Pesticide Residues in Medicinal Plants

REVIEW ARTICLE

Pesticide Residues in Medicinal Plants andPhytomedicines

Vania G. Zuin and Janete H. Y. Vilegas*Universidade de Saõ Paulo, Instituto de Quı´mica de Saõ Carlos, Saõ Carlos, SP, Brazil

Pesticides, which are mainly applied on crops for the protection of plants against a range of pests, havebeen found in crude medicinal plants as well as in infusions, decoctions, tinctures and essential oils. Thisfact has caused concern in various segments of society and scientific investigation has been demanded toassess the health hazards more accurately. The present review covers more than 30 years (1963–1998) ofpublished methods of analysing pesticide residues in medicinal plants, with special emphasis on the rele-vance of these matrices, the legislation, the risks involved in using material containing uncontrolledamounts of residues and the possible effects of technological factors on the proportion of pesticide trans-ferred from the raw material to the end product. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords:pesticide residues; chromatographic analysis; medicinal plants; phytopharmaceuticals; toxicology.

INTRODUCTION

The use of medicinal plants in both crude and preparedforms has increased greatly. The World Health Organiza-tion has estimated that 80% of the global populationrelies chiefly on traditional medicine for health care andthere are reports that about 51% of all drug preparationsin industrialized countries derive from plants, acting assources of therapeutic agents or models for new syntheticcompounds, or as raw material for semisyntheticproduction of highly complex molecules (Farnsworthetal., 1985; Whitaker and Evans, 1987; Bruhn, 1989;Gentry, 1993).

According to Pifferi and Vitali (1993), the past twodecades have been characterized by a resurgence ofinterest in the development of new drugs and the re-establishment of old ones from plant sources—not onlyas part of the ‘green fashion’, or the ‘dangerous appeal’ ofalternative phytotherapy—but principally due to theprogress made in isolation technology, analytical in-strumentation and computer data processing, which havecontributed to the reduction in scepticism about herbalmedicines.

However, the complexity of these drugs and theirinherent biological variation, make it indispensable,though difficult, to evaluate their safety, efficacy andquality (Schilcher, 1987). Owing to the great variabilityin plant chemical composition that results from factors towhich plants are exposed during their growth, storage andthe different stages of manipulation, characterization and/

or standardization of phytopharmaceuticals are necessary(Baueret al., 1993). Standardization of herbal prepara-tions should allow the knowledge of their compositionand prevent, or at least make less likely, the consumptionof drugs of questionable quality. The latest Pharmaco-poeias, based on recent studies and attempts at standard-ization, prescribe methods, usually chromatographic, forthe analysis and quality control of medicinal products ofplant origin (Ma et al., 1991; Koupai-Abyazaniet al.,1992; Vilegaset al., 1995; Bauer and Tittel, 1996;Matysik, 1996; Gye`resiet al., 1997; Moraeset al., 1997;Raffaelli et al., 1997).

Standardized phytopharmaceuticals must have aknown content of active or characteristic substances.Exogenous substances must be below specified limitsrecommended by regulatory agencies (Evans, 1989).Depending on the type of preparation, organolepticfeatures, moisture and ash content, physical propertiesand adulterants are checked to confirm identity anddetermine purity (Parodiet al., 1993; Bauer, 1998). Thepresence of chemical adulterants (e.g. diazepam, chlor-diazepoxide and caffeine) in herbal products wasreported by Laiet al. (1995). The authors point out thatthe adulterated herbal medicines they analysed (exten-sively consumed in China and other Asian countries) aretypically sold as ‘wonder drugs’ without any indication ofthe contents or manufacturing location.

Microbiological contamination and foreign materialsare important quality criteria in the testing of medicinalplants. As with any product from agricultural or wildsources, medicinal plants can be contaminated by organicor inorganic substances of natural or synthetic origin,such as insects, microorganisms, e.g. fungi and theirmycotoxins (Roy and Chourasia, 1990; Kumar and Roy,1993; Chourasia, 1995; Efuntoye, 1996; Azizet al., 1998;Halt, 1998), heavy metals (Wong and Koh, 1986; Wonget al., 1993; Chizzola and Franz, 1996; Sathiyamoorthyetal., 1997; Figuraet al., 1998), radioactive materials

PHYTOTHERAPY RESEARCHPhytother. Res.14, 73–88 (2000)

Copyright# 2000 John Wiley & Sons, Ltd.

* Correspondence to: J. H. Y. Vilegas, Laboratorio de Cromatografia,Instituto de Quı´mica de Saõ Carlos, Universidade de Saõ Paulo, Caixa Postal780, 13560-970, Saõ Carlos, SP, Brazil.E-mail: [email protected]/grant sponsor: CAPES.Contract/grant sponsor: CNPq.Contract/grant sponsor: FAPESP.

Received 5 May 1999Revised 23 July 1999

Accepted 13 September 1999

(SimonandGraham,1996;KaravaevaandMolchanova,1998), fumigation residues (Chaigneau,1983, 1986;ChaigneauandMuraz,1986;Goldberg,1986)andplantprotectionsubstances,i.e. pesticides,the subjectof thepresentreview(Ruminska,1965;DebskaandLutomski,1979;Robinet al., 1979a;DebskaandGnusowski,1980;Nastasaet al., 1981;Mautneret al., 1982).

PESTICIDE RESIDUES IN PLANT MATERIAL

Typesof pesticidesand their toxic effects

Pesticidesare single substancesor mixtures used toeliminate undesirable vegetable and animal life inagriculturalandurbanecosystems.Theycanbeclassifiedaccordingto their chemicalcomposition,function andmodeof actionin organisms.Chemically,thecompoundscanbe divided into threegroups:biological (vegetable,bacterialor fungal),inorganicandorganicpesticides.Thelast group, which is the largest and has pronouncedphysiological activity, is constituted mainly by theorganochlorine,organophosphorus,carbamateand tria-zine compounds(Barlow, 1985).

Theorganochlorinepesticides,e.g.1,1,1-trichloro-2,2-di-(4-chlorophenyl)-ethane (DDT) are an extensivegroup of compoundsthat have been widely used asinsecticides.Owing mainly to their persistencein humanbeings,their usehasbeenrestrictedor evenbannedinmany countries.The toxicity of individual compounds,and hence the potential hazard to animals includinghumanbeings,varieswidely. Aldrin, dieldrin andendrinareconsideredto bethecompoundsthatcommonlycausepoisoning. In the main these insecticides producesymptomssuch as the over-stimulationof the centralnervous system,which may be worsenedby solventeffects.

The organophosphoruspesticides, e.g. parathion,malathion and diazinon, are potent cholinesteraseinhibitorsandcanbevery toxic. CNSsymptomsincluderestlessnessas well as depressionof the respiratoryorcardiovascularsystem.Repeatedexposuremay have acumulative effect although these pesticides are, incontrast to organochlorines,rapidly metabolizedandexcretedandarenot appreciablystoredin body tissues.Among someof the other groupscited, the carbamates(N-substitutedestersof carbamicacid)arecholinesteraseinhibitors. They differ from the organophosphoruspesticidesbecausetheir inhibitory effect is generallylessintenseandmorerapidly reversed.Furthermore,theydonotseemto entertheCNSasreadily,soseverecentraleffectsareuncommon(Reynolds,1989).

LD50 is theamountof a toxicantnecessaryto kill 50%of thetestpopulationwithin a specifiedperiod.It is usedto estimateoral anddermalpesticidetoxicity, in termsofweightof thechemicalperunit bodyweight(mg/kg).Forexample,pesticidessuchas(2,4 dichlorophenoxy)aceticacid (2,4-D), dicamba, parathion and carbofuran areincluded in the highly toxic class(LD50 0–50mg/kg).However, LD50 is not sufficient alone to evaluatetoxicity, becauseit gives information only about theacute toxicity of a compoundin relation to a certainanimalspeciesanddoesnot describepossiblealterationsthat may occur due to prolongedexposure,i.e. chronictoxicity. Chronic toxicity refers to the capacity of a

substanceto causepoisonouseffectsover the long-term,at low-level exposure(Barnardet al., 1997).Data fromanimalor humanexposuretestshaveindicateda numberof examplesof chronic toxicity, including carcinogeni-city causedby compoundssuchas(2,4 dichlorophenox-y)aceticacid (2,4-D, triazines,endrin,linuron, rotenone,captanand maleic hydrazide,teratogenicityby cyana-zine,carbaryl,endrin,rotenone,andbenomyl,mutageni-city by dillate, trifluralin, dimethoate,carbaryl,rotenone,benomylandmaleichydrazide,neurotoxicityby ethyl(p-nitrophenyl)thiobenzenephosphate(EPN)andreproduc-tive effectsby dibromochloropropane(DBCP),dimetho-ate,rotenoneandbenomyl.

Theabusiveanddisorderedemploymentof pesticideshas causeda seriesof problemsof an ecological andpublic healthnature(Suganavam,1996).Someof theseare the appearanceof new pests, extermination ofpollinator insects, bioaccumulationand existence ofresiduesin the atmosphere,lithosphereandhydrosphere(Plimmer, 1996; Zuin et al., 1999). The fate andbehaviour of pesticides in a soil–plant–atmospheresystem, which is mediated by water dynamics, aredeterminedby inherent propertiesof thesechemicals,their formulation type, application technique,climaticconditions,soil characteristicsandplant morphology.Insucha system,pesticideswill undergoa fairly limitednumberof reactionsthroughphysical,physico-chemical,chemicalor biochemicalprocessessuchas adsorption,desorption,ion-exchange,free-radicalreactions,oxida-tion, reduction, hydrolysis, alkylation, dealkylation,decarboxylationandisomerization(Kips, 1985).

Severalstudieshavebeencarriedout in orderto assessand predict the potential contaminationof vegetableproducts.Thesefocuson aspectsincludingthepathwaysof pesticideuptakefrom the soil by plants(Beitz, 1989;Behrendtand Bruggemann,1993; Schroll et al., 1994),the penetrationand photodegradationof pesticidesinplantsurfacesaswell astheir volatilization from foliage(Bentson, 1990; Dorfler et al., 1991; Schreiber andSchonherr,1993;Kubiaketal. 1995;Kirschetal., 1997),the bioconcentrationandkinetic releaseof pesticidesinplant leaves(Bacci et al., 1990; Zongmaoand Haibin,1997), the developmentof mathematicalmodels toestimate the maximum potential pesticide residue inplantmaterialin thefoodchain(Pfleegeretal., 1996)andthe effect of plant protectionresidueson the chemicalreactionsthattakeplacewithin vegetabletissuesandviceversa (Harvey, 1986; Lydon and Duke, 1989), amongothers.

Useof pesticidesin the cultivation of medicinalplants

In practice,the large-scalecultivation of medicinal aswell as food plants, is not possiblewithout pesticides.Becauseof high costs,‘organic’ (pesticide-free)cultiva-tion is only possibleon a small scale—orthe risk ofsevereattackby pestsmustbeaccepted.Thequantityofraw material coming from organic plantationsor fromwild areasis at the moment insufficient to satisfy thedemandof the herbal drug market (Schilcher,1985a;Bisset,1994).Consequently,the ideal situationnot onlyfor medicinal but also for food plant productionis theimplementationof integratedpostmanagementbasedonthe rational useof pesticide-basedtechniques(Ausher,

74 V. G. ZUIN AND J. H. Y. VILEGAS

Copyright# 2000JohnWiley & Sons,Ltd. Phytother.Res.14, 73–88(2000)

1997). The application of pesticidesunder controlledconditions can be done by methodsthat allow theireffective, safe and economicuse (Zweig and Aspelin,1983).For example,Kiraly (1996)showedthat fusariumwilt diseasehadbeenonly partially controlledby regularfungicides such as benomyl. However, the use ofintegrated control measures,a pesticide–lime–nitrateregime,stimulatedplantdiseaseresistanceandinhibitedor evenkilled thepathogens.

Pesticidesor evengrowth regulatorsmay interferetolarger or smaller extents with secondarycompoundbiosynthesisin higherplants,altering the percentageofthe active compounds (Vomel and Mautner, 1982;Czarnecki and Zalecki, 1990; Omer et al., 1997).Reichling and collaborators (Reichling et al., 1977;Vomel et al., 1977; Reichling, 1979a,1979b) investi-gatedthe influenceof five herbicideson flower produc-tion and on the composition of the essentialoils ofMatricaria chamomillaL.. When treatedwith variousherbicides,this plant suffered alterationsin the totalcontentof essentialoils aswell asin theproportionof themain compounds (bisabololoxide B, bisabolol andbisabolonoxide).Schilcher (1977) also confirmed thatthe growth of Matricaria chamomilla L. and thecompositionof theessentialoils werestronglyinfluencedby the type of herbicideand the amountand period ofapplication, which can probably be explained by amechanismof action basedon the inhibition of nucleicacid synthesis.

Contrasting results were obtained by Pank andcollaborators (Pank and Neczypor, 1978; Pank andEnnet,1980,1988;Panket al., 1978,1979,1980,1981,1982,1983,1984,1986,1987a,1987b),who testedmorethan 60 herbicides in a long-term study in order todevelopchemical killing methodsto control weedsinseveralmedicinal plant fields. The authorsconcludedthat,althoughthe utilization of thoseherbicideshadledto a considerablereduction in weed cover and aconsequentdiminutionof manualwork neededfor weedcontrol, the useof chemicalsdid not result in any yieldreduction or morphological variation and did notinfluencethe productionof essentialoils, including therespectiveactiveprinciples.

Theeffectof herbicidesonthequalitativepropertiesofmedicinalplants(MelissaofficinalisL., SalviaofficinalisL. and Menthax piperita (L.) Huds. cv. Perpeta) wasinvestigatedby Vaverkovaet al. (1995a,1995b,1997).The treatmentof medicinalplantswith herbicides,e.g.terbacil and linuron, did not influencethe essentialoilcontentof the treatedplants,comparedwith thosenottreated.Similarly, the application of a herbicide for-mulationdid not causedetectablechangesin therelativeproportion of main and secondarycomponentsof theessentialoils.

In the sameway, the influenceof a wide rangeofherbicidesand growth regulatorson the growth andessentialoil contentof sageandpeppermintwasevaluatedby El-Keltawi andCroteau(1986a,1986b,1987).Exceptfor oil yieldreductionin thecaseof severestunting(e.g.bymalazide application), no direct relationship betweentissuegrowthandessentialoil formationin thesespecieswasobserved.Theresultsobtainedby Maas(1979)withthe applicationof herbicidesin AnethumgraveolensL.,Thymus vulgaris L., Matricaria chamomilla L. andOcimumbasilicum L. demonstratedthat the effects ofherbicideresiduesontheproductionof activecompounds

by medicinal plants cannot be defined for non-homo-geneousgenetic herbal material, since the response,tolerance and breakdown rate of pesticidesmay bedifferent for each plant. Recently,emphasishas beengiven to genetictransformation,in particularto increasethe productionof secondarymetabolitesandto generatemedicinal plants resistant to herbicides,diseasesandpests.Accordingto Bajaj (1998),biotechnologicaldevel-opmentshavefar-reachingimplicationsfor the improve-mentof medicinalherbs.

It has to be noted that besidesdirect contamination,which would be the resultof pesticideapplicationto theplants of interest, medicinal plants may also besusceptible to indirect contamination. Pluta (1989)attributedthe considerablepesticidecontentin most ofthe studiedherbalraw materialto global contaminationof theenvironment.In somecasesthis maybecausedbyaccidentalcontamination,e.g. hygienic intervention inforests, a very common procedure in developingcountriesto eliminatepestsandrestraintropicaldiseasessuchasmalaria.However,Lorussoet al. (1985)considerthat global environmentcontaminationcanonly explainthe presenceof organochlorinecompoundsin medicinalplants and not that of organophosphoruscompounds,owing to their relatively fast degradation.Thus, theorganophosphorusresidues,as well as other pesticideclasses, come from the continuous use of suchcompounds at some stage of phytopharmaceuticalproduction.

According to Bisset (1994) pesticide residuesarealways found in both foodstuffs and medicinal plants,whetherthe living plantsare treatedwith pesticidesornot, sincetheseenvironmentalcompoundshavespreadmoreor lessworld-wide.In fact, thereis a largenumberof publicationson such residuesin food (Dorea et al.1996;Lancaset al., 1996)andcuriously—although theproblemsof pesticideresiduesin medicinal plantsanddrugpreparationsarenot entirelydifferent from thoseofvegetablefood (Schilcher,1985b)—thereis still littleinformationavailableregardingmedicinalplants(BagchiandPuri, 1985).

While theuseof pesticidesfor drugandfood plantsisregulatedin many countries, legislation is lacking orlargely ignoredin severaldevelopingcountries.Unlikeindustrializedcountriesthat haveestablishedan exten-sivebodyof law to protectthepublic from occupational,environmentalandconsumerhazards,manydevelopingcountrieshavenotyetdoneso.Thedisparitiesin nationalregulation and other factors controlling hazardousproductsrequirethe recognitionof internationalregula-tion for pesticideresiduelimits for all the public andprivate entities that intervene or are involved in thedistribution and utilization of medicinal plant products(Finkelman,1996).

SETTING MAXIMUM PESTICIDE RESIDUELIMITS FOR HERBAL DRUGS

The WHO (WHO, 1992) has made a series ofrecommendations,on thebasisof nationalconsultations,to promotethe establishmentof an internationallist ofpesticides suitable or unsuitable for utilization oncultivated medicinal plants.The WHO has also estab-lishedmaximumresiduelimit (MRL) for thesebiocides

PESTICIDERESIDUES 75


Table 1. Main analytical methodsfor determination of pesticideresiduesin medicinal plants and phytopharmaceuticalsResidue range

Pesticidea Matrixb Sample preparationc Determinationd (mg/kg)e Reference

OC Infusions of Tilia cordata Mill. LLE followed by CC (Florisil/anhydrous Na2SO4)

GC-ECD 0.0029±0.369 Lino andSilveira, 1997b

OC Dry and powdered Tilia cordataMill., Melissa of®cinalis L., Malvasylvestris L., Citrus aurantium L.,Matricaria chamomilla L., Cassiaangustifolia Vahl, Cassia senna L.and Cassia obavata Colladon

Maceration (acetonitrile, n-hexane or H2O), Ultra-Turrax orultrasonication homogenization,centrifugation followed by CC(C18, Florisil or Florisil/anhydrous Na2SO4)

GC-ECD 0.02±0.5 Lino andSilveira, 1997a

OC Commercial bags of mint, vervain,lime tree, tea and camomile

Infusion, SPE (C8) followed byCF3COOH, CCl3COOH treatmentor Cl2 oxidation

GC-MS 1 MoltoÂ et al.,1994

OC Commercial infusion bags oflinden, camomile and black tea

Grinding (plant material plussandplus anhydrous Na2SO4),heating (hexane:acetone), LLEfollowed by CC (Florisil/anhydrous Na2SO4)

GC-ECD 0.0001±0.321 FernaÂ ndez etal., 1993

OC Commercial samples of Frangulaecortex, Quercus cortex,Calendulae¯os, Crataegi fol. c. ¯or.,Malvae ¯os., Tiliae ¯os.,Matricariae ¯os., Sambuci ¯os.,Verbasci ¯os., Betulae folium,Farfarae folium, Fragariae folium,Melissae folium, Menthaepiperitaefolium, Uvae ursi folium,Myrtilli folium, Rosmarini folium,Rubi fruticosi folium, Rubi idaeifolium, Salviae folium, Carvifructus, Crataegi fructus, Foeniculifructus, Rosae pseudofructus,Absinthii herba, Cardui benedictiherba, Equiseti herba, Millefoliiherba, Thymi herba, Urticae herba,Violae tricolor. herba, Althaeaeradix, Liquiritiae radix, Taraxaciradix c. herba and Valerianae radix

Soxhlet (hexane/Si gel/anhydrous Na2SO4), CC (celite)and LLE

GC-ECD 0.001±1.051 Benecke andOrtwein, 1992

OC `KuÈ mmelfruÈ chte' aceration and agitation(acetone), LLE and CC (Florisil/Na2SO4)

GC-ECD 0.05±0.54 Gabrio et al.,1990

OC `BirkenblaÈ tter', `Heidekraut',`BrombeerblaÈ tter',`Heidelbeerkraut', `Ginsterkraut',`Johanniskraut', `Brennesselkraut',`Rainfarnkraut', `Schafgarbenkraut'and Àckerschachtelhalmkraut'


GC-ECD 0.008±77.99 Benecke et al.,1989

OC Commercial pre-packed medicinalplant mixtures

Maceration (H2O:acetone andcelite), LLE followed by CC(basic alumina) or CC (celite)and H2SO4

GC-ECD 0.012±0.330 Alamanni et al.,1988

OC Tinctures SPE (C18) GC-ECD 0.010±0.500 GuÈ beli andClerc, 1988

OC Commercial pre-packed mixturesof `Camomilla', `Tiglio', `RosaCanina', `Verbena', `Liquirizia',`Finocchio', Àrancio ®ore', Àrancioscorza', `Melissa', `Valeriana',`Luppolo', `Menta Anice', `Timo',`Senna', `Boldo', `Carciofo Anice',Àlburno', `Brughiera Lavanda',`Salvia', Èucalipto Liquirizia',`Gramigna', `Guajaco', Ìssopo',`Parietaria', `Menta piperita',`Salsapariglia', Ànice Stellato,Ànice verde' and `Carvi'

Maceration (H2O:acetone andcelite), LLE followed by CC(basic alumina) or CC (celite)and H2SO4

GC-ECD 0.11±0.51 Alamanni et al.,1987b

OC Commercial samples of Ùvaursina', `Boldo', `Senna',`Camomilla', `Biancospino',`Rabarbaro', `Frangula', `Passi¯ora',`Fiori d'arancio' and `The'

Maceration (H2O:acetone andcelite), LLE followed by CC(basic alumina) or CC (celite)and sulphuric acid

GC-ECD 0.010±0.095 Alamanni et al.,1987a



Table 1. ContinuedResidue range


OC Flores Chamomillae, FloresMalvae, Flores Crataegi, FoliaCrataegi cum Floribus, FructusAnisi tot., Herba Hyperici, HerbaMillefolii, Herba Thymi, HerbaUrticae, Semen Lini, Folia Menthaepip., Folia Melissae, Flores Tiliaeand Herba Equiseti

Powdered plant material: Plantplus sand or celite mixture oncolumn glass (petroleum etheror acetonitrile) followed by CC(deactivated Si gel/anhydrousNa2SO4) Infusions: LLE and CC(deactivated Si gel/anhydrousNa2SO4)

GC-ECD < 0.001±0.950 Ali, 1987

OC Dried and powdered samples ofFlores Chamomillae and RadixValerianae


GC-ECD andGC-MS

0.013±9.164 Benecke et al.,1986a

OC Dried and powdered Flor. Verbasci,Flor. Chamomillae, Fruct. Foeniculi,Fruct. Carvi, Fol. Salviae, Fol.Melissae, Fol. Menthae pip., Fol.Thymi, Herba Millefolii, Rad.Althaeae and Rad. Valerianae


GC-ECD 0.10±0.5 Benecke et al.,1986b

OC Ginseng Radix, Angelicae Radixand Cnidii Rhizoma

Maceration (acetone:H2O)followed by CC (Biobeads SX3and Si gel)

GC-ECD andTLC

0.004±1.18 Kwon et al.,1986

OC Infusions of Herba Equiseti, FloresHibisci, Flores Chamomillae, FoliaSennae, Fructus Carvi, FoliaBetulae, Folia Menthae pip.,Fructus Foeniculi, Fructus Anisi andFructus Sennae

LLE and CC (deactivated silicagel/anhydrous Na2SO4)

GC-ECD 0.001±0.600 Ali, 1985

OC Dried Flores chamomillae Maceration (hexane), CC (celite)followed by chemicaltransformation

GC-ECD 0.01±0.5 Benecke et al.,1985

OC Cassia senna L. and Cassiaangustifolia Vahl.

Comparison between severalmethods e.g. ultrasonicationand CC

GC-ECD 0.001±1.86 Stahl and Rau,1984

OC Herba Equiseti, Semen Lini, FloresHibisci, Flores Chamomillae, FoliaSennae, Fructus Carvi, FoliaBetulae, Folia Menthae piperitae,Fructus Foeniculi, Fructus Anisi andFructus Sennae

Plant plus sand mixture oncolumn glass and CC(deactivated Si gel/anhydrousNa2SO4) or maceration withagitation (n-hexane), LLE andCC (deactivated silica gel/anhydrous Na2SO4)

GC-ECD 0.01±3.0 Ali, 1983b

OC Powdered Equiseti herba, Linisemen, Hibisci ¯os, Chamomillae¯os, Betulae folium, Foeniculifructus and Anisi fructus

Plant plus sand on columnglass and CC (deactivated Sigel/anhydrous Na2SO4) ormaceration with agitation (n-hexane), LLE and CC(deactivated Si gel/anhydrousNa2SO4)

GC-ECD 0.02±3.00 Ali, 1983a

OC Camomile ¯owers (Matricariachamomilla L.)

Maceration (NaCl 2% solutionfollowed by benzene),centrifugation followed by CC(Florisil)

GC-ECD-FID 0.008±0.017 Molinari et al.,1981

OC Dried material and infusions ofFlosaurantii, Fol. Verbenae odorateand Fol. Menthae pip. Electiss.

Maceration, LLE and CC(Florisil)

GC-ECD 0.001±0.668 Schlumpf andStettler, 1981

OC Powdered Flores Chamomillae,Folia Sennae and Fructus Phaseoli

Plant plus quartz sand mixtureon column glass (petroleumether) followed by CC(deactivated Sigel/anhydrousNa2SO4)

GC-ECD 0.002±0.47 Fehr and Ali,1980

OC Dried and powdered Tiliaplatyphyllos Scop., Anthemisnobilis L., Malva of®cinalis L.,Citrus aurantium L. var. amara,Crataegus oxyacantha L., Lippiacitriodora H. B., Matricariachamomilla L., Melissa of®cinalisL., Mentha piperita L., Salviaof®cinalis L. and Ribes nigrum L.

Maceration(acetonitrile:petroleum ether),LLE followed by CC (BiobeadsSX3)

GC-ECD 0.004±0.20 Robin et al.,1979b





OC Dried and powdered Anthemisnobilis L., Citrus aurantium L. var.amara, Malva of®cinalis L.,Crataegus oxyacantha L., Lippiacitriodora H. B., Melissa of®cinalisL., Matricaria chamomilla L.,Mentha piperita L., Ribes nigrumL., Salvia of®cinalis L. and Tiliaplatyphyllos Scop.

Maceration(acetonitrile:petroleum ether),LLE followed by CC (BiobeadsSX3)

GC-ECD 0.004±0.20 Robin et al.,1978

OC Powdered Fol. Menthae piperitaeand Flos. Chamomillae

Maceration (petroleum ether)followed by CC (charcoal/celite/anhydrous Na2SO4/alumina oracid alumina)

TLC and GC-ECD

0.01±0.5 Henneberg andLutomski, 1972

OC Dried and powdered CortexChinae, Cortex Frangulae, CortexQuercus, Flores Arnicae, FloresCalendulae, Flores Chamomillae,Flores Sambuci, Flores Tiliae, FoliaBetulae, Folia Digitalis, FoliaHamamelidis, Folia Hederae, FoliaMelissae, Folia Menthae and FoliaRosmarini

Maceration with/withoutagitation or heating (n-heptane),H2SO4 treatment, CC(anhydrous Na2SO4/Si gel/H2SO4) and LLE

TLC 0.05±6.30 Luckner andLuckner, 1972

OC andOP

Angelicae gigantis Radix,Atractylodis Rhizoma alba andCurcumae Rhizoma

Maceration (acetonitrile:H2O)and H2SO4 treatment

GC-FID 0.25±12.5 Yoon et al.,1998

OC andOP

Camomile SFE (CO2) GC-ECD, GC-FPD and GC-MS

0.2±0.6 Carisano andRovinda, 1995

OC andOP

Plant material and hydro alcoholicextracts (Crataegus and Senna)

SFE (CO2) GC-NPD andGC-ECD

Erdelmeier,1993

OC andOP

Commercial samples of `MentaPiperita', `Fiori d'arancio', `Lavanda®ori', `Salvia foglie', `Tiglio ®ori',`Rosmarino foglie', `Verbenaodorosa foglie', Ànice semi', `Timovolgare', Èufrasia', Meliloto',`Melissa sommitaÁ ', `Finocchio semi'and mixture of herbs

Maceration (H2O:acetone andcelite), LLE followed by CC(Florisil)OP and CC (celite orbasic alumina) with/withoutH2SO4 treatmentOC

GC-FPD andGC-ECD

0.01±5.93 Muccio et al.,1981

OC andOP

Matricaria chamomilla L. ofdifferent origins

Maceration (methanol), LLE, CC(Alumina, Fuller's earth andFlorisil)

GC-ECD andGC-NPD

0.019±5.68 Reichling et al.,1979

OC andOP

`Mauve', `Menthe', Òranger',`Thym', `Millefeuilles', `Romarin',`Sauge', `Serpolet', `Tilleul' and`Verveine'

Maceration (ethylacetate:dichloromethane), LLEand CC (Florisil)

GC-FPD andGC-ECD

0.002±1.42 Illes et al., 1976

OC andOP

Infusion and oil of Mentha piperitaL.

Infusion: LLE, CC (Florisil) Oil:direct injection after dilution

GC-NPD andGC-ECD

0.0005±8.0 Friedrich andBuÈ ter, 1975

OC andOP

Fresh and dried Mentha piperita L. Blending (celite and acetone),LLE and CC (Florisil)

GC-NPD andGC-ECD

0.05±50 Friedrich andBuÈ ter, 1974b

OC andOP

Peppermint hay and oil Hay: Maceration (n-hexane:isopropyl alcohol or HClconc.), LLE followed by H2SO4

treatment and sulphonation orCC (celite/anhydrous Na2SO4)Oil: charcoal treatment andsulphonation, LLE and CC(celite) or only CC (charcoal/attasol-Dicalite)

SP and GC-MCD

0.01±61 Starr et al.,1963

OP Fresh and dry Mentha sp.,Majorana hortensis L. andMatricaria chamomilla L.

Maceration (acetone), LLEfollowed by SPE (Florisil)

HPLC-UV andGC-MS

0.024±0.23 Ahmed et al.,1998

OP Dry ¯owers, fresh ¯owers andleaves of chrysanthemums

Blending (acetone), ®ltration(through GC-NPD celite layer),coagulation and LLE

GC-NPD 0.036±2.00 Wu and Fan,1992





OP Humulus lupulus L., Artemisiavulgaris L., Erica cinerea L., Melissaof®cinalis L., Mentha piperita L.,Laurus nobilis L., Salvia of®cinalisL., Cinnamomum zeylanicum Nees,Malva sylvestris L., Peumus boldusMolina, Rosa gallica L.,Sarothamnus scoparius Koch,Citrus aurantium L., var. amaraLink., Citrus aurantium L., var.dulcis Pers., Crataegus oxyacanthaL., Tilia sylvestris Desf., Lippiacitriodora H.B. and K.

Maceration(acetonitrile:petroleum ether)with agitation, LLE and CC(micro-styragel 100 AÊ )

HPLC-UV 0.04±5 Atindehou etal., 1981

UC Dried M. of®cinalis L., M. piperita L.and E. angustifolia Moench

Ultra Turrax homogenization(acetone:H2O), LLE followed byCC(Florisil/anhydrous Na2SO4)

GC-NPD andGC-MS

0.02±0.051 Tekel et al.,1998a

UR Roots of Echinacea angustifoliaMoench

Maceration (acetone), LLEfollowed by (Florisil/anhydrousNa2SO4)

GC-NPD andTLC

0.02±0.40 Tekel et al.,1998b

UC Melissa of®cinalis L. Maceration (acetone), LLEfollowed by (Florisil/anhydrousNa2SO4)

TLC 0.005±0.01 Tekel et al.,1994

CB Calendula of®cinalis L. Maceration, LLE andsubsequent CC or derivatizationfollowed by distillation

GC-NPD andGC-ECD

0.05±2.55 Pank and Ennet,1988

TR Dried and powdered`Eibischwurzel', `SalbeiblaÈ tter',`SpitzwegerichblaÈ tter',`FenchelfruÈ chte', `Angelikawurzel',`KorianderfruÈ chte',`KuÈ mmelfruÈ chte',`PfefferminzblaÈ tter',`ThymianblaÈ tter'and`Schafgarbenkraut'

Maceration with agitation,centrifugation or Soxhlet(chloroform), CC (alumina) andLLE

GC-NPD 0.02±10 Gabrio andEnnet, 1982

TR Thymus vulgaris L. Maceration with agitation(chloroform) and CC (alumina)

GC-NPD 0.01±0.66 Pank et al., 1982

TR Plantago lanceolata L. Maceration (methanol), LLE andCC (alumina)

TLC and GC-NPD

0.05±2 Pank and Ennet,1980

TR Matricaria chamomilla L., Menthapiperita L., Salvia of®cinalis L.,Achillea millefolium L. and Thymusvulgaris L.

Maceration (methanol), LLE andCC (basic alumina)

TLC 0.02±0.05 Reifenstein andPank, 1975

CD Peppermint leaves (dried andfresh) and marsh-mallow leaves(fresh)

Blending (acetone), hydrolysisfollowed by steam distillation,LLE, diazotization and couplingreaction

SP 0.17±0.44 Debska et al.,1979

OC, OPand PY

Lippia citriodora, Mentha aquatic xMentha spicata andChamaemelumnobile

Maceration and blending(acetone oracetonitrile) followedby CC (Ultra styragelOP or SigelOC, PY)

GC-ECD andGC-NPD

0.05±1.5 Nunes et al.,1997

OC, OP,TR andPY

Mallow and tea Maceration (petroleumether:acetone) and CC (Florisil/Si gel/anhydrous Na2SO4)

GC-ECD andGC-NPD

0.02±0.08 Cioni andBartolucci, 1994

OC, OPand PY

Dried Livesticum of®cinali K. andTanacetum balsamita L.

Maceration (acetone) orinfusion followed by LLE andCC `sandwich' (combination ofanhydrous Na2SO4/Si gel/celite/charcoal)

HPLC-UV, SP 0.006±0.180 Miellet, 1982

OP, PY,PH andBZ

Peppermint and monarda oil(Mentha piperita L. and Monarda®stulosa L.)

Direct injection or LLEBZ GC-ECD,GC-NPD andHPLC-UV

0.01±24.83 BeÂ langer, 1989

TR, BTand UR

Melissa of®cinalis L. Maceration with agitation(chloroform) and CC(alumina)TR; Maceration(methanol), derivatization andCC (Florisil)BT; Soxhlet(ethylether), derivatizationfollowed by distillation andLLEUR

GC-NPD,GC-FPD and CT

0.02±33.8 Pank et al.,1987b



in cultivated or wild medicinal plants as well asappropriatemethodologiesfor their analysis.

The MRL is calculatedafter safety tests in humanbeings,which indicatetoxicologically acceptablelevelsaccordingto themostreliableassaysavailableat thetime(FAO/OMS,1994).Owingto thespecificrequirementsofpharmaceuticalpreparations,MRL determinationcannotbe basedsolely on the CodexAlimentarius,WHO/FAOrecommendations,which are designedto cover onlyfoodstuffs. The toxicological evaluation of pesticideresiduesin herbal drugs must take into account theintakeof materialby a patient(e.g.routeof administra-tion, dose and duration of treatment),and the hugedifferences in patterns of consumption within thepopulation,as well as the sensitivity of groupssuchasbabies,children,pregnantwomen,old andvery ill peopletowardsthesebiocide compounds(Krstulovic and Lee,1997).

Manyproblemsmayoccurin establishingthetolerable

contaminantlimits in productssuchasphytopharmaceu-ticals (Maclntosh et al., 1996). Besides consideringbioaccumulation,bioreactivityandsynergicinteractionsin the toxicological assessmentof a specific pesticide,Hapke (1982) also mentionedthat a highestnon-toxicdose, obtainedby experimentsin animals, cannot bedirectly convertedto a correspondingdosefor humans.The non-observableeffect level (NOEL), which isdefined as the highest dose (mg pesticide/kg bodyweight/day)that producesno observabletoxic effectsinthe most sensitive species, is derived from chronictoxicity testsandis usedto settheacceptabledaily intake(ADI) for humans:

ADI � NOELx �Safetyfactor �1=100 to 1=2000�� 1�TheADI, describedasthedaily intakeof achemicalovera lifetime that causesno appreciablerisks to humanhealthaccordingto our presenttoxicologicalknowledge,includesa variablesafety factor, e.g. 1/100, applied in



BT, UR,BD andTR

Mentha piperita L. Maceration (methanol),derivatization and CC(Florisil)BT; heating (soda),distillation, derivatization andCC (Alox V)UR; maceration(methanolUR or acetoneBD),LLEUR and CC (e.g. Florisil)UR,

BD; maceration with agitation(chloroform) and CC(alumina)TR

GC-FPD, GC-MCD, GC-ECDand GC-NPD

0.01±0.70 Pank et al., 1986

TR, URand BD

Carum carvi L. Maceration with agitation(Chloroform) and CC(alumina)TR; Soxhlet (ethylether), derivatization followedby distillation and LLEUR;maceration (methanolUR,acetoneBD), LLEBD and CC(Florisil)UR, BD

GC-NPD, CTand GC-ECD

0.01±0.05 Pank et al., 1984

UR, BPand BD

Achillea millefolium L. Maceration (methanolUR, BP oracetoneBD), LLE and CC(e.g.Florisil)UR, BP, BD

GC-ECD 0.01±0.66 Pank et al., 1983

BP andUR

Salvia of®cinalis L. Maceration (methanol) andCCBP; Soxhlet (ethyl ether),derivatisation followed bydistillation and LLEUR

GC-ECD andCT

0.018±0.678 Pank et al., 1981

CB, URand BP

Valeriana of®cinalis L. Maceration (ethanolCB,methanolBP), LLECB andsubsequent CCCB, BP; Soxhlet(ethyl ether), derivatizationfollowed by distillation andLLEUR

TLC, CT andGC-ECD

0.005±1.00 Pank et al., 1980

CB, BD,PX andDT

Matricaria chamomilla L. Maceration (methanol), LLE andCC (Florisil or Alumina/Fuller'searth)

GC-ECD andGC-NPD

<0.5±1.9 VoÈ mel et al.,1977

a OC, organochlorines; OP, organophosphorus; PY, pyrethroids; TR, triazines; CB, carbamates; UR, ureas; UC, uraciles; CD,carboxanilides; PH, phthalimides; BZ, benzimidazoles; BT, benzothiadiazoles; BD, benzamides; BP, bridged diphenyls; PX, phe-noxys; DT, dinitroanilines.b To avoid incorrect denomination, the terminology used for some plant materials was maintained as in the language of theoriginal paper cited.c LLE, liquid-liquid extraction; SFE, supercritical ¯uid extraction; SPE, solid phase extraction; CC, column adsorption chromato-graphy.d GC-ECD, gas chromatography ± electron capture detector; GC-NPD, gas chromatography ± nitrogen-phosphorus detector;GC-FPD, gas chromatography ± ¯ame photometric detector; GC-FID, gas chromatography ± ¯ame ionization detector; GC-MS,gas chromatography ± mass-spectrometry detector; GC-MCD, gas chromatography ± microcoulometric detector; HPLC-UV,high-pressure liquid chromatography ± ultraviolet detector; SP, spectrophotometry; CT, colorimetry; TLC, thin layer chromato-graphy.e Pesticide range (mg pesticide/kg matrix) studied (forti®ed samples) or determined (commercial herbal drug materials).



uncomplicatedcaseswhereall therequiredtoxicologicaldata are available.According to the WHO (1992), theratio betweenmedicinal plant material and food con-sumptionshouldnot exceed1% of the permissibletotalintake of pesticidesfor humanbeings.For example,itwould not be admissible if 30–50% of the ADI forpesticide residues,primarily derived from food anddrinking water,were to be accountedby the additionalconsumptionof vegetabledrugs.

Theestablishmentof residuelimits asregulatoryactionlevels should be recognizedas a referencepoint forprosecution or other legal action. McLean (1996)proposedthattheMRL doesnotdefinepreciselyaspecificlevelwhichrepresentsahealthhazard,andtheoccasionalconsumptionof a commodityexceedingthe MRL for apesticidedoesnot representdangerto humanhealth.

SomePharmacopoeias,e.g. the Brazilian Pharmaco-poeia,neitherdefinetheMRL nor recommendanalyticaltests for pesticides. However, the fact that thesecontaminantsare not included in the Pharmacopoeiadoesnotmeanthattheyshouldbetolerated(Farmacope´iaBrasileira,1996). The EuropeanPharmacopoeia(Phar-macope´e Europeenne,1997) fixes limits for a seriesofpesticides,which fall in the range of mg pesticide/kgplantmaterial(ppm),andforbidstheapplicationof somecompoundssuchasethyleneoxide.However,if thelimitof a specific pesticide does not appear in the ECdirectives it might be calculatedusing the followingformula:

MRL� ADI x WMDI x �100x �safetyfactor�� 2�

whereMRL is themaximumresiduelimits (mg/kg),ADIis the acceptabledaily intake (mg compound/kgbodyweight),valueof FAO/WHO,W is thebodyweight(kg),andMDI themeandaily intakeof drug (kg).

Somelimits higher than thosefixed by the EuropeanPharmacopoeiacanbetoleratedin exceptionalcases,e.g.when a plant requires a special managementforcultivation. Determinationtests for pesticide residuescanbe partially or totally eliminatedwhenthe completetreatmenthistoryof a lot is knownandconductedwithincontrolledandpreciseprocedures.

Whenthedrugis usedto prepareextracts,tincturesorother phytopharmaceutical formulations in which themanipulationmay influencethe pesticideconcentrationin the final product, the MRL is calculatedusing theexpression:

MRL� ADI x W x EMDI x �100x �safetyfactor�� 3�

whereE is the extractioncoefficientof pesticide,whichdependson the methodof preparationand needsto beexperimentallydetermined.

Equation(3) aswell asthecalculationof E valuesweredescribedby Ennetet al. (1978) and later discussedinanotherpublication(Ennet,1989).Likewise,Schilcheretal. (1987) pointed out that the evaluationof a certainpesticidemustberelatedto thefinal drugpreparationandnot to the initial raw plant material.Theseauthorsalsodetailed analytical methodsfor pesticideresiduesanddescribedtheir principalproblems,whicharemainly dueto the extensiveuse of wild-growing herbs that maycontainseveralunknowncompoundsamongthe largernumberof commercializedbiocides(about400).

METHODS FOR THE DETERMINATION OFPESTICIDE RESIDUES IN HERBAL DRUGS

General considerations

As previouslymentionedby BagchiandPuri (1985),inpractice the huge numberof medicinal plants and theimmensequantityof pesticidecompoundsutilized makethe whole phenomenonmultifactorial, and the develop-ment of a completeanalytical method for a range ofpesticides(multiresidue analysis) rather difficult. Thepublished methodsare not essentiallydifferent fromthose proposed for foodstuffs, such as vegetables(Cetinkaya,1988; Torres and Luque de Castro,1996;Zongmao and Wang, 1996; Lehotay and Valverde-Garcia, 1997), tea (Cetinkaya and Thiemann, 1985;PetersenandJensen,1986;Buchholz,1988;Wan,1989,1990;Silva andThiemann,1991)spices(Sullivan,1980)or beverages(Brennecke,1989, 1991; Mortimer et al.,1995), so it is very common to find researchgroupsworking with both matrices(Mestreset al., 1974,1975;Lorussoet al., 1984;Tekel andHatrik, 1996;Muccio etal., 1997).Nevertheless,Gabrioetal. (1988)stressedthatthe analytical methods establishedfor pesticides infoodstuffscannotbe appliedunconditionallyto medic-inal plantsandtheir products.As phytopharmaceuticalssometimescontaintotally dissimilarconstituents,speci-fic methodsfor each one must be developedin thesecases.

Generally the methodology of pesticide residueanalysisincludesthreemain stages:extraction(usuallyfollowed by a clean-up procedure), separation anddetection of pesticides,with special attention to thecorrectidentificationandconfirmationof the compound(Molinari et al., 1981; Wagler, 1988). The mainanalytical methodsreported between1963 and 1998,concernedwith thedeterminationof pesticideresiduesinmedicinalplantsandpreparationsmadefrom them,areshownin Table 1. This table neither includesPharma-copoeialmethods,nor is intendedto beexhaustive.

As seen in Table 1, the majority of the paperspublishedduring 1963–1998areof Europeanorigin, asa consequenceof the long tradition of usingphytophar-maceuticalsin that continent.A considerablenumberofthesestudiescomefrom Germany,particularlyfrom theformer GermanDemocraticRepublic(DDR), andfocuson organochlorine pesticide analysis, although thedetermination of two or more different classes ofpesticides,aswell asotherpollutantslike polychlorinatedbiphenyls (PCBs), was common. PCBs include 209possibleindividual compoundsvarying in the numberandpositionof chlorinesubstitutions(1–10atoms)onthephenylgroups,e.g.aroclors.

Over severaldecadesof research,Schilcher(1983a)found organochlorine compounds to be the maininsecticidespresent in most medicinal plant samplesstudied.Possibleexplanationsfor this fact,aswell asforthe great number of publications concerningorgano-chlorinedeterminations,maybetheir widespreaduseformany years,during cultivation and/or after harvestingandstorage,their continuing—althoughforbidden—useuntil now and their long biological half-life. Theorganophosphoruscompoundswere seldom found inherbaldrugsanddrugpreparations,owing to their lowerpersistenceandbioaccumulationcomparedwith organo-



chlorines,andalsobecauseof their high toxicity, whichmakessuchcompoundsnot very popularin the cultiva-tion of medicinal plants. Except in somecases,otherclassese.g. carbamateswere determinedin only smallquantitiesin herbdrugmaterial.

Thestudyof severalpollutantsandrelatedproblemsinherbaldrugsby thesameresearchgroupis verycommon.Beneckeandcollaborators(Beneckeet al., 1985,1986a,1986b,1987,1988,1989; Beneckeand Ortwein, 1992)establishedmethodsfor the determinationof organo-chlorinepesticidesandPCB. It is well known that PCBcan easily interfere in the analysis of DDT-typecompoundsand vice versa, owing to their structuralresemblanceand,consequently,similar chromatographicproperties.Theidentificationof PCBresiduesin samplesof FlosChamomillae1 couldbeachievedby theretentionbehaviourin gaschromatographyanalysisusingdifferentcolumns,by GC-MSor throughchemicaltransformation(KOH or CrO2 treatment)to avoid interferencein theanalysisof DDT compoundsandPCBin thismatrix.Thedeterminationof PCBandorganochlorinepesticideswasperformedlikewiseby Gabrioetal. (1990)andAlamanniet al. (1987a,1987b,1988),who alsofoundthepresenceof pathogenicmicroorganismsin vegetabledrugs. Ali(Ali, 1983a,1983b,1983c,1985, 1987; Fehr and Ali,1980),apartfrom determiningpesticides,alsoobservedother critical impurities such as heavy metals e.g.thallium, leadandcadmiumin 11 widely usedmedicinalplants from 20 countries.As well as the problem ofpesticide application in medicinal plant cultivation,especially for Folia MenthaePiperitae, Friedrich andcollaborators(FriedrichandButer,1974a,1974b,1974c,1975;FriedrichandSchneider,1976)investigatedtheuseof fumigation agentsin SennaeFructus Angustifoliaeduringstorage.

Official methods(WHO, 1992;Pharmacope´e Europe-enne, 1997) do not differ significantly from thoseobtainedby thepresentbibliographicalresearch.Accord-ing to the former, if the pesticideto which the plantmaterialhasbeenexposedis a knowncompoundor canbe identified by suitable means, a well-establishedmethodfor the determinationof this particularpesticideresidue,or evenof a specificclassof suchcompounds,shouldbeemployed.However,whentheplantmaterialisof unknownprovenanceandseveralgroupsof pesticidesmaybepresent,it mightbenecessaryto usemethodsthatmeasuretotal organic chlorine, phosphorus,arsenicorlead for, respectively,chlorinatedhydrocarbons,phos-phateandpesticidescontainingarsenicor lead.

Since GC and/or HPLC were consideredabsolutelyessentialfor analyticalwork with pesticideresiduesbySchilcher et al. (1987) and Gabrio et al. (1988), thenecessaryinvestigationscould not be carried out in asimple laboratory. One of the commonestand leastexpensiveseparationtechniques,thin layer chromato-graphy, is only suitable for disproportionatelylargesamples,andmoreoverit is only applicablefor averyfewpesticides,with higherMRL values,soin practiceTLC isexcluded.As has been cited (Bisset, 1994), reputableherbal drug wholesalersexamine their products forresiduesin appropriatelaboratories.

Samplepreparation methods

The methodsmost frequently describedin referencesfoundbetween1963and1998includemacerationof themedicinalplantswith an appropriateextractionsolvent(usually acetone), a clean-up step (mostly columnchromatographywith Florisil) andananalyticalchroma-tographicseparationtechnique(essentiallyGC) coupledwith a selectiveand sensitivedetector(mostly electroncapturedetector).Someaccountsof colorimetric, bio-logical andbiochemicalassayswerealsofound(Panketal., 1980,1981,1984,1987b;Miellet, 1982;BagchiandPuri, 1985).

The analysis of pesticides and their degradationproductsin medicinal plants, particularly multiresiduedeterminations,hasbeenconsideredproblematic.This isdue to the matrices,which are mostly dried plants inpowderedform, the large diversity of compoundswithpolarity closeto thoseof the pesticidesand also to thesensitivityto solvolysisandoxidationof somepesticideslike organophosphoruscompounds(Pharmeuropa,1993;Nuneset al., 1997). It is important to rememberthatsometimesa method suitable for one genus may beinappropriatefor another(e.g. plants containingmuci-lage), which demandsadditional adaptiveprocedures.Actually, the identification and quantificationof pesti-cides in herbal drugs is difficult, even when highresolutionor high performancechromatographicmeth-odscoupledwith spectroscopictechniquesareused,asasample pretreatment is required to reduce matrixinterferencesbefore the final determinations(Molto etal., 1994).More recently,techniquessuchasSFEhavebeenproposedto minimisethe stepsof sampleprepara-tion (Erdelmeier,1993;CarisanoandRovida,1995).

Changesto pesticidelevelsduring preparation ofphytopharmaceuticals

Like LutomskiandDebska(1974),Pluta(1988a,1988b,1989,1990)alsostudiedvegetabledrugsfor contamina-tion by pesticidesin Poland. During 1980–1984,thecompoundsmostfrequentlyfoundin herbalraw materialas well as in granulatedproductsand vegetableblendswere p,p'-DDT and its metabolitesp,p'-1,1,-dichloro-2,2-di-(4-chlorophenyl)-ethane(p,p'-DDD) andp,p'-1,1,-dichloro-2,2-di-(4-chlorophenyl)-ethylene (p,p'-DDE)(>0.1–> 10ppm). Changesin the concentrationlevelsof organochlorinecompoundsin raw materials weredirectly reflectedin thelevelsof thesecompoundsin finalphytopharmaceuticals,and this was strongly noticeablein the caseof herbblends.A probablereasonlies in thecomposition of the preparationsas well as in thetechnologicalprocess,i.e. the productionmode.It wasfound that the quantity of compoundstransferredfromraw materialto the final preparationdependsmainly onthe type and quantity of the solvent, the temperature,the duration of heating and the properties of plantmaterials, i.e. chemical composition and degree ofcomminution.The highesttransfer(passage)of organo-chlorine compoundsto a correspondingphytopharma-ceuticalwereobservedwith alcoholextracts,whereasinthe caseof aqueousextractsthe extractedamountsweremuch lower. In the case of tinctures the transferamountedto approximately90%,for dry extractsnearlyto 60%,for infusionsto about25%andfor decoctionsto

1 The terminology used for plant material is maintained as in the originalreference, sinceseveralpublicationsincludedonly popularand/orcommercialnames,without botanical correct designation(seealsofinal comments).



only 14%.Nonetheless,theauthorwarnsthatdespitethediminutionof danger,wheninfusionsanddecoctionsarepreparedwith a poor solvent for halogenderivatives,even low concentrationscan be hazardousfor humanorganisms weakened by disease and with reducedimmunity.

ZongmaoandXuefen(1990),whoconsidertheresiduelevel in the final preparationmoreimportantthanin theplant material,showedthat thepesticideresiduesin rawmaterial pass into the infusion at a concentrationdependingmainlyonthewatersolubility of thepesticide.Somepesticides,suchasDDT, dicofol andpyrethroids,areextractedonly in small amountsduring the infusionprocesswhile others,e.g.dimethoateandmalathion,canbe extractedto more than 50%. Wan et al. (1991) alsomentionthe dependenceof the pesticideextractionrateon watersolubility anddescribedthewholeprocessasareversibleequilibrium betweenadsorptionand dissolu-tion. The influenceof chemicaldegradationduring theinfusiondoesnot seemto be important.

Residuelossesof a hexachlorocyclohexane(a-HCH),b-HCH, g-HCH, hexachlorobenzene(HCB), heptachlorand its epoxide,p,p'-DDE, p,p'-DDD, o,p'-DDT, p,p'-DDT, a-endosulfan,b-endosulfan,endosulfansulphate,aldrin, dieldrin andendrin,whenTilia cordataMill. wassubmitted to three different infusion processes,werestudiedby Lino andSilveira(1997b).Accordingto theseauthors,besideswatersolubility andfugacity influence,the infusion procedurewas a determining factor inpesticideloss, not only due to the different forms ofcontactbutalsobecausechemicaldegradationtookplacewhenpesticidesweresubmittedto the effect of boiling.Thiswasalsoconfirmedby ZongmaoandHaibin (1988).

The behaviour of residual organophosphoruspesti-cidesin infusionsof someleaveswasexaminedduringleachingor cookingby Nagayama(1996)andtherateofdecreasewas closely related to the octanol–waterpartition coefficient, Kow, according to the regressionformula log Lr = 2.25ÿ 0.312� log Kow, whereLr is thetranslocatedpesticideconcentrationin thefinal beverage(%). A similar investigationwascarriedoutby Zimmerliand Blaser (1982). The transferof organochlorineandorganophosphoricacid ester insecticides from driedFolium melissae,Flos aurantii, Folium verbenaeodor-atae, Flos chamomillae and Folium menthae to theinfusionwas,within certainlimits, nearlyindependentofthepesticidecontentof theherb,thetypeof plantanditscontentof essentialoils. Thus,the transferratio, Y (%),describedby Y= 100� (5.62� Lÿ0.42� 1)ÿ1, whereL iswatersolubility andhasastronginfluenceonthevalueofY. The Y valuesfound for two naturally contaminatedsamplesshoweda satisfactoryagreementbetweenthecalculatedandmeasuredvalues.

Somephytopharmaceuticals may havea larger pesti-cidecontentthantheiroriginal rawmaterial,which is thecasefor lipophilic compoundsin essentialoils.Starretal.(1963) showed that the quantity of residue found inpeppermintoil dependspartly on the severity of thedistillation,i.e. theamountof steamused.Sincethisplantcontains approximately 0.5% oil when mature, oilresidues200-fold thosefound in the plant could occurasa resultof severedistillation.DDT residuesin mint oilcould thusbe asgreatas2300ppm (mg/kg) if completetransferoccurredduringdistillation.

The pesticide tolerance limits (according to thePharmacopoeiasor other official regulations) were

occasionallyexceededin someof thesamplesdescribedin Table1. BeneckeandOrtwein(1992)statedthatonly9% of drugsanalysedfrom 1986to 1989containedmorethan0.5mg/kg of HCH andDDT, which wasthe MRLstipulatedby the Pharmacopoeia of the former DDR. Inan extensivestudy basedon 2654 samples,Schilcher(1982) showedthat a considerableproportion did notconformto regulationsgoverningmaximumpermissibleamounts. However, it should not be forgotten thatpreparationsfrom medicinal plants normally containonly 1/5th to 1/3rd of pesticideresiduespresentin thecrudedrugandtheADI valuesareveryseldomexceeded(Schilcher,1983b;SchilcherandHabenicht,1998).

Lino and Silveira (1996) reported the use of con-taminatedphytopharmaceuticalsasoneof thesignificantsourcesof humanexposureto organochlorinepesticides.This route of intake, together with others, has beenclaimed as responsiblefor more than 10% of totalpesticideingestion.However,such pesticideintake byhumanbeingsdoesnotseemto representaproblemwhentheconsumptionin phytopharmaceuticals is considerablylower thanthat from food sources.

Voitcu etal. (1995),analysingpesticidesin 12samplesof medicinal plants and four pharmaceuticalproductsfrom Rumaniandrug factories, warned of the conse-quencesof the existenceof even small amounts ofpesticide residue.These small quantities of pesticideresiduehavetheir own biologicaleffectsandcanchangethe therapeuticeffect. Thus, the presenceof pesticidescan theoretically influencethe quality of treatmentbyphytopharmaceuticals.

Removalof pesticidesfrom medicinal plants

Alternative methodologiesto reducepesticidelevels inmedicinal plants have been reported in the literature(Allorde andPlumel,1984).Decontaminationof naturaldrugsfrom organochlorineinsecticideswasproposedbyStahlandRau(1984).DDT andHCH in CassiasennaL.and Cassia angustifolia Vahl. leaves and pods wereremovedby supercriticalfluid extractionusing CO2 at100bar.Theactivecompounds,i.e. thesennosides,werenot extractedandremainedin thedrug,from which theycouldbeeasilyobtainedby furtherextractionwith water.

Insteadof using pesticidesor fumigants during thestorageof medicinalplantsandprepareddrugs,Gerardetal. (1990)suggestedan alternativeprocedureto preventpestattacks.With the help of a Carvex-double-chamber(apparatusthat pressurizesCO2 up to 40 barsat roomtemperaturefor 1.2h),CO2 wassuccessfullyusedto treatraw materialandprepareddrugsin theoriginal package.Theobviousadvantagesof thismethodarethatCO2 doesnot leave residuesbehind, is toxicologically harmless,highly efficient, simple to use,completelysecurein itsmanagementandofficially testedandauthorized.

FINAL COMMENTS

The relatively small numberof paperspublishedon thesubjectof pesticidedeterminationin medicinalplantsandtheir final preparationsshould be mentioned. Somereasons,aspreviouslycited,maybedueto thecomplex-ity of thematrixaswell asthetediousandlonganalytical



procedures.Most of the papers grouped here wereacquired with difficulty due to their limited access(periodicalsof restrictedcirculation and/or in a widevariety of languages).In someof them, the scientificnameof the medicinalplant investigatedwasnot given.In other cases,the analytical procedurefor the samplepreparationwasnot satisfactorilydescribedin thepaperssurveyed,which led automaticallyto additionalresearchon the respectivereferencematerialcited.

Despitethesearguments,this review showsthat it isimperativeto dosupplementarystudiesandorganizedataobtainedfrom the most diverseand reliable sourcesin

this areaof pesticideresearch.Thus,the collectionof aconsiderableamountof availableinformation regardingthe issueof pesticideresiduesin medicinalplantsanditssystematization,asin thepresentreview,is believedto beof importance.

Acknowledgements

The authorswish to thankCAPES,CNPqandFAPESPfor financialsupportandfellowships.The contributionof FernandoM. Lancastothe preparationof this review is gratefully acknowledged.

REFERENCES

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