P arasitic serious health problem in most diseases represent a developing countries. In par- ticular, head lice represent a major problem especially [or primary school children. The higher prevalence among infested children correlates directly with those in lower socio-eco- nomic groups within the community.
In addilion to long hair, family size, age, crowding and personal hygiene contribute to the higher rates of pediculosis among this social group (Sinniah et al., 1983).
Treatmenl of lice with pediculi- tide compounds such as Permethrin,
0 A THE POTENTIAL - EFFECTIVENESS
OF ESSENTIAL OILS IN THE CONTROL OF HUMAN HEAD LICE IN
MOROCCO
MOUHSSEN LAHLOU, RACHED BERRADA, ABDELAZIZ AGOUMI, MOHAMMED HMAMOUCHI
Twenty-four essential oils isolated from Moroccan aromatic plants and 15 qf their isolated components were tested in vitro on Pediculus humanus capitis (head lice) for licicidal and niticidal properties. Four oils were found to be effective in the laboratory when a#&ed in two ways: microatmosphere and direct application. The time required by an essential oil to act potently is defiendent upon its composition and the technique used.
malathion and DD’l; is unsuccessful, suggesting that the head louse has probably developed resistance (Arzube, 1968; Sagaidakovskii, 197 1; Sinniah and Sinniah, 1982; Chosidow et al., 1994; Remy-Kristensen et al., 1995).
‘I‘he search for new natural insecticidal compounds derived from plants repre- sents an important scientific challenge.
Plants are a good source of biolog- ically active natural products. These natural compounds are all biodegrad- able, renewable and less toxic.
Aromatic plants or their extracts have been described as a source of treatment for various infectious diseases, and also evaluated for their antiparasitic prop- erties (Hmamouchi et al., 1998; 2000).
Many tests arc required for evalu- ating the effectiveness of pediculicides (Agourni et al., 1987; Rousset et al., 1988; Rousset and Agoumi, 1989;
Agoumi, 1992; Combescol et al., 1996;
Agoumi and Bou Kachabine, 1999).
l‘he potential effectiveness of essential oils and their main components as a treatment for head lice has been reported previously (Gauthier et al., 1989; Veal, 1996).
This article describes the method- ology employed to detect the niti- tidal and licicidal effects on Pediculus humanus cufiitis by 24 essential oils obtained from Moroccan plants and fifteen of their active oil
components.
Plant collection and essential oil extraction
Plants were collected between 1992 and 1994. horn different regions of Morocco. They were identified taxo- nomically at the National Scientific Institute, Department of Vegetal Biology Rabat. A representative speci- men of each species was deposited in the herbarium of the Faculty of Medicine and Pharmacy, Rabat. All of the plants tested are presently used in the indigenous systems of medicine (‘IBble 1) (Charnot, 1945; Bellakhdar, 1978; Hmamouchi, 1999).
Samples of air-dried leaves (150 g) of each individual plant species were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus recommended by the French Pharma- copoeia. CC analysis of the oils thus obtained was performed using a fused silica capillary column (25 m x 0.25mm), coated with OV-101, with the oven temperature programmed from 50 to 250°C at a rate of 5Wmin.
GUMS analysis was carried out on a Hewlett-Packard Capillary GC-quadru- pole MS system (model 5970) fitted with a 25 m x 0.23 mm f&ed-silica col- umn coated with DB-I and using the same gas chromatographic parame- ters. Identification of the compounds was carried out by the comparison of both the GC retention times and of MS data against those of the reference standards (Fluka).
Lice and nits
Head lice, Pediculus humanus ca$itis and their nits were collected from infested children aged between 3 and 12 years who were hospitalized in Rabat Children’s Hospital. Nits and lice (I 0 of each) were tested using identical pyrex glass Petri dishes (5.5cm diameter, 1.5 cm height).
Biological tests
Tests were carried out according to Gauthier et al. (1989), using two tech- niques:
1. M~cmatmosfil~ere - In this method, the whole oil or component evaporates into the atmosphere of a closed Petri dish, saturating it with vapour, whereby the volatile oil can exert an inhibitory effect on lice and hatching nits.
Different volumes of the essential oils (3.10-50 ~1) or individual isolated components (10 ~1) were dispensed into a small container within the Petri dish. Nits and lice were deposited on fil- ter paper using a u-shaped glass-rod.
Three perforations were made in the lids of the Petri dishes to allow for vcn- tilation then covered with moist gauze.
The Petri dishes were incubated at 28°C in 70% relative humidity (optimum con- ditions for the development of nits and lice) for 9 days. Ihe lice were observed every hour thcrcafter for 6 h and then at 24-h, after which time death could not necessarily be attributed to the effect of the volatile components or whole oils. The nits were observed each day for 9 days until hatching. The resul- tant larvae were tested for viability by placing them on the back of a hand and noting whether they began to feed.
2. Direct application - In this technique, 0.5 ~1 of the essential oil or one of their main components was applied directly onto t,he nits and lice, then observed.
The percentage mortality was correct- ed using Abbott’s (1925) formula:
n% deaths
= [(test - control)/control] x 100
Active doses of essential oil at dilution factors of 1:2, 1:4 and 1:10 (in alcohol) were also tested using the direct application technique. All tests were duplicated and the average of the two assays taken as the final result. In all cases, lethal doses that were 100% effective (LD,,,) were
determined.
Screening of essential oils Lice
Tests on lice using the microatmos- phere technique with 50~1 (‘Fable 2), 25, 12.5 and 6.25 ~1 of essential oil were performed. Using 3.10 p,l of essential oil (Table 3), the results demonstrated the effectiveness of the essential oils from Chenofiodium amhro- sioides, Mentha pulegium and T!~ymus broussonettii (LD,,, 1 h = 3.10 pl), fol- lowed by Ruta chalepensis (LDloo 2 h = 3.10 ~1). Using the direct applica- tion technique, the lice were dead with- in 15 min of exposure to an application of 0.5 kl.
Nits
Using the microatmosphere technique, tests were done using 50 ~1 (Table 4), 25, 12.5 and 6.25 ~1 of essential oil. With 3.10 ~1 (Table 5), the essential oils from Cheno$odium ambrosioides, Mentha bulegium, Thyus broussonettii, Otiganum xm$actum and Ruta chalefiensis were the most potent among those tested (LDloo
= 3.10 ~1). These oils were found to be 3s equally potent using the direct appli- ration technique (Tdble 5).
Niticidal tests performed with diluted active essential oil (Table 6) demonstrated the effectiveness of essential oils from Chenopodium, ambro- sl:ol:des, Mentha @legl:um and Kuta chalepensis. There was zero percent hatching of nits in both the microat- mosphere and direct application test methods. With a 1:4 dilution of essen- tial oil from Thymus broussonettii, 20% of the nits hatched using the direct appli- cation method. At a 1 :lO dilution, 20% of nits hatched using the micro- atmosphere method and 30% using the direct application. With a I:4 dilution of essential oil from Origanum compacturn, 20% of the nits hatched using the microatmosphere test method.
a-pirrene
CH,O OH eugenol
OH
carved
linalol
P3 0
CHO
anisaldehyde
c
‘0
menthone
i’
CHO
citronella1
c menthol OH
9 \
fkaryophyllene
72 Ii OH
63 /
myrtenol
r- , CHO
:_
0 I ,&cineole
c \
limonene
cr”
fcnchone
neral Fig. I Main essential oil components tested on Pediculus humanis cupitis nits and lice
Activity of essential oil components on lice and nits
In order to search for the active compo- nents responsible for niticidal and licicidal activity and to try and explain the correlation between chemical com- position and activity, we studied the main components of each oil (Fig. 1) that had shown toxicity towards nits and lice:
1.
2.
3.
4.
5.
alcohols: linalol, (-)-menthol, carveol, myrteuol and geraniol aldehydes: anisaldehyde, euge- nol, citronella1 and citral (geranial and neral)
hydrocarbons: limonene, (6)- tmns-caryophyllene and cu-pinene kctoncs: (-)-mcnthone and (-)- fenchone
oxide: I,%cineole.
The main components of t.he essential oils used in the trials were determined by CC/MS (Tables 7-12).
The results of licicidal activity are
shown in Table 13. ‘l‘he products can be classified in five groups:
I.
2.
3.
4.
5.
products showing no toxicity, i.e.
anisaldehyde
products revealing a licicidal activ- ity only after long exposure, i.e.
geraniol and (-)-truns-caryophyl- lene (LD,,, G h = 10 ~1)
substances demonstrating a weak licicidal action, i.e. citral and 01- pinene (LDIoo 4 h = 10 f.r,l) those showing a medium licicidal effect, i.e. myrtenol, carve01 and eugenol (LDroo 3 h = 10 ~1) components revealing a high licicidal activity, i.e. (-)-menthol, (-)-fenchone, (-)-menthone, 1,8- cineole, linalol, citronella1 and limonene (LDloo l-2 h = 10 ~1).
For niticidal tests, Table 14 clearly shows the importance of the alcohol, aldehyde and ketone compounds using the microatmosphere and direct appli- cation methods. The nits were highly sensitive to all these components.
Hydrocarbon compounds like (-)-truns- caryophyllene and limonene were more active in the distemper technique (0% nits hatched) than in the micro- atmosphere method.
In addition to phenolic and pheno- lit ether compounds, ketones and the oxide l&cineole have also demonstrated their effectiveness against lice (Veal, 1996). The components l,&cineole, linalol, men- thone, menthol and limonene have already been reported as being larvicides and ovicides (Laurent et al., 1997). Compounds such as a-pinene, 1,X cineole and linalol have already been shown to have a potent effect on head lice (Gauthier et al., 1989).
The essentiat oils from Men&
pulegiurn and Thymus broussonettii have showed efficacy in previous
” 2001 HARCOURT PUQLlSHLRS LTD
100 100
A. 0
100 . . .
antimicrobial tests (Tantaoui-Elaraki et al., 1993; Vokov et al., 1993) whilst the essential oil of Chenopodium ambro- sioides had a fungicidal activity when tested against dermatophytes (Kishore et al., 1993). Its component ascaridol exhibits potent antiparasitic and anthelmintic properties (Kliks, 1985).
We can conclude that essential oils from M&ha pulegium and Thymus brous- yonettii (Lamiaceae), Chenofiodium ambro- rioides (Chenopodiaceae) and Ruta chalepensis (Rutaceae) possess the most powerful licicidal and niticidal activities towards the head louse, Pediculus humanus capitis, as shown by results from the in vitro experimental methods of microatmosphere and direct applica- tion. This activity was evident firstly by their lower lethal concentrations and contact time on lice, and secondly, by their inhibition of nit hatching over the 9-day of tests.
‘l‘he in vitro tests must now be fol- lowed by in viva investigations using
children infested with head lice. The active essential oils found during this study could be used in their active dilu- tions within lotions, shampoos, sprays or pomades. The effectiveness of the treatment would take into account both the clinical viewpoint (presence of reinfestation or not) and the parasito- logicalviewpoint (survival of lice and viability of hatched larvae after treat- ment). ‘Tolerance to the product would have to be taken into consideration in addition to any adverse cutaneous, ocular or general effects.
The authors wish to thank Professor M.
Bentatou of the Scientific Institute of Rabat for plant identification and Professor J.M. Bessiere of Phyto- chemistry, Laboratory of Montpellier, for the chemical analysis of the cssen- tial oils.
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