94.84%), C. commutatus (94%), C. martinii (93.4%), C. pendulus (89.56%) and CPK2S-RRL Jammu (89.18%). Of all the essential oils C. martinii prevented all mosquito biting within 6 ft. distance, followed by C. commutatus, C. nardus, C. pendulus and the hybrid species all being effective up to four feet distance. Some essential oils such as those of C. pendulus, C. commutatus and the Lemongrass variety of CKP2SRRL Jammu gave a feeling of slight itching on the sites of mosquito feeding on the hands in the laboratory evaluation. Mosquito repellent activities of Cymbopogon species are comparable to those of Ocimum, Betula, Geranium and Pinus (Chokechaijaroenporn et al., 1994). Although, the essential oils of Cymbopogon are of high volatility when compared with DEET and DMP, nonetheless, some of the species examined here adequately protect from mosquito bites for a good period of time. In fact the protection rate from mosquito feeding and landing in at least four species, viz., C. nardus, Jamrosa, C. commutatus and C. martinii can be paralleled with those of Achillea millefolium and DEET or DEPA (Tunon et al., 1994; Rao and Rao, 1991). Gas chromatographic and GC-MS analyses manifested the occurrence of several active compounds possibly known for their potential, repellent activities against a variety of blood sucking insects (e. g. camphene, myrcene, limonene, trans-caryophyllene, methyl eugenol and 1,8-cineole) (Masui and Kochi, 1974; Hubert and Weimer, 1985; Nutting et al., 1974). It is, however, interesting to note from our investigations that the two species, viz. C. nardus and C. commutatus which are found to greatly repel the vector mosquitoes contain geraniol (65.6% to 74.2%) as a major chemical constituent. Certain other less known repellent compounds linke (Z)-~-ocimine and (E)-~-ocimine are also present in small proportions. Camphene, limonene and myrcene containing species were found to have less repellency. Elemicin containing Jamrosa proved a good repellent to vector mosquitoes. It is, therefore, of further interest to ponder upon the role of such compounds which have earlier got little attention from repellency point of view (Table 5).
329
Prof. M. L. Sharma, Punjab University, Chandigarh for identifying the Cymbopogon species. For his cogent suggestions on statistical matters, authors are grateful to Mr. T. Ramnath, Assistant Director, DMRC. Thanks are also due to Dr. ]. C. Chalchat, Laboratory of Chemistry of Essential Oils, University Blaise Pascal, Clermont, France for his help in chemically analyzing some of the plant species studied in this paper.
References Bowen, M. E: The sensory physiology of host seeking behaviour in mosquitoes. Ann. Rev. Entomol. 36: 139-158, 1991. Chokechaijaroenporn, 0., Bunyapraphatsara, N. and Kongchuensin, S.: Mosquito repellent activities of Ocimum volatile oils. Phytomedicine 1: 135-139, 1994. Hubert, T. D., Weimer, O. E: Anti-repellent terpenoids from Melampodium divaricatum. Phytochemistry 24: 11971198, 1985. Masui, K., Kochi, H.: Camphor and tricyclodecane in deodorants and insect repelling compositions. Japan Kokai 74: 100239. 1974. Through Chem. Abstr. 48: 165913 F (1976). Nutting, W.L., Blum, M. S., Fales, H. M.: Behaviour of the north American termite, Tenuirostritermes tenuirostris, with special reference to the soldier frontal gland secretion, its chemical composition, and its use in defence. Psyche 81: 16, 1974. Through Chem. Abstr. 82: 1995 H (1975). Rao, S. S., Rao, K. M.: Insect repellent N,N-diethylphenylacetamide: an update. J. Med. Entomol. 28: 303-306, 1991. Rutledge, L. c., Gupta, R. K., Piper, G. N., Lowe, C. A.: Studies on the inheritance of repellent tolerances in Aedes aegypti. J. Amer. Mosquito Control Assoc. 10: 93-100,1994. Shahi, A. K., Sen, D. N.: The grasslands: their distribution, existence and utilization, especially under arid environment. Indian Rev. Life Sci. 10: 3-26, 1990. Sharma, V. P., Mehrotra, K. N.: Malaria resurgence in India: a critical study. Soc. Sci. Med. 22: 835-845, 1986. Sukumar, K., Perich, M.]., Boobar, L. R.: Botanical derivatives in mosquito control: a review. J. Amer. Mosquito Control Assoc. 7: 210-237, 1991. Tunon, H., Thorsell, W., Bohlin, L.: Mosquito repelling activity of compounds occuring in Achillea millefolium L. (Asteraceae). Econ. Botany 48: 111-120, 1994. Tyagi, B. K.: Alternate control strategies for mosquito vectors of human diseases in India. [In: Perspectives in Entomological Researches. Ed. O. P. Aggarwal], Scientific Publishers, Jodhpur: 359-372, 1994. Tyagi, B. K.: Control of malaria vectors in India. Indian Rev. Life Sci. 12: 211-238, 1992.
Address Acknowledgement
Authors are thankful to Dr. R. C. Chaudhary, Deputy Director & Officer-In-Charge, Desert Medicine Research Centre, Jodhpur for critically going through the manuscript. Thanks are also due to Dr. S. N. Sharma, RRL, Jammu-Tawi for his help in extracting and providing essential oil samples, and to
B. K. Tyagi, Deputy Director, Desert Medicine Research Centre, Post Box 122, New Pali Road, Jodhpur342005, India Tel.: 0291-741022; Fax India-0291-741022; e-mail: [email protected]