Potential of essential oils in schistosomiasis control in Morocco  

Dr M. Lahlou, Laboratory of Biochemistry Cellular and Molecular Biology FSU: Biology and Health Faculty of Sciences Ain Chock, Km 8 POB 5366, Ma&-if 20101 Route of El Jadida Casablanca, Morocco

E-mail: lahloumoh@hotmail.com

S

known as bilharziasis, is a para- sitic disease that dates back to antiquity. It is endemic in 76 countries and while approximately 200 million people are infected with the disease, over 600 million are exposed (Marston

& Hostettmann, 1991). In its various forms, schistasomiasis is second to malaria as the worlds most widespread parasitic disease and has increased sig- nificantly owing to the spread of irri- gated agriculture. Propagation is via

POTENTIAL OF ESSENTIAL

OILS IN

SCHISTOSOMIASIS CONTROL IN

MOROCCO

MOUHSSEN LAHLOU AND

RACHED BERRADA

Twenty-eight essential oils isolated from Moroccan aromatic plants were tested against the molluscan intermediate host of urinary schistosomiasis, Bulinus truncatus. Ten oils showed an interesting activity on snails. The most potent were from Citrus aurantium L. var. valencia Late and Otiganum compacturn

Benth. (L&=0.28 (0.21-0.38) ppm and 0.44 (0.32-0.60) ppm, respectively). The relationship between chemical composition of tested essential oils and their molluscicidal activity are discussed. It was found that among tested main components, thymol and geraniol exhibited potent

knockdown effect on snails (LCSO = 0.13 (0.1 O-O. 16) ppm and 0.15 (0.09-0.24) pp m, respectively). 0 2001 Harcourt

Publishers Ltd

contaminated water and occurs

principally during childhood in the area concerned.

In Morocco, only Schistosoma haematobium is reported to be endemic, as well as in 52 eastern Mediterranean and African countries (WHO, 1987).

The establishment of irrigation

schemes has resulted in an increase of the prevalence of the disease; essen-

tially in the summer when human

water contact is frequent. The inter- play of three factors of age, sex, and ethnic affiliation largely defines the demographic patterns of the preva-

lence and the intensity of infection.

Both of these increase in young

Moroccan children reaching a peak at age 1 O-14 years and then declines, the intensity of infection more rapidly than the prevalence.

It is well known that molluscs represent the most common category of intermediate hosts for digenetic trematodes. Moreover, many species of digeneans are pathogenic parasites

of humans and domestic animals.

Consequently, it has been recognized for many years that one method of controlling digenean helminthoses is

THE 1NTERNATlONAL JOURNAL OF AROMATHERAPY 9001 volO)noe 0

Numerous research groups are presently involved in screening plants

for diverse pharmacological and

I biological < activities. One aspect is the current search for plant-derived prod- ucts, which are relevant to the control of the tropical diseases schistosomiasis, those that have schistosomicidal, cerca- ricidal or molluscicidal activities.

lesting for molluscicidal extracts and pure compounds is relatively easy to perform and provides not only a possi- bility of combating schistosomiasis but also gives a lead into the discovery of

other important biologically and

biodegradable active substances

(Marston 8c Hostettmann, 1991). On the other hand, treatment of water bodies with molluscicidal compounds is considered an impor-tant element in an integrated strategy for morbidity

I

:’

So, the control of human schistosomia- sis involving control of the gastropod hosts is comparatively more important since the alternative and/or simultane- ously employed method is limited LO interrupting the parasite’s life cycle in the human host.

‘l’he rising costs of synthetic mol- luscicides, the concern about the devel- opment ofresistance to them and their toxicity LO non-target organisms has led to an increasing interest in plants and plant-derived compounds for the Lontrol of schistosomiasis (Wurzel et al., 1990). ‘The use of plants with

molluscicidal properties can be a

Gmple, inexpensive and effective

method of controlling snail intermedi- ate hosts of schistosomiasis in both

rural and farming endemic areas

(Pretorius CL al., 1988; Chifundcra et al., 1993).

control, but as the use of synthetic molluscicidcs is impeded by the high costs, there is a demand for inexpen- sive alternatives (Lemmich et al., lYY5).

Several molluscicides of plant ori-

gin (natural products) have been

screened worldwide for this purpose, identilied, and the active principles iso- lated (WHO, 1983; Combes & Cheng, 1986; Hmamouchi et al., 3998, 2000;

Lahlou et al., 20Olb). For essential oils, little attention has been paid to their molluscicidal action except for those from Cedrw deodara, Azadirachta indica and Embelia ribes reported molluscicidal on Lymnaea ucumkuzta and Indofilanorbis exustus (Singh et al., 1995).

In continuation of our pro-

gramme on the valorization of

Moroccan aromatic plants for antipar- asitic properties (Lahlou et al., 2001a), we report a novel method to study mol- luscicidal properties of 28 essential oils

obtained from Moroccan aromatic

plants and 23 active plant terpenes on Huliwus truncates, a real vector of trans- mission of urinary schistosomiasis.

Their extracts have already been

studied on the same snail species (Hmamouchi el al., 2000).

Plant collection and essential oil extrac~~~~~

Plants were collecled in period between lYY2 and lYY4 in different

region of Morocco (Al Hoceima,

Errachidia, Ouazzane, Marrakech,

Rabat and ‘1Bounate). They were taxo- nomically identified at the National Scientific Institute, Rabat. A voucher

specimen of each sample was

deposited in the herbarium of the Faculty of Medicine and Pharmacy of Rabat. All plant tesled are used in the

indigenous systems of medicine

(Hmamouchi, 1999) (Table 1).

Sarnpies of 1.5Og oF air-dried

plant material we1.e subjected to

t~ydrod~st~ilatioil for 1 hours using a

Clevcnger apparatus recommended by

I+ench pharmacopoeia.

Chemical analysis

Fach sample was analyzed by gas

chrornatograph and performed on a

fused silica capillary column (25m x 0.25mm), coated with OV-101; oven

temperature was programmed from

50” Lo 250°C al a rate of 5”C/min.

CC/MS analysis was carried out on a

Hewlett-Packard Capillary GC-quadru- ple MS system (model 5970) fitted with a 25 IO x 0.23 mm fused-silica column coated with DE-1 and using the same

gas chromatographic parameters.

Identification of the compounds was carried out by the comparison of the retention times and MS data with those of reference compounds.

MOlEUSCS

Snails of BuZinz~.Ls trunca.~rrs species were collected from El Attaouia region (South of Morocco). They were raised

in the laboratory under controlled

tempemure (25°C i 1°C) and relative humidity (70% RH kj%). \vater was oxygenated in permanency.

Chemicals

Niclosamide (2,5’-Dichloro-4’-nitr~~s~ : ali-cylanilide) (Bayer, Casablanca) was / used as reference compound according

/ to the WHO (1983) instruction. The

other pure compounds tested came

from Aldrich, Fluka and Merck.

Biological tests

Molluscicidal tests were carried out

according to the method recom-

mended by WHO (1965, 1970) and

modified according to Duncan and

Sturrock (1987), as indicated in our works (Hmamouchi et al., 1998, 2000;

Lahlou et al., 2001b). In this method, five snail of uniform size (4-6 mm shell height) were tested. The exposure time was 24. hours followed by a recovery

period of 24 hours in distilled warcr.

Death was ascertained by examining immobilized snails under a dissecting microscope for the absence of heart beat (WHO, 1965; 1970). Essential oils and their main components were dis- solved in 0.2% of ‘liveen 80 as indi- cated for antimicrobial tests (Benjillali

et al., 1986). Solutions were then dispersed in water by application of 2 KC/s ultra-sound frequency for <5 ’ seconds. ‘Lhis technique was modified fi-om our work (Lahlou et al., 2001b).

Milky clcal- solurions were thus

obtained and various concentratiorls were prepared. Systematic controls with the same ‘liveen 80 concentration were included.

Statistical analysis

Lethal concentrations LC~~~ and LCSO

and 95% confidential limits were

determined using the method of

Litchfield and Wilcoxon (1970). ‘rests were done in triplicate.

(+)-lkxdooi L-Menthol

(-)-Trans caryophyllene Myrtenol

c c

A

p-Cymme

0 3”

Fig. 1 Main esseztiul oil compounds tested on B. huncatus snails

~cree~~~g c3f essential oil5 for

r~oLlascic~~aL activity

For the primary screening ol’ essential oils, the results of Table 2 generally show that 25% of all rhe plants did not demonstrate any molluscicidal activity.

Among active plants, particular

interest is accorded to the oils of Citrus

~uruntium L. var. valencin Late and Olignnum compacturn Benth. which exhibited a potent knockdown effect on Bulinus truncatzu at less than 24 hours and at the lowest concentrations

(IX,0=O.28 (0.21-0.38) ppm and

0.44 (0.32-0.60) ppm, respectively).

They were successively followed b>

Ruta cha&e&s L. (LC& =0.52 (0.39-0.68) ppm), Chenu@dium ambm- sioides I,. (LCSo=0.54 (0.38-0.76) pp) and Anthewzis nob&s L.

(LC,” = 0.56 (0.42-0.74) ppm). Other plants had a median molluscicidal action like Citrus limonunz (I,.) Burm;

F’elargonium cajitatum (L.) Zherit; Citrus aurantiurrr L. ssp. brrgamia Wright et Arrr.; C. auruntium L. var dulce Pers

and Cinnamomum zeylanicrrm Nees (LCjo < 1 .OO ppm). Other plants tested presented a lower activity when

compared with the precedents

(LCio > 1.00 ppm).

/ Activity of terpenes on B. truncatus / The main components of these active

essential oils were determined by

CC/MS (Xables 3-7). In order to search for the active compounds responsible for the molluscicidal properties, we studied the major components present in each oil (Fig. 1, and designated in bold in Gbles 3-7) that had shown loxicity towards snails.

‘lhe activity of these terpenes was

compared with niclosamide (an

anthelminthic drug) taken as a control as recommended by WHO (1983). The results are shown in Table 8. The products can be classified in four

groups:

i. Products showing no toxicity, such as 1, S-cineole (eucalyptol), car- vone, L-menthone, L-menthol, (-)- fenchone and linalool.

ii. Substances demonstrating a weak molluscicidal action such as limo- nene, pulegone, citronellol, car- veol, eugenol, myrtenol, (-)-tmns

caryophyllene; citronella1 and

citral (I& > 1. 00 ppm).

iii. Those showing a medium mollus- cicidal effect like p-cymene, car-

vacro1, myrcene, P-pinene and

ol-pinene and (LC,, < 1 .OO ppm).

iv. Those revealing a high molluscici- da1 activity such as camphene (LC,,=O.24 (0.17-0.35) ppm), geraniol (I,Cjo = 0.15 (0.09-0.24) ppm) and thymol (ISSo = 0.13 (0.1 O-O. 16) ppm).

7‘he relation between composition and activity leads us to suppose that molluscicidal activity of the essences from the aromatic plants studied may be attributable both to their majol components (alcoholic, phenolic, ter- penic or ketonic compounds) and to the minor ones present in these oils. It is possible that they may act together synergistically.

Nevertheless, even the compo- nents rated as having ‘high activity’ did not differ much from the results obtained using the control synthetic molluscicide niclosamide (IX& = 0.12

(0.08-0.18) ppm). Therefore, the

results of molluscicidal activiries of

these oils and different rerpenes are

high enough by comparison of our

work (Lahlou et al., 200 I b) to consider relying solely on them as molluscicidal.

‘l‘he technique used for dissolution of tested oils and their main components

using a minimum quantity of the

‘tensio-active’ Tween 80 (0.2%) in addi- tion to t.he physical method for their dispersion (ultra-sound apparatus), led for the first time to a good solubility, diffusion and dispersion of the essen- tial oil in water, thus ensuring a better conlact of these oils and/or their

constituents with tested snails.

Therefore, the quantity of ‘Iken 80 used for dispersion of an essential oil had important influence on its biolog- ical activity.

However, the toxic effects of these oils and their constituents to the B. tmncatus molluscs had been indi- cated by two main reactions that differ

in their mechanism of action as

TOXIC,I~Y OF MAIN COMPONENTS OF TESTED ESSENTIAL

OILS ON 6. TRUNCAtU.5 SNAILS

molluscicides. Either the mollusc draws in the shell after ejection of the haemolymph, or the mollusc inflates and extends out of the shell by rupture of osmotic equilibrium which is under neurohormonal control (McMullough

8.~ Mott, 1983). Other studies

(Appleton, 1985) have shown that

exposure of the snails to molluscicides resulted in a severe disturbance of the snails’ ionic and water balance, apparently due to a change in the permeability of cell membranes, par- ticularly of epithelial cells throughout its body.

Considerable attention has recently paid to the biodegradable substances as antiparasitic agents. It is becoming increasingly evident that certain natural products elicit activity in a number of biological systems; essen- t,ially those acting as intermediate hosts of parasitic diseases. Further studies on the tests for cercdricidal activity and toxicity of the active oils on non-target organisms are under consideration.

We would like LO thank Pr

M. Hmamouchi of the Faculty of

Medicine and Pharmacy of Rabat for his collaboration.

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155-167.

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Phytochemical screening and

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71-72.

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Schistosomes are the only members LI/ the digenetic trematodes or jlukes whose larvae

;benetrate directly into the jinal host (humans for example) a$er release ,from sn,ails. Th,ey enter through the dermis and are carried vin the blood to the liver where they mature and ,jorm mating pairs.

S. jafionicum and S. mansoni then relocate to the mesenteric veins around the .small intestine and lay egg3 wilhin 6-12 weeks.

The sfiecies endewric in Morocco, S. haema- to&m, relocates to the veins surrounding the bladder:

Dermatitis can develop during pene- traiilfion of the lar?ine and may be seuere on ,reFeated irlfe(:tion. Allergic symptoms

iiks ,fever; lym~hadeno~ath~ and

hefiatomegaly a@ear during the larva,1 i developnentnl stqe in, the liver; bu,t the major problems occur once the e,qgs begin to migrate through the tissues to the outside world.

In ,urinary schr.~tosow~iasi.~ (S. haema- tobium), the eggc migrate through the bladder wall causing haenzorrhage, injlam- mation and possibly malignant changes.

The eggs ore excreted in the urine but SOUP may br carried in tiae bloodsireana to

lodge in other tis.sues like the liver; lungs and central nervous system. A severe inJlammatory v@onse ensues that may lead to m,any complications such as portal hyper- tension.

The drug of‘ choice i.s praziquantel and it has no pharmacological effects on.

humans at theraputic doses. It acts by interfering with adult and larval sch,isto- some calcium homeostasis, leadivvg to their death. The problem is that dead worms or their products may be carried back to the liver and initiate intense injlammatory reactions. Although all the worms may be

killed, in, advanced cases the pathology is irreversible.

It is recognized that control of this dis- ease is best achieved by breaking the trans- mission cycle, either by avoidnnce of infected water or destruction of the inaermediate host, the snails.