Ptr(01)_1465.pm5

PHYTOTHERAPY RESEARCH
Phytother. Res. 18, 435–448 (2004)
METHODS TO STUDY THE PHYTOCHEMISTRY AND BIOACTIVITY OF ESSENTIAL OILS Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1465
REVIEW ARTICLE
Methods to Study the Phytochemistry and
Bioactivity of Essential Oils
Mouhssen Lahlou*
Laboratory of Biochemistry, Cellular and Molecular Biology, Department of Biology, FSU: Biology and Health, Faculty of
Sciences Ain Chock, Casablanca, Morocco
Many essential oils are extracted, analysed and their main components are identified, characterised and then
published without any biological testing whatsoever. Their useful biological activities can remain unknown for
years. Yet, the search for these activities often increases our knowledge of the potential use of oils in thera-
peutics. Therefore, there is a real need for a simple, reliable and reproducible methods to study the bioactivity
of essential oils and their constituents which can detect a broad spectrum of action or specific pharmacological
activities in aromatic plants. These methods can then be employed by natural product chemists, pharmacologists
and biologists to conduct their scientific research and to valorise natural products. Standardisation of some of
these methods is therefore desirable to permit more comprehensive evaluation of plant oils, and greater
comparability of the results obtained by different investigators. Copyright 2004 John Wiley & Sons, Ltd.
Keywords: essential oils; phytochemistry; bioactivity; methodology.
the study of biological and pharmacological activities INTRODUCTION
of these products, as the value of an essential oil inaromatherapy has to be related to its chemical com- Essential oils are valuable natural products used as raw materials in many fields, including perfumes, Most papers do not often cite the geographic origin cosmetics, aromatherapy, phytotherapy, spices and and the exact composition of the essential oil studied, nutrition (Buchbauer, 2000). Aromatherapy is the thera- which somewhat negates their findings. No considera- peutic use of fragrances or at least mere volatiles to tion is given to the basic question as to what the prin- cure, mitigate or prevent diseases, infections and ciples responsible for the therapeutic action are (Janssen indispositions by means of inhalation (Buchbauer et al., 1993a). This has recently attracted the attention of many Essential oils are complex mixtures comprising many scientists and encouraged them to screen plants to study single compounds. Each of these constituents contrib- the biological activities of their oils from chemical and utes to the beneficial or adverse effects of these oils.
pharmacological investigations to therapeutic aspects.
Therefore, the intimate knowledge of essential oil Hopefully, this will lead to new information on plant composition allows for a better and specially directed applications and new perspective on the potential use application (Buchbauer, 2000). Considering all the aforementioned differences in essential oil composition, Many papers on the biological activity of essential it is clear that only a detailed knowledge of the con- oils have been published. The data, however, show much stituents of an essential oil will lead to a proper use in discordance between the same essence. The reasons cosmetics by perfumers and cosmetic chemists. How- for this variability can be understood if we take into ever, such a detailed knowledge can only be obtained account all the factors influencing the chemical com- by means of carefully performed capillary-GC experi- position of the oils, namely, climatic, seasonal and geographic conditions, harvest period and distillation This paper is aimed primarily at summarising the technique, among others (Panizzi et al., 1993). The phytochemical study and the bioactivity methods to effect of plant maturity at the time of oil production investigate essential oils from aromatic plants as and the existence of chemotypic differences can also potential biological and pharmacological resources.
drastically affect this composition (Lahlou and Berrada, Some consideration of the special, practical problems 2003). These variations are of distinct importance in of testing volatile oils (poor water solubility) are alsogiven, including suggestions for overcoming these pro-blems. The study also provides a set of guidelines forresearch on biological, pharmacological and toxico- * Correspondence to: M. Lahlou, Laboratory of Biochemistry, Cellular logical activities of plant oils with the aim of assessing and Molecular Biology, Department of Biology, FSU: Biology and Health, whether an essential oil possesses a specific biological Faculty of Sciences Ain Chock, Km 8, POB 5366, Maârif 20101, Route ofEl Jadida, Casablanca, Morocco.
and pharmacological effect or whether it has a wider Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
Copyright 2004 John Wiley & Sons, Ltd.
the reference standards (with known source); as pre- PHYTOCHEMISTRY
viously reported (Lahlou et al., 2000b; 2001b,d; Lahlouand Berrada, 2003).
Plant collection
Analytical conditions and procedures used should There is often a lack of information on the distribution • apparatus of oil analysis (make and model number of the oil in different plant parts. Such information may have predictive value, but is not available due to the failure of most investigators to systematically study all parts of plants. For this reason, the investigated parts • the temperature programming conditions includ- richer in essential oil (cones, fruit, leaves, bark) should ing injector temperature, detector and column tem- be selected on a rational scientific basis and with solid peratures; in addition to mass spectra (electronic justification (preliminary chemical screening). Further- more, plants showing an aromatic character shouldbe collected. Examples of such plant families are Sometimes identification by GC/MS must be con- Anacardiaceae, Apiaceae, Asteraceae, Chenopodiaceae, firmed by retention indices (Kovats Indices) on two Cupressaceae, Gentianaceae, Lamiaceae, Myrtaceae, columns of different polarity, as reported in our earlier Pinaceae, Piperaceae, Poaceae, Rutaceae, Verbenaceae work (Lahlou, 2003); or on the same column, but at a different temperature, as indicated by Denayer andTilquin (1994); and claims for the identification of newconstituents should be supported by co-injection with Essential oil extraction
authentic compounds. Data should thus include essen-tial oils optical rotation, density and refraction index ‘Essential oils are products, generally, of rather com- (Lahlou et al., 1999; 2000a).
plex composition comprising the volatile principles On the other hand, compounds which are not easily contained in the plants, and more or less modified separated by GC, and molecules structurally similar like during the preparation process’ (Bruneton, 1995). They stereo-isomeric compounds of essential oils are ana- are essentially obtained by hydrodistillation (the plant lysed by 13C NMR as reported by Tomi et al. (1997).
material is heated in two to three times its weight of This technique is also applied to the study of the chem- water with indirect steam from outside the still) as ical intraspecific variation and could also be used in opposed to steam distillation (the plant material is the quality control of volatile oils.
extracted by direct steam, produced in the still, or byindirect steam, produced outside and fed into the still),hydrodiffusion (low-pressure steam (< 0.1 bar) replacesthe volatile from the intact plant material by osmotic BIOACTIVITY PROCEDURE
action) or CO extraction (Buchbauer, 2000); in addi- tion to expression of the pericarp (or cold pressing) Animal models for pharmacological and toxicological
which is a special method for Citrus (Rutaceae) peel oils extraction (Baaliouamer et al., 1992; Dellacassaet al., 1992) from fresh or dried material.
Many countries and organisations have legislation and The microwave irradiation [or microwave assisted guidelines for the care and use of animals for pharma- process (–MAP–)] has also been developed and reported cological and toxicological research purposes (Guide by many authors as a technique for extraction of essen- for the Care and Use of Laboratory Animals, 1985; tial oils in order to obtain a good yield of the essence International Guiding Principles for Biomedical and to reduce time of extraction (Paré et al., 1989; Collin Research Involving Animals, 1985; Interdisciplinary et al., 1991; Bouzid et al., 1997; Brosseau, 1997; Chiasson Principles and Guidelines for the Use of Animals et al., 2001; Ghoulami et al., 2001). This technique has in Research, 1988; Use of Laboratory Animals in Bio- also been applied for the extraction of saponins from medical and Behavioural Research, 1988). Moreover, it some medicinal plants (Safir et al., 1998). The MAP is the responsibility of researchers to ensure that their process uses microwaves to excite water molecules in practices conform with those relevant to them. Train- the plant tissues causing plant cells to rupture and ing and research involving animals should incorporate release the essential oils trapped in the extracellular procedures which are designed and performed with tissues of the plant (Bélanger et al., 1991).
due consideration of current scientific knowledge, the Another technique consists of extracting oils using a relevance to human or animal health, the advancement mechanical and thermochemical reaction (Bouzid et al., of the science of toxicology and the potential benefit 1997). Yield data of these oils is therefore determined to society. Nevertheless, these researches must contain precise details on animals, especially the choice of theadequate laboratory animal model for a specific phar-macological study. Thus, the species, (e.g. Psamomis Chemical analysis
obesus rats, Meriones shawi, Wistar rats, hamster, dogs,monkeys, rabbits, Swiss mice, Sprague-Dawley), should Chemical analysis of essential oils is generally performed be carefully selected and the number of animals kept using GC (quantitative analysis) and GC/MS (qualita- to the minimum required to achieve reproducible and tive analysis) (Keravis, 1997). Identification of the main scientifically valid results. Experiments that require components is carried out by the comparison of both the use of animals must be conducted in accordance the GC retention times and MS data against those of with the Guiding Principles in the Use of Animals in Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
METHODS TO STUDY THE PHYTOCHEMISTRY AND BIOACTIVITY OF ESSENTIAL OILS Toxicology, which were adopted by the Society of Toxi- with agar was considered homogeneous as a true solu- tion in ethanol. Furthermore, MIC (minimal inhibitoryconcentrations) for different bacterial species in thepresence of agar appears to be significantly lower than Dispersion and solubility of essential oils
those observed in the presence of Tween 80 or ethanol(Remmal et al., 1993b).
Generally, essential oils are poorly soluble in water,and this causes many problems for studying their bio-logical and pharmacological properties. In order to Laboratory conditions and experimental techniques
overcome these problems, many authors have recom-mended the use of various solvents in the dilution of Results of biological tests of essential oils are often essential oils such as acetone, alcohol, ethylene glycol, different and dependent upon experimental conditions ethanol, methanol, DMSO and DMF (Dayal and per comparison to the literature data. These are both Purohit, 1971; Allegrini et Simeon de Buochberg, related to the laboratory conditions (temperature, 1972; Martinez Nadal et al., 1973; Beylier-Maurel, 1976; photoperiod, etc.), materials; in addition to the models Morris et al., 1978; Conner and Beuchat, 1984; Lahlou taken for biological experimentation (age, life cycle).
et al., 2000b); or using an emulsifier detergent or Moreover, the method of extraction of essential oils, ‘Tensioactif’ like Tween 20 or Tween 80 in different influences their chemical composition, and thus, can percentage (Allegrini et Simeon de Buochberg, 1972; have repercussion regarding their biological properties.
Allegrini et al., 1973; Pellecuer et al., 1976; Beylier, Techniques of solubility of these oils are among others 1979; Benjilali et al., 1984; 1986; Lahlou et al., 1999; problems. Normalisation of methods and laboratory 2000a; 2001a,b; Lahlou, 2003). Furthermore, Chalchat conditions, in addition to the techniques used (extrac- et al. (1991) compared the results obtained for the tion, dissolution and dispersion of oils, culture medium) determination of the MIC of essential oil constituents for testing biological and pharmacological activities using two modern methods in liquid media, one involv- of essential oils at the definitive stage of laboratory ing Tween 80 as emulsifier and the other using noble screening are desirable in order to provide a common Agar as stabiliser. The authors concluded that the two basis for the comparison of results obtained in various parts of the world on different organisms tested under On the other hand, Remmal et al. (1993a,b; 2001) demonstrated that ethanol, Tween 80, Tween 20 and Whenever pharmacological bioassays are investigated Triton X100 present a depress effect of antimicrobial in persons presenting with some heart-vascular diseases activity of tested oils in a solid medium, in addition to (hypertensive diabetes) or in healthy volunteers (for their antagonist effect in a liquid medium. The authors the study of the effects of fragrances on autonomic suggested the use of 0.2% of agar suspension. In this nervous system parameters and self-evaluation, for ex- case, minimal inhibitory concentration (MIC) and mini- ample), studies in these cases should take into account mal lethal concentration (MLC) found by these authors sex, age, weight and socio-cultural level of persons who for different bacterial species in the presence of agar took part in the experiments, in addition to the number were significantly lower than those observed in the pres- of subjects, which allows homogeneous statistical analy- ence of Tween 80 or ethanol. This demonstrates the sis. Furthermore, experiments should always take place fact that solvents and detergents often used in antimi- at the same time of day (the same conditions). Some- crobial studies significantly decrease the antibacterial times, 24 h prior to the beginning of each experiment, activity of tested oils. The use of agar suspension to subjects had to abstain from food and beverages con- disperse essential oils also resulted in obtaining lower taining the substances tested. Moreover, all subjects MIC (Lens-Lisbonne et al., 1987) than those previously should give written informed consent to all aspects of reported by Morris et al. (1978) and Simeon de the study. A second group of subjects should be used Buochberg (1976) for the same micro-organisms and the same oils dispersed with ethanol and Tween 80,respectively.
Balansard (1990) and Remmal et al. (2001) proposed Correction of percentage mortality
another method to standardise emulsion of essentialoils essentially for antimicrobial tests in order to main- Lethal effect of essential oils or their components on tain their viscosity stable in aqueous medium. This tech- biological organisms should take into account mortality nique consists of adding 50 to 100 µl of oil to a sterile in the control. Thus, in cases where control deaths tube containing 10 ml of noble agar. After centrifuge occurred, the data should be corrected using Abbot’s agitation, the exact quantity of oil was determined (1925) formula, as reported in our works (Lahlou after tube weighing; the volume of agar solution was et al., 2000b; 2001d; Lahlou, 2002; Lahlou and Berrada, calculated in order to avoid desired concentrations of the emulsion (2 to 4 mg /ml). These emulsions wereeasily used without particular technical problems forall evaluation of tested oils.
Statistical analysis
The study undertaken by Santos et al. (1997) on anti- bacterial activity of some essential oils did not utilise Lethal concentrations/doses LC50, LC90 (or lethal Tween 80 (detergent) or ethanol (alcohol) to disperse doses LD50, LD90) and 95% confidential limits could the essential oils with agar as these have been reported be determined according to Dupont (1970) using the to interfere with the estimation of antimicrobial activity method of Litchfield and Wilcoxon. They can also be of essential oils (Remmal et al., 1993b). The dispersion calculated using the Reed and Muench method (1938) Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
or using probit analysis method as described by Finney In order to facilitate the essential oils’ dispersion in (1971), as reported in our works (Lahlou, 2001; Lahlou the aqueous medium, the Tween 80 was used at 1.0% et al., 2001a,b,c,d; Lahlou, 2002; 2003; Lahlou and (Lahlou et al., 2001a). The results obtained indicated Berrada, 2003; Lahlou et al., 2003).
an interesting molluscicidal activity of these compounds Lethal doses LD50 could also be calculated using at much high concentrations (0.20 103 ppm ≤ LC ≤ the INRA-INSA ‘Toxicologie’ analysis software which 2.85 103 ppm for oils and 0.12 103 ppm ≤ LC ≤ 2.68 uses the probit method developed by Bliss (1935).
103 ppm for the main components). When the Tween The results of biological activities could be presented 80 was used at 0.2% only, and the solutions obtained as mean ± SEM, with n indicating the number of observa- were then dispersed in water by application of 2KC/s tions. Values can be analysed using a Student’s t-test, ultra-sound frequency for 5 sec, milky clear solutions ANOVA or non-parametric tests, where appropriate, were obtained and various concentrations were pre- and are considered to differ significantly when p < 0.05.
pared. The results obtained from this second technique In cases where data are insufficient for these tech- (Lahlou et al., 2001b) are high enough in comparison to niques, the dose-response data should be transformed that of the latter and tested oils and their constituents into a straight line by means of a logit transforma- acted at low concentrations (0.28 ppm ≤ LC ≤ 2.84 ppm tion (Swaroop, 1966); lethal concentrations are derived for oils and 0.13 ppm ≤ LC ≤ 2.93 ppm for the main from the best fit lines obtained by linear regression Thus, the technique used for dissolution of tested Results of other pharmacological or physiological oils and their main components using a minimum activities are usually expressed as means ± the standard quantity of the ‘tensio-active’ Tween 80 (0.2% only) in error of the mean used in order to construct a dose- addition to the physical method for their dispersion response curve. The significance of the results should (ultra-sound apparatus), has led for the first time to a be assessed by means of unpaired or paired Student’s good solubility, diffusion and dispersion of the essen- t-tests, Mann–Whitney U-tests, Dunn’s test and one- tial oil in water, thus ensuring a better contact of these way (doses) or two-way (treatment × doses) analysis of oils and/or their constituents with tested organisms variance (ANOVA) (Lahlou et al., 1999; 2000a).
(snails in this case). Therefore, the quantity of Tween80 used for dispersion of an essential oil had an import-ant influence on its biological activity.
Factors affecting the bioactivity of essential oils
The correlation composition-activity. Biological activ-
Studies of biological and pharmacological activities of ity of an essential oil is in strict direct relation to its essential oils/constituents may take into account the chemical composition. Thus, in our screening plants used in local folk medicine for biological properties(Lahlou et al., 2000b; Lahlou, 2001; Lahlou et al., Effect of treated plant part. In order to indicate a
2001a,b,d; Lahlou, 2002; 2003; Lahlou and Berrada, difference in activity between two different parts of 2003), we noted a difference in activity of tested oils the same plant, many studies have been performed.
and their main components on different biological In particular, for molluscicidal tests (Lahlou et al., models studied. Essential oil, in its totality, acted less 2001b), oil obtained from leaves of Citrus aurantium than the major constituents. In these studies, and in var. amara Link (Rutaceae) was devoted on activity order to search for the active components responsible to B. truncatus snails at tested concentrations. Whereas, for the biological activity, we studied the main com- oil of the fruit of the same plant exhibited potent ponents of oils that have shown toxicity to these bio- molluscicidal activity at lower concentration (LC = 1.46 logical models. The relation between composition and activity leads us to suggest in these cases that bio- In fact, there is a lack of information on the distribu- logical activity of the essences from the aromatic plants tion of the biological activity in different plant parts studied may be attributable both to their major com- essentially related to the difference in distribution of ponents (alcoholic, phenolic, terpenic or ketonic com- active compounds (or active principles) which are more pounds) and to the minor ones present in these oils.
frequent in some plant parts than in others. Although It is possible that they may act together synergistic- many other compounds do occur independently, dif- ally to contribute to the toxicity of the totality of the ference in chemical composition of these oils and gives Technique used. During our in vitro study for licicidal
Effect of the solvent/detergent. The most useful solvents
and niticidal activities of some Moroccan essential oils in laboratory are toxic to biological organisms and and their main components or plant oil chemotypes on consequently interfered with the activity being studied Pediculus humanus capitis (head lice), two methods were (Lahlou, 2001). The choice of an adequate solvent (or investigated: microatmosphere and direct application detergent) is indispensable before envisaging a bioassay (Lahlou et al., 2000b; Lahlou and Berrada, 2003). It using an essential oil. Thus, in order to demonstrate was found that some oils tested were most active on the effect of the detergent (quantity), used for disper- lice using the microatmosphere technique [Chenopod- sion of an essential oil on the results of biological tests, ium ambrosioides (Chenopodiaceae), Mentha pulegium a comparative study was carried out for the same ‘tensio- and Thymus broussonettii (Lamiaceae) (LD active’ Tween 80 when used differently in two different 3.10 µl]. Whereas others were found to be much pieces of research on Bulinus truncatus snails (Lahlou less active using this method [essentially Lavandula et al., 2001a,b). In this case two very different sets of stoechas (Lamiaceae), Chrysanthemum viscidehirtum (Compositae), and Cedrus atlantica (Pinaceae)]. While, Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
METHODS TO STUDY THE PHYTOCHEMISTRY AND BIOACTIVITY OF ESSENTIAL OILS during niticidal tests and using the microatmosphere The perfume concentration in most cosmetic prod- way, essential oils from C. ambrosioides, M. pulegium, ucts ranges between 0.5% and 2%, thus being too small T. broussonettii and O. compactum, in addition to Ruta for any risk of toxicity from essential oil-constituents, chalepensis were found to be the most potent among with the exception of some more or less toxic terpenic those tested (LD = 3.10 µl), other oils such as ketones, such as thujone or pulegone (Buchbauer, those from Citrus limon and C. sinensis (Rutaceae), Pinus pinea (Pinaceae), Myrtus communis (Myrtaceae),Cinnamomum zeylanicum (Lauraceae), Pelargoniumsetaceum and Chrysanthemum viscidehirtum (Com- Reference compounds
positae) acted more potently using the microatmospheretechnique (0% of nits hatched) than in direct applica- All biological and pharmacological tests using natural tion (10–30% of nits hatched). Essential oil from products must include proper controls, and in this Melaleuca viridiflora (Myrtaceae) was more active on respect, comparison with an agreed reference standard nits essentially in direct application (0% of nits hatched), compound, under similar laboratory conditions, is whereas 30% of nits hatched in microatmosphere of essential and indispensable. It is recommended that this standard should be as follows (Table 1).
Biological material. During our study on licicidal and
niticidal activities of some Moroccan essential oils
Comparison with literature data
(Lahlou et al., 2000b), we observed that those derivedfrom Mentha pulegium and Thymus broussonettii This step is important in order to conduct a scientific (Lamiaceae), Chenopodium ambrosioides (Chenopodia- laboratory research with a rigorous procedure in order ceae), and Ruta chalepensis (Rutaceae) possess the most to compare all findings with those reported in the litera- powerful licicidal and niticidal activities towards head ture for other compounds studied under identical lice, Pediculus humanus capitis. Other oils were found laboratory conditions. This also indicates whether the to be active only on nits like Origanum compactum results found are within the accepted ranges (or limits) (Lamiaceae) until volume of 3.10 µl. On the other hand, as reported in the literature or are more or less inter- using the direct application technique, louses were found esting. Activity of oils from aromatic plants of the same to be more sensitive and died within 15 min of exposure family are rigorously comparable. However, there is to an application of 0.5 µl of all tested oils.
some difficulty related to the conditions of experiment-ation which means that the results are sometimes rela- Choice of the doses/concentrations. Paracelsus’ state-
tive. In general, using the same plant species oil should ment remains true down the ages: ‘All substances are result in similar biological or pharmacological activities poisons; there is none which is not a poison. The right under identical laboratory conditions around the world.
dose differentiates a poison and remedy’ (Paracelsus, Moreover, it is necessary to check in the literature pp. 1493–1541). Thus, it is important to note that this to ascertain if tested plant oils have been previously choice must not be hazardous. In addition, this charac- investigated for the same properties.
ter is most important for the study of bioactivity. Fur-thermore, these concentrations should be in accordancewith the limits already documented in the literature(other works for identical activity using the same com- METHODOLOGY
pounds or identical kind of plant). For this reason, inall our tests performed on biological organisms, when Antiparasitic activities
an essential oil or the main components tested was foundto be toxic at a concentration, other appropriate lower Molluscicidal activity. Schistosomiasis is a parasitic
concentrations (or dilutions of active ones) were pre- disease in most developing countries. Fresh water snails pared and performed in order to find the minimal toxic act as the intermediate host; so control of these dis- concentration which caused 50% or 90% toxicity or eases is best achieved by breaking the transmission cycle, lethal doses/concentrations LD50-LD90/LC50-LC90.
either by avoidance of infected water or destruction of For example, in niticidal and licicidal tests (Lahlou the intermediate host, the snails (Lahlou et al., 2001b).
et al., 2000b; Lahlou and Berrada, 2003), different Bioassays were performed using Bulinus truncatus snails volumes of the oils (3.10–50 µl) were tested.
according to our works (Hmamouchi et al., 1998, 2000; Since essential oils are highly concentrated fluid Lahlou, 2001; Lahlou et al., 2001a,b; 2002; Lahlou, 2003).
substances, they are rarely used in an undiluted form.
Thus, five snails of uniform size (4–6 mm shell height) Before application, oils are first blended with a carrier were tested in distilled water containing each concen- oil. This blending dilutes the essential oils so that they tration of tested product. The exposure time was 24 h are safe, and also helps to slow down the rate of evapo- followed by a recovery period of 24 h. Death of snails ration, to spread them evenly, and to increase their was ascertained by examining immobilised snails under eventual absorption into the skin. For this reason, a dissecting microscope for the absence of heart beat.
active niticidal oils obtained from Chenopodium ambro- sioides (Chenopodiaceae), Mentha pulegium, Thymusbroussonettii and Origanum compactum (Lamiaceae), Licicidal and niticidal activities. Tests on head lice,
in addition to Ruta chalepensis (Rutaceae) were tested Pediculus humanus capitis and their nits were performed in diluting the low potent volume (3.10 µl) at dilution in identical Pyrex glass Petri dishes (5.5 cm diameter, factors of 1:2, 1:4, and 1:10 in alcohol 95° (Lahlou 1.5 cm height) according to our works (Lahlou et al., 2000b; Lahlou and Berrada, 2003), using two techniques: Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
Copyright 2004 John Wiley & Sons, Ltd.
Table 1. Standard compounds recommended for biological and pharmacological tests
WHO, 1970; Gebremedhim et al., 1994; Lahlou, 2001; Lahlou et al., 2001a,b,c;Lahlou, 2003 Temephos, abate, malathion, allethrin, deltamethrin, WHO, 1981; Laurent et al., 1997; Jantan et al., 1999; Lahlou, 2001; Lahlou et al., bromophos, fenitrothion, fenthion, chlorpyrifos Streptomycin sulphate, cephalosporin C, penicillin G, El Mahi et al., 1997; Santos et al., 1997; Larhsini et al., 1999; Akinpelu and gentamicin, tetracycline, actinomycin D, rifampicin, ampicillin, Olorunmola, 2000; Khan et al., 2000; Takaisi-Kikuni et al., 2000; Ebi, 2001; piperacillin, kanamycin, chloramphenicol, streptomycin, Erdemoglu and Sener, 2001; Marquina et al., 2001; Ogunwande et al., 2001; tobramycin, enrofloxacin, benzyl penicillin, cloxacillin, Rojas et al., 2001; Schlemper et al., 2001; Atindehou et al., 2002; Chowdhury et al., 2002; Asha et al., 2003; Chowdhury et al., 2003; Fleischer et al., 2003a,b;Gupta et al., 2003; Copland et al., 2003; Kumarasamy et al., 2003a,b;Omer and Elnima, 2003; Perez et al., 2003; Somchit et al., 2003 Griseofulvin, ampicillin, amphotericin B, chloramphenicol, Testa et al., 1991; Rahalison et al., 1994; El Mahi et al., 1997; Akinpelu and econazole, nystatin, sulconazole, itraconazole, miconazole, Olorunmola, 2000; Khan et al., 2000; Ebi, 2001; Erdemoglu and Sener, 2001; 5-flucytosine, propiconazole, tioconazole, fluconazole, Kariba et al., 2001; Marquina et al., 2001; Rojas et al., 2001; Rub Nawaz et al., ketoconazole, benlate, nabam, clotrimazole, uconazole, 2001; Ogunwande et al., 2001; Chowdhury et al., 2002; 2003; Lahlou et al., 2002; Fleischer et al., 2003a,b; Panagouleas et al., 2003; Perez et al., 2003; Somchitet al., 2003; Omer and Elnima, 2003; Vollekov et al., 2003 Praziquantel, santonin, pyrantel pamoate, mebendazole, Keita et al., 1990; Awad and Probert, 1991; Ndamba et al., 1994; Martin et al., 1997; piperazine citrate, biltricide, metronidazol (Flagyl®), albendazole, Munoz et al., 2000; Ishih et al., 2001; Teixeira et al., 2001; Shuhua et al., 2000; levamosol, gentian violet metronidazole, pentamidine, sodium Onyeyili et al., 2001; Isah et al., 2003; Tapia-Pérez et al., 2003 stibogluconate (PentostamR), chloroquine, emetine,sulfamonomethoxine, pyrimethamine Dao nil (glibenclamide), streptozotocin, sulfamid, biguanid, Chattopadhyay et al., 1993; Skim et al., 1999; Kameswara et al., 2001; Chakrabarti et al., 2003; Ohiri et al., 2003 Atropine, dicyclomine, neostigmine, nordihydroguairetic acid, Santos and Rao, 2001; Baggio et al., 2003 Boskabady and Ramazani-Assari, 2001; Boskabady and Khatami, 2003 Phenylbutazone, carrageenin, indomethacin, dichlorfenac, Susplugas et al., 1993; Choi et al., 2000; Muko and Ohiri, 2000; Badilla et al., 2003; Dongmo et al., 2003; Mandal et al., 2003; Medhi et al., 2003; Sayyah et al., 2003 Badilla et al., 2003; Rahman et al., 2003 Atropine sulfate, diphenoxylate, chlorpromazine, loperamide, Chaves et al., 1998; Biswas et al., 2002; Sairam et al., 2003; Hajhashemi et al., 2000 Morphine, paracetamol, acetylsalicylic acid, lorazepam, Susplugas et al., 1993; Alaoui et al., 1998; Aziba et al., 2001; Sayyah et al., 2003 METHODS TO STUDY THE PHYTOCHEMISTRY AND BIOACTIVITY OF ESSENTIAL OILS (i) Microatmosphere: in this method, the whole oil or reversed position after laying down the essential component evaporates into the atmosphere of a oil on a filter paper in the middle of the dish cover.
closed Petri dish, saturating it with vapour, whereby The oil evaporates in the atmosphere of the dish the volatile oil can exert an inhibitory effect on lice and the volatile phase can exert its inhibitory effect on the inoculated micro-organisms.
(ii) Direct application: in this technique, 0.5 µl of the (iii) This latter technique was modified by Benjilali essential oil or one of their main components was et al. (1984). The authors used Pyrex glass Petri applied directly onto the nits and lice and the dishes with exactly the same shape where the conidiospores were inoculated in radial lines. ThePetri dishes were turned upside down and the filter Atiprotozoal oils. Mikus et al. (2000) examined 12
paper was put in the middle of the cover and essential oils and eight terpenes for antiprotozoal soaked with variable amounts of tested oil. For activity. They were screened for antitrypanosomal and each 3–6 strains series, and for each oil, six Petri antileishmanial activity by an in-vitro growth inhibition dishes were prepared. The partial inhibitory effect assay, whereby the protozoa were seeded into wells of essential oil was determined after incubation containing different concentrations of the test sub- by comparison of the growth observed (length stances. After allowing 72 h for growth, efficacy was and width) to that of the control. This technique evaluated colorimetrically via an oxidation-reduction presents some advantage when compared to the indicator and expressed as the ED50 (effective dose), original method of Kellner and Kober. First, it is the inhibitory concentration that reduced the growth inexpensive since it allows the study of several strains using the same Petri dish. Second, theradial inoculation offers the advantage of easilyestimating the inhibition effect by measuring the Antimicrobial tests
extent of growth and thus, to compare severalstrains under the same conditions for their In recent years due to an upsurge in antibiotic-resistant infections, the search for new prototype drugs to (iv) Beylier-Maurel’s method (1976) has also been combat infections is an absolute necessity and in this modified by Benjilali et al. (1986) to study antimi- regard plant essential oils may offer great potential and crobial effects of other oils in a solid medium. This hope. These products have frequently been reported to technique, unlike the preceding one, permitted be antimicrobial agents (Martinez Nadal et al., 1973; testing per direct contact with the micro-organism, Franchomme, 1981; Benjilali et al., 1984, 1986; Tantaoui- the totality of essential oil constituents. In addi- Elaraki et al., 1992; 1993; Panizzi et al., 1993; Remmal tion, the minimum inhibitory concentration (MIC) et al., 1993a; Remmal, 1994; Chanegriha et al., 1994; Lattaoui et al., 1994; 1997; Lacoste et al., 1996; Tantaoui- (v) Moreover, according to Santos et al. (1997), anti- Elaraki, 1997). According to the latter author, the study bacterial screening of essential oils from Psidium of antimicrobial activity of essential oils presents some and Pilocarpus species of plants was performed by agar dilution method (National Committee forClinical Laboratory Standards, 1990) and the MIC essential oils present a complex chemical was determined for each essential oil using a two- fold serial dilution technique (Hufford et al., 1975).
(ii) they are poorly soluble in water like their The essential oils were added at concentrations ranging from 50 to 3000 µg/ml to melted and (iii) some of their constituents are volatile; cooled Muller Hinton agar and then distributed to (iv) they must be used in low doses.
sterile Petri dishes. The test bacterial organismswere inoculated in sterile saline (0.85%) on a 0.5 The antimicrobial activity of essential oils has been MacFarland standard (108 colony forming units demonstrated by numerous researches. Thus, many (CFU)/ml) and this suspension was diluted 1:10 so antimicrobial methods have been suggested, most as to obtain a final concentration of 107 CFU/ml.
One micro-litre of this suspension was delivered The first method, reported by Tantaoui-Elaraki to the agar surface resulting in the final desired et al. (1992), is a combination of the Beylier- inoculum of approximately 104 CFU per spot. The Maurel’ technique (1976), and modified by Benjilali Petri dishes were then incubated at 37 °C for 18– et al. (1986). The technique allows assessment of 20 h and after this period they were observed for the Minimal Inhibitory Concentration (MIC) and the lowest concentration that inhibits visible growth the Minimal Lethal Concentration (MLC) of the oil. It consists of cultivating the micro-organisms Therefore, it is important to mention the sensitivity on Millipore membranes of 0.45 µm porosity placed or resistance of tested micro-organisms (Gram-positive on agar media containing different concentrations and Gram-negative bacterium, yeast, mold or fungi) toward tested essential oils. In this case, the micro- (ii) The second method is derived from the micro- organisms can be classified into three groups: atmosphere technique of Kellner and Kober,reported by Allegrini and Simeon de Buochberg (1972). This second technique consists of cultivat- (ii) highly resistant micro-organisms, having a weak ing the micro-organisms to be tested in Petri dishes on agar medium and incubating these dishes in (iii) micro-organisms with a medium over-all sensitivity.
Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
Antiviral oils
down was observed daily for 4 days and comparedwith controls.
Besides their antibacterial and antifungal properties, (iii) For ovicidal tests, 10 eggs of T. infestans were essential oils had also been reported to possess inter- introduced in each dish where the filter paper esting antiviral activity. Bammi et al. (1997) demon- disks impregnated with a known volume of oil strated the effect of five essential oils on Epstein-Barr was deposited. The hatching of the larvae and the virus (EBV) (Viridae) which caused the infectious effect on them (knock-down or mortality) were mononucleosis associated with Burkitt lymphoma and observed every day until the control eggs com- naso-pharynx carcinoma. The study aimed the effect of these oils on the expression of EBV viral capside anti-gen (VCA) in the Marmouset B95-8 lymphoblastoid The results of this study indicated the effective- cellular line using the indirect immuno-fluorescence ness of 20 oils on nymphs and eggs when the impreg- technique. The results showed a cytotoxic effect of nated paper tests were used. These tests proved to tested oils at a dilution factor lower than 1:500. More- be the most sensitive and were therefore chosen for over, the valour of cellular viability was not affected.
studying the action of 12 terpenes present in those Treatment of B95-8 cells with 1:1000 dilution of Thymus oil increased the fluorescence intensity of VCA-positive cells in two separate experiments. In three other Growth inhibitors and antifeedants oils/constituents.
tests, only fluorescence intensity was increased by oil Asarones isolated from the essential oil of Acorus from Thymus sp., while the percentage of the fluorescent calamus L. rhizomes (Araceae) were potent growth cells did not increase significantly.
inhibitors and antifeedants to the variegated cutworm,Peridroma saucia Hubner (Koul et al., 1990). Experi-mental diets contained cis- or trans-asarone at concen- Insecticidal activity
trations of 60–2000 ppm fresh weight of diet. Larvalweights and mortality were recorded after 7 and 10 Essential oils are also reported to have insecticide days for first-instar larvae and after 2, 4, 6 and 8 days properties, essentially as ovicidal, larvicidal, growth inhibitor, repellency and antifeedant (Saxena and Nutritional experiments were carried out with fourth- Koul, 1978; Dale and Saradamma, 1981; Schearer, 1984; instar larvae as these larger larvae and their frass could Krishnarajah et al., 1985; Nath et al., 1986; Koul, 1987; be more accurately weighed than that of the first three Isman et al., 1990; Shaaya et al., 1991; Lahlou et al., instars. In the first experiment, 20 larvae/concentration 2001d). The influence of certain oils and their constitu- were provided with either compound at dietary con- ents on the reproduction of some insect species and on centrations of 250 –1000 ppm. Relative growth per unit morphological changes in others has also been discussed weight of the insect at the outset of the experiment (Saxena and Rohdendory, 1974; Ramos and Stefen, (RGRi) and relative consumption per unit weight of the insect at the outset of the experiment (RCRi) werecalculated on a dry weight basis after three days of Larvicidal and ovicidal oils/constituents. According to
Laurent et al. (1997), 63 essential oils isolated from Cis- and trans-Asarone were also topically applied to Bolivian plants were tested on Triatoma infestans for fourth-instar larvae in 1 µl acetone at doses of 5, 10, 20 ovicidal and larvicidal properties. This insect is respon- and 30 µg/larva, with appropriate controls treated with sible for transmission of Chagas’ disease to humans in acetone alone. Twenty larvae were treated with each the region extending from the arid Peruvian highlands compound at each dose and were then allowed to feed to the very dry northeastern Brazilian regions, and the on untreated diet for 72 h before insects, remaining food, plains of Argentina. Three types of test were used: and frass were dried at 60 °C to constant weight, and topical application on insects; nymphs on impregnated paper and eggs on impregnated paper. In all tests, the Antifeedant activity was assayed using a leaf disk essential oils were used as ethanol solutions with con- choice test. The 2.0-cm2 disks were punched out from cabbage leaves and treated on each side with 15 µl ofaqueous asarone solutions emulsified with Triton X- For insects test, 1 µl of each solution was applied 100 (0.1%) to give concentrations ranging from 1.0 to directly over the abdomen of 10 fourth instar 45.0 µg/cm2. Controls were treated with 15 µl of the nymphs of T. infestans. After observing daily for a carrier alone. The leaf disks were dried at room week, the nymphs were treated again with 5 µl temperature and then fourth- or fifth-instar larvae were of the same solution. Two sets of controls were introduced into each arena containing two treated utilised. One control group was treated with and two control disks in alternate positions. Experi- ethanol only, while the other was not treated. The ments were carried out with two larvae per Petri effect of the application was observed for another dish with five replicates for each treatment. Consump- week and compared with the controls.
tion was recorded using a digitising leaf area meter (ii) For nymphs, 200 µl of each ethanol solution of after 20 h for fourth-instar and 6 h for fifth-instar essential oils were deposited over filter paper disks which were dried at room temperature for 5 min Gross dietary utilisation (efficiency of conversion of and placed in Petri dishes, then 5 fourth instar ingested food, ECI) was decreased when the diet was nymphs of T. infestans were introduced in each supplemented with cis-asarone or when this compound dish. Insect control groups were treated in the same was topically applied to fourth-instar larvae. Inhibition way but dosed only with ethanol. The effect knock- of growth occurred even at a moderate topical dose Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
METHODS TO STUDY THE PHYTOCHEMISTRY AND BIOACTIVITY OF ESSENTIAL OILS (5 µl/larva) primarily as a result of decreased efficiency Concentration-effect curves for each agent tested of conversion of digested food (ECD), even though were performed by cumulative additions to the bath the approximate digestibility (AD) of the food was in increasing concentration. In experiments examining unchanged. Oral or topical treatment with trans-asarone the relaxation of the basal tonus of the ileum, paired also significantly inhibited larval growth, but in this segments of ileum were set up: one piece was exposed case the effect can be strictly attributed to decreased to the oil and the other received no treatment. Relaxa- consumption, as dietary utilization (ECI) was not tion due to test substance was taken to be the differ- ence between the tonus of control and test segment.
Finally, in order to quantify the effect of EOCN onhigh K+-induced contractions, the preparations were Antidiarrhoeal savoury
exposed to the EOCN for a 5 min period after whichthe bath solution was changed for one containing 60 mM Hajhashemi et al. (2000) investigated essential oil from Satureja hortensis for treatment of some gastrointestinal Other methods investigated the effects of the essen- problems. Isolated rat ileum was maintained in a water tial oil of Croton zehntneri either on isolated smooth bath and spasmogenic agents, either potassium chlo- muscle preparations of the guinea-pig on skeletal mus- ride (KCl) or acetylcholine (Ach) were added in vary- cles, in addition to anethole and estragole (Albuquerque ing concentrations. They caused a dose-dependent et al., 1995; Coelho-de-Souza et al., 1998). The effects contraction of the tissues. The spasmolytic activities of of essential oil of Mentha x villosa were also studied dilutions of savoury essential oil were then determined on skeletal muscle of the toad (Fogaça et al., 1997) in addition to the effects of eugenol on excitation- The oil was also investigated for in-vivo tests on mice.
contraction coupling in skeletal muscle (Leal-Cardoso The test animals were orally given a 10% solution of savoury essential oil (1 mg/100 g) diluted in Tween 20,whilst the control animals were given either nothing ora solution of the emulsifier. Thirty minutes later, all of Antinociceptive effect of essential oil
the mice were given castor oil in an attempt to inducediarrhoea but the animals in the essential oil group had The antinociceptive effect of leaf essential oil from Psidium guajava and its major constituents, β- The savoury essential oil was considered as pasmolytic caryophyllene and α-pinene was assessed by Santos towards the ileum by countering the effects of depo- et al. (1998) using chemical (formalin and acetic acid) larisation caused by KCl and inhibiting the activation and thermal (hot-plate) nociceptive tests in adult male Acetic acid-induced writhing test. The number of
Effects of essential oils/constituents on muscle:
writhes per mouse were counted during a 10 min intestinal myorelaxant and antispasmodic effects
period, starting 10 min after i.p. administration of0.6% acetic acid (10 ml/kg). Vehicle (2% Tween 80 in The effect of the essential oil of Croton nepetaefolius saline, 10 ml/kg), essential oil (100, 200 and 400 mg/kg) (EOCN), a medicinal plant from the north-east of Bra- or its major components, β-caryophyllene (400 mg/kg) zil, and its constituents cineole, methyl-eugenol and were administered orally 45 min before acetic acid terpineol, were studied on intestinal motility in-vivo and on in-vitro mechanical activity of intestinal smoothmuscle (Magalhàes et al., 1998).
Formalin test. Mice were injected with 20 µl of 1%
formalin into the subplantar space of the right hind
In vivo experiments. Each experimental mouse received
paw and the duration of paw licking was determined a quantity of charcoal 30 min after receiving EOCN or 0–5 min (first phase) and 20–25 min (second phase) vehicle administered intragastrically. After a further after formalin. The Psidium essential oil suspended 20 min the animals were killed and the small intestine in 2% Tween 80 was administered orally at doses of removed. The distance travelled by the marker was 100, 200 and 400 mg/kg, 45 min before formalin injec- measured and expressed as a percentage of the total tion at a dose of 10 ml/kg. Control animals received intestinal length. In addition, some animals were used an equal volume of vehicle. To investigate the pos- to determine the effect of EOCN on castor oil-induced sible mechanisms involved in the antinociceptive effect of the essential oil, animals received caffeine(20 mg/kg, i.p.) or naloxone (1 mg/kg, s.c.) 15 min In vitro experiments. Male guinea-pigs were killed and
before oral administration of essential oil (400 mg/kg).
small segments of intestine and the gastro-oesophageal, The effect of β-caryophyllene and α-pinene, the two pyloric and ileo-caecal sphincters were removed by major components of plant oil were also tested at a The tissues were mounted vertically in organ baths containing tyrode solution bubbled with air. In experi- Hot-plate test. Mice were preselected by placing them
ments with ileum, following an equilibration period of individually on the hot-plate maintained at 51° ± 0.5 °C 1 h, two standard contractions were induced by adding and animals that showed a reaction time greater than 60 mM potassium chloride to the bath. The majority of 20 s were discarded. The reaction time was measured the results presented were then normalised as a per- before and 30, 60 and 90 min after oral administra- centage of the mean of these initial contractions.
tion of essential oil (100, 200 and 400 mg/kg), its major Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
components β-caryophyllene (400 mg/kg), α-pinene addition, serum samples of the mice were analyzed by (400 mg/kg) or vehicle. A cut-off time of 45 s was used gas chromatography-mass spectroscopy (GC-MS), gas chromatography-fourier transform infrared (GC-FTIR),and gas chromatography-flame ionization detection(GC-FID) to identify and quantify potent compounds Gastroprotective effect of essential oil constituent
effective in increasing or decreasing the motility of miceby inhalation alone. Fragrance compound motility data According to Santos and Rao (2001), the gastroprotec- were correlated with data of the single-odor detection tive effect of 1,8-cineole on ethanol-induced gastric thresholds to compare volatility and motility effects.
mucosal damage in rats was performed as follow: Gastric Mucosal Damage. Gastric mucosal damage was
induced in conscious rats by intragastric instillation of
CONCLUSION
ethanol. The test drug 1,8-cineole was administered atdoses of 25, 50, 100 and 200 mg/kg body weight by oral It can be concluded that biological and pharmacolog- gavage before ethanol. The animals were killed 60 min ical activities of essential oils must take into account after ethanol and their stomachs removed and opened different parameters and factors which can affect re- along the greater curvature to observe the lesions sults of these studies (species, ecological factors and macroscopically. Lesion severity was determined by environmental conditions). Thus, each plant species measuring the area of lesions with a transparent grid presents a profile which it will express differently among placed on the glandular mucosal surface.
these factors. For this reason, a few published studieson essential oils have aimed at elucidating the degree Gastric Wall Mucus. The ethanol-induced changes on
of specificity of the effect of these compounds among gastric mucus was indirectly analyzed by determining the various types of their bioactivity.
the amount of Alcian blue bound to the gastric wall.
Moreover, the studies presented in this paper demon- The mucus-dye complex was extracted by placing the strated the general utility of the bioassays using com- segments in 10 ml of 0.5 M magnesium chloride for pounds of diverse structures and a complex chemical 2 h. Four milliliters of dye extract was mixed with an composition of such essential oils. The knowledge of equal volume of diethyl ether. The quantity of Alcian the methods for testing essential oils/constituents is blue extracted per gram of glandular tissue was then therefore indispensable in discovering the spectrum of action of these natural products, their modes of actionand their therapeutic applications.
Gastric Secretary Studies. The pylorus of each rat was
Nevertheless, it is worth noting that essential oils are ligated under light ether anesthesia to study the basal very heterogeneous mixtures of single substances, bio- gastric secretion. Cineole and cimetidine were adminis- logical actions are primarily due to these components tered intraduodenally immediately after pylorus ligation.
in a very complicated concert of synergistic or antago- Control animals were given the vehicle instead of test nistic activities. Mixtures of such chemicals show a broad drugs. The animals were killed after 4 h. The stomachs spectrum of biological effects and pharmacological prop- were removed and the volume of gastric juice was erties. Moreover, these compounds seem to possess a distinct molecular formula, a certain molecular weight,and certain physicochemical properties.
Gastric Mucosal Nonprotein Sulfhydryls (NP-SH).
Several factors such as phonological age of the plant, Gastric mucosal NP-SH were measured and the percent humidity of the harvested material, and the glandular stomachs from rats treated with 1,8-cineole method of extraction have been identified as possible or vehicle were removed and homogenised in ice-cold sources of variation for the chemical composition, 0.02 M ethyl-enediaminetetraacetic acid. The homogen- toxicity and bioactivity of the extracts.
ate was mixed and then centrifuged. The supernatants It is hoped that research institutes and universities were mixed and the sample was shaken. The absorb- will continue their efforts to discover other new natural active compounds derived from aromatic plants anddevelop their action spectrum on biological materialsand pharmacological animal models to find other ac- Sedative effects of essential oils and fragrance
tive natural molecules with potent therapeutic action compounds
devoid or less toxic than synthetic ones. It is also desir-able that preclinical research on essential oils is a nec- In order to screen and quantify a series of fragrance essary part of the drug discovery and development compounds and essential oils for their sedative pro- process, especially given that there is no single forum perties, groups of four mice each were exposed under and focus for guidance on determining the validity and standardised conditions to these compounds (Buchbauer et al., 1993b). Thus, the motility of the animals was Finally, we should maintain our efforts in consider- ascertained after inhalation and also after caffeine- ing and valorising our natural patrimony, as well as induced overagitation to collect more detailed in- conducting more scientific research on aromatic plants formation on activating or sedating effects of several from chemical analysis, biological, toxicological and compounds and their aromatherapeutical usage. In pharmacological investigations to therapeutic aspects.
Copyright 2004 John Wiley & Sons, Ltd.
Phytother. Res. 18, 435–448 (2004)
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