Ethnopharmacology of Lippia Alba Thierry Hennebelle A, Sevser Sahpaz A, Henry Joseph B, Franc¸Ois Bailleul A,∗ a Laboratoire De Pharmacognosie, E.A

Available online at www.sciencedirect.com

Journal of Ethnopharmacology 116 (2008) 211–222

Review Ethnopharmacology of Lippia alba Thierry Hennebelle a, Sevser Sahpaz a, Henry Joseph b, François Bailleul a,∗ a Laboratoire de Pharmacognosie, E.A. 1043, Université de Lille 2, Faculté de Pharmacie B.P. 83, 59006 Lille cedex, France b Association pour la promotion des PLAntes MEDicinales et AROMatiques (APLAMEDAROM) de Guadeloupe, F-97142 Les Abymes, Guadeloupe, France Received 10 October 2007; received in revised form 29 November 2007; accepted 29 November 2007 Available online 8 December 2007

Abstract Introduction: Chemical, ethnopharmacological and pharmacological research on Lippia alba (Mill.) N.E. Brown and the evidence that exists for its various usages have been looked for, focusing on high quality studies. Ethnopharmacological investigation: The species is mainly used against digestive and respiratory ailments, and as a sedative and antihypertensive remedy. Chemical constituents: Seven chemotypes exist for the essential oil, the non-volatile compounds are iridioids, phenylethanoids, ﬂavone glycosides and biﬂavonoids. Biological activities and ethnopharmacological appraisal: Some positive, although partial, results have been obtained on sedative and anxiolytic activities. Real effects in other traditional uses can mainly be explained by anti-infectious and analgesic properties, at the moment. Conclusion: Well conducted biological studies are still needed for several indications of this species. Its use as a sedative deserves a clinical investigation. The chemical variability of the species seems important both in the essential oil and in non-volatile compounds, so future research on the pharmacological properties of these extracts should provide more chemical data which will increase their validity. © 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lippia alba; Verbenaceae; Digestive; Respiratory; Sedative; Hypertension; Anti-infectious

Contents

1. Introduction ...... 212 2. Botanical data ...... 212 3. Ethnopharmacological investigation ...... 213 4. Chemical constituents ...... 213 5. Biological investigation ...... 214 5.1. Anti-infectious properties ...... 215 5.1.1. Antibacterial and antifungal activity ...... 215 5.1.2. Antiviral activity ...... 219 5.1.3. Antiprotozoal activity ...... 219 5.2. Effects on the nervous system ...... 219 5.2.1. Neurosedative activity ...... 219 5.2.2. Analgesic and anti-inﬂammatory activities...... 219

Abbreviations: AFSSaPS, Agence Française pour la Securit´ e´ Sanitaire des Produits de Sante;´ BHA, butyl-hydroxyanisole; BVDV, bovine viral diarrhoea virus; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EC50, effective concentration 50%; HIV, human immunodeficiency virus; HSV, herpes simplex virus; IC50, inhibiting concentration 50%; LD50, lethal dose 50%; MIC, minimal inhibitory concentration; LSU, level of significative use; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NCI, National Cancer Institute; PMA, phorbol myristate acetate; TEAC, trolox equivalent antioxidant capacity; TRAMIL, TRAditional Medicine for the IsLands; VSV, vesicular stomatitis virus. ∗ Corresponding author. Tel.: +33 320 964039; fax: +33 320 964039. E-mail address: [email protected] (F. Bailleul).

5.3. Cardiovascular activity ...... 220 5.4. Miscellaneous ...... 220 5.4.1. Antioxidant activity ...... 220 5.4.2. Cytotoxic activity ...... 220 6. Toxicity ...... 220 7. Ethnopharmacological appraisal ...... 220 8. Conclusion ...... 221 References ...... 221

1. Introduction It is a shrub, with a quadrangular branch, reaching 1.7 m in height. The leaves are membranaceous, petiolate, pubescent, Lippia alba (Mill.) N.E. Brown is an aromatic shrub belong- opposite or ternate, and its limb shows variable forms with a ing to the Verbenaceae family, which is widely used all over pointed apex, cuneiform or decumbent basis and serrated or South and Central America for different purposes. In particu- crenated border (except on the basis). The flowers (3–5 mm) are lar, Lippia alba has been used for centuries in French overseas white or pink. The fruit is composed of two indehiscent nucleus, departments (French Guiana, Guadeloupe, Martinique) despite each containing one brown seed (Adams, 1972; Fournet, 2002; the fact that, when slavery was still authorised in France (that is Howard, 1989). to say until 1848), black slaves did not have the right to use or An incredible number of synonyms are to be found for Lip- sell medicinal plants, lest they poisoned white settlers. As the pia alba. They belong to the genera Lippia, Lantana, Phyla, French Pharmacopeia was created during the slavery period, no Verbena and Zapania (ISB, 2007). The explanation may lie in medicinal plants from the overseas departments were included at the variability of the species and more generally in the com- that time, except for those used by white people. In spite of the plexities inherent in the Verbenaceae family. Seven infraspecific abolition of slavery, traditional remedies of black populations taxa of the species Lippia alba (Mill.) N.E. Brown have been had no official status until very recently. Lippia alba and Senna established: Lippia alba f. alba Moldenke, Lippia alba f. inter- alata (L.) Roxb. (Fabaceae) were the first two plants from overseas departments to be approved by the French Drug Agency (AFSSaPS) for inclusion in the French Pharmacopeia (Robard, 2003). This work is also of interest because of the impending inscription of Lippia alba in European (French and Spanish) Pharmacopeias. The aim of this work is to list and compare papers that have dealt with ethnopharmacological, biological and pharmacological studies of this species, so as to synthesise existing scientific evidence on the different uses of this plant. A discussion of the available data on activities and chemical composition ensues.

2. Botanical data

Lippia alba (Mill.) N.E. Brown (Fig. 1) belongs to the Verbe- naceae family, which also includes other medicinally important plants like Aloysia triphylla (L’Herit.)´ Britt. and Verbena officinalis L. Lippia alba is abundantly present between the south of the United States of America (Florida) and the north of Argentina. It is also present in India (Singh et al., 2000) and Australia (Day and Mc Andrew, 2003). The local and traditional names are numerous in Latin America, because of widespread traditional use, and are generally derived from its aromatic smell or medicinal properties. The most common name in Brazil is cidreira, but this can also be used for 17 other lemon-scented herbs with similar uses (Matos et al., 1996). In Spanish-speaking countries and French overseas departments, it is often named after other aromatic species, e.g.: salvia morada (Senatore and Rigano, 2001), orégano de cerro (Stashenko et al., 2003), an´ıs de España (Pino Alea et al., 1996), mirto (Alan´ıs et al., 2005), verveine blanche, sauge du Brésil (Sastre and Breuil, 2007). Fig. 1. Lippia alba (Mill.) N.E. Brown (Verbenaceae) (photograph by H.J.). T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 213 media Moldenke, Lippia alba f. macrophylla Moldenke, Lippia One survey was specifically dedicated to the species Lippia alba f. scabra Moldenke, Lippia alba var. globiflora (L’Her.)´ alba. Near Oriximina´ (state of Para,´ Brazil), people use two Moldenke, Lippia alba var. carterae Moldenke and Lippia alba different infraspecific taxa of Lippia alba: Lippia alba f. alba var. lanceolata (Griseb.) Mulgura´ (IPNI, 2007). Moldenke (erva-cidreira), which is cultivated, and Lippia alba f. intermedia Moldenke (carmelitana), which is harvested in the 3. Ethnopharmacological investigation wild at certain periods of the year. Both were cited as having sedative and digestive properties, but the major use agreement Numerous papers have presented ethnopharmacological for cidreira was far better (MUA = 92.0%) than for carmelitana studies dealing with Lippia alba (among other plants). A selec- (MUA = 66.7%). Lippia alba f. alba has sometimes been used tion of these studies is presented here. Cited papers (except against respiratory ailments and hypertension (Oliveira et al., where noted) have been selected on the basis of their qual- 2006). ity, in accordance with the criteria for ethnopharmacology and The main medicinal usages found in these selected well- ethnobotanical studies in the “Rules of 5” of the Journal of conducted ethnopharmacological surveys (see Table 1) are in Ethnopharmacology (Verpoorte et al., 2006), with a special accordance with those that are cited in other ethnopharmaco- attention to the use of frequency of citation (or related quan- logical studies and in other papers or books. The four main uses titative parameter). are against digestive, respiratory, cardiovascular troubles and as A study, performed around three cities in the state of Sao˜ a sedative medicine. Nevertheless, numerous other usages are Paulo, showed a widespread use of Lippia alba: it was the sev- cited and the versatility of this plant makes it a species of high enth most cited species by plant users and traditional healers potential economic interest (Albuquerque et al., 2007a,b). (29.5%). Leaves are used as an infusion against states of excite- ment, hypertension, digestive troubles, nausea and cold, to heal 4. Chemical constituents wounds locally and as a syrup against cough and bronchitis. An infusion of the roots is also used against bad colds and coughs Most of the investigators studied the essential oil, by con- (Di Stasi et al., 2002). In two studies carried out in the state ventional methods of analysis, i.e. gas chromatography, in most of Bahia, Lippia alba was one of the two most frequently cited of the cases coupled with mass spectrometry. The composition species. In the village of Sapucaia, Cruz das Almas, it is used of the essential oil is very variable, suggesting the existence as a sedative and also against hypertension, flatulence and pain of a high number of chemotypes. We have recently endeav- (Rodrigues and Guedes, 2006). In Itacare,´ it is used against stom- oured to establish a system classifying all published analyses ach ache and digestive troubles with high concordance between in seven chemotypes, on the basis of the composition and of informants (Pinto et al., 2006). In a Mixe community, in Mex- possible common biosynthetic pathways between different oils. ico, the leaves of Lippia alba were frequently cited by traditional Briefly, chemotype I had citral, linalool, ␤-caryophyllene as the healers as active against gastrointestinal troubles (Heinrich et al., main constituents (four subtypes); in the case of chemotype II, 1992). In San Isidro, in the municipality of San Jose´ de Pare- it was tagetenone; the most common case for chemotype III was Boyaca,´ Colombia, Lippia alba is used as a painkiller, against limonene with a variable amount of carvone, but biosynthetically digestive troubles, diarrhea, stomach pain, flu and cough. It is related monoterpenic ketones (i.e. dihydrocarvone, piperitone, one of the ten species with the highest LSU (level of significative piperitenone) could be found instead of carvone (two subtypes); use), according to TRAMIL: it was cited by 20% of interviewed the four other chemotypes corresponded to isolated analyses: people (Toscano-Gonzalez,´ 2006). chemotypes IV (myrcene), V (␥-terpinene), VI (camphor-1,8- In the papers mentioned above, Lippia alba has always been cineole) and VII (estragole) (Hennebelle et al., 2006a). Since the considered as very useful, and is frequently one of the most cited publication of this classification, we have found two new analy- species. In contrast, in the following surveys, although Lippia ses of the essential oil of Lippia alba (Barbosa et al., 2006b; Silva alba appears, it seems to be of relatively little interest, quanti- et al., 2006): in both cases, citral was the major compound, with tatively speaking. In the Quilombola de Olho D’agua´ dos Pires a low amount of linalool (<5%), both therefore corresponding community, in Esperantina (Brazil), an infusion of the leaves is to subtype Ia. used against sore throat and flu (Franco and Barros, 2006). In In the case of structural characterisation of non-volatile com- the municipality of Igarassu (state of Pernambuco), local spe- pounds of Lippia alba, most investigators have studied the cialists use Lippia alba against anaemia and digestive troubles leaves. The presence of three iridoids, geniposide (1, Fig. 2), (Gazzaneo et al., 2005). In the Afro-Caribbean community of theveside (2) and shanzhizide methyl ester (3), was reported Livingston (Guatemala), the leaves of Lippia alba are used in (Heinrich et al., 1992; Von Poser et al., 1997; Barbosa et various forms against cough (decoction as a beverage), skin dis- al., 2006a). All three compounds were found again in one eases (macerate is used for washing), flatulence (infusion as a study, along with geniposidic acid (4), caryoptoside (5), 8-epi- beverage), nausea and vomiting (the plant is smoked), headaches loganin (6) and mussaenoside (7)(Hennebelle et al., 2006b). The (the plant is crushed and used as a poultice). The oral route is phenyletanoid/phenylpropanoid constituents characterised by the most frequently cited (Giron´ et al., 1991). In Ponta Grossa, two different groups were isonuomioside (8), decaffeoylverbas- Porto Alegre (Brazil), although Lippia alba is rather rarely men- coside (9)(Barbosa et al., 2006a), verbascoside (10), 2-acetyl tioned (low use value), the consistency of the use of its leaves as verbascoside (11), isoverbascoside (12), calceolarioside E (13), a sedative is considered as good (Vendruscoloand Mentz, 2006). forsythoside B (14) and cistanoside F (15). The flavonoid 214 T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222

Table 1 Reported uses of Lippia alba in ethnopharmacological surveys Type of use Population or geographic zone Part used and method References

Digestive Digestive troubles in Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, infusion Di Stasi et al. (2002) general Igarassu, state of Pernambuco (Brazil) Leaves, n.r. Gazzaneo et al. (2005) Oriximina,´ state of Para´ (Brazil) Leaves of Lippia alba f. alba, Oliveira et al. (2006) leaves and flowers of Lippia alba f. intermedia, infusion or decoction San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Itacare,´ Bahia State (Brazil) n.r. Pinto et al. (2006) Nausea and/or vomiting Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, infusion Di Stasi et al. (2002) Livingston (Guatemala) Leaves, smoking Giron´ et al. (1991) Stomach pain Mixe, Oaxaca, Tehuantepec isthmus Leaves Heinrich et al. (1992) (Mexico) San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Itacare,´ Bahia State (Brazil) n.r. Pinto et al. (2006) Flatulence Sapucaia, state of Bahia (Brazil) n.r. Rodrigues and Guedes (2006) Livingston (Guatemala) Leaves, infusion Giron´ et al. (1991) Diarrhea San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Respiratory Respiratory ailments in Oriximina,´ state of Para´ (Brazil) Leaves of Lippia alba f. alba, Oliveira et al. (2006) general infusion or decoction Bronchitis Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, syrup Di Stasi et al. (2002) Sore throat Quilombola de Olho D’água dos Pires, Leaves, infusion Franco and Barros (2006) Esperantina, state of Piau´ı (Brazil) Flu Quilombola de Olho D’água dos Pires, Leaves, infusion Franco and Barros (2006) Esperantina, state of Piau´ı (Brazil) San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Cough Livingston (Guatemala) Leaves, decoction Giron´ et al. (1991) Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, syrup Di Stasi et al. (2002) Roots, infusion San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Cold Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, infusion Di Stasi et al. (2002) Sedative Vale do Ribeira, State of Sao˜ Paulo (Brazil) Leaves, infusion Di Stasi et al. (2002) Sapucaia, state of Bahia (Brazil) n.r. Rodrigues and Guedes (2006) Pontes e Lacerda and Comodo (state of Mato Leaves, infusion or syrup Coelho and da Silva (2003) Grosso) Oriximina,´ state of Para´ (Brazil) Leaves of Lippia alba f. alba, Oliveira et al. (2006) leaves and flowers of Lippia alba f. intermedia, infusion or decoction Ponta Grossa, Porto Alegre (Brazil) Leaves Vendruscolo and Mentz (2006) Cardiovascular Hypertension Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, infusion Di Stasi et al. (2002) Sapucaia, state of Bahia (Brazil) n.r. Rodrigues and Guedes (2006) Oriximina,´ state of Para´ (Brazil) Leaves of Lippia alba f. alba, Oliveira et al. (2006) infusion or decoction Miscellaneous Anemia Igarassu, state of Pernambuco (Brazil) Leaves, n.r. Gazzaneo et al. (2005) Headache Livingston (Guatemala) Leaves, poultice Giron´ et al. (1991) Pain Sapucaia, state of Bahia (Brazil) n.r. Rodrigues and Guedes (2006) San Jose´ de Pare-Boyaca´ (Colombia) n.r. Toscano-Gonzalez´ (2006) Skin diseases Livingston (Guatemala) Leaves, macerate for washing Giron´ et al. (1991) Wounds Vale do Ribeira, state of Sao˜ Paulo (Brazil) Leaves, local use Di Stasi et al. (2002) n.r.: not recorded. glycosides so far known to occur in the leaves are apigenin- 5. Biological investigation 7-glucuronide (16), luteolin-7-glucuronide (17), clerodendrin (18), luteolin-7-diglucuronide (19) and 3-acetylclerodendrin Lippia alba is probably one of the most studied species in the (20)(Hennebelle et al., 2006b). Two biflavonoids (21, 22, Fig. 2) Lippia genus from a pharmacological point of view. The subjects were also isolated (Barbosa et al., 2005). of experimental works performed on this species are generally Only one work so far has been dedicated to the chemical consistent with its traditional uses. Because of the chemical vari- composition of the roots of Lippia alba and led to the character- ability of the plant the main identified compound(s) is (are) cited isation of mussaenoside, theviridoside (23) and gardoside (24) when available. Papers were selected on the basis of the use of (Sena Filho et al., 2007). appropriate positive controls. T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 215

Fig. 2. Isolated chemical constituents of Lippia alba.

5.1. Anti-infectious properties There is no agreement on the level of acceptance for plant when compared with standards; therefore, some authors con- 5.1.1. Antibacterial and antifungal activity sider only activity comparable to antibiotics, while others Antiseptic activity has been the most studied field of inter- consider even higher values. It has been suggested that an MIC est for Lippia alba. Antibacterial and antifungal activities per value of 100–200 ␮g/ml could be considered as good for plant species are shown in Table 2. Only one study totally respected material (Borges-Argaez´ et al., 2007). Such values were reached the criteria of the “Rules of 5”, applied since 2006 (Verpoorte et with various root extracts against Micrococcus luteus (acetone, al., 2006) in the Journal of Ethnopharmacology: a positive con- methanol, chloroform) and Bacillus subtilis (chloroform) and trol was used and the MIC of both an essential oil (76% linalool; with ethanol or hydroethanolic leaf extracts against Micrococcus 0.60 mg/ml) and a positive control (nistatin; 0.05 mg/ml) were luteus and Candida krusei (Aguiar, 2006; Holetz et al., 2002), reported (Duarte et al., 2005). In other works, the determina- which are not frequent pathogens in immunocompetent humans. tion of MIC was lacking (with only a determination of growth Nevertheless, slightly higher MIC values (250–500 ␮g/ml) inhibition zone, in comparison with a positive control) or the were obtained with a 40% carvone essential oil, acetone MIC of the positive control was not given (although its use was and chloroform root extracts against Staphylococcus aureus mentioned). (Aguiar, 2006; Pino Alea et al., 1996). Moderate activities 216 T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 Aguiar (2006) Pino Alea et al. (1996) Oliveira et al. (2006) Holetz et al. (2002) Aguiar (2006) Sena Filho et al. (2006) Pino Alea et al. (1996) Holetz et al. (2002) Aguiar (2006) Oliveira et al. (2006) Sena Filho et al. (2006) Aguiar (2006) Pino Alea et al. (1996) Oliveira et al. (2006) V, M ≥ g/disc) ␮ g/disc) < Ka g/disc) < Ka g/disc) < Ka ␮ ␮ ␮ L/disc) L/disc) > V, M L/disc) > V, M ␮ ␮ ␮ V, M 11 mm (“+”) at ” (50 and 100 mg/ml) ” (50 and 100 mg/ml) ”(2000 g/disc) g/disc) g/disc) g/disc) g/disc) g/disc) ≤ ≥ − − − ␮ ␮ ␮ ␮ ␮ ␮ (30 (30 50 mg/ml, < T 1 mg/ml 1 mg/ml (30 (10 (10 (10 2 GI + at 100 mg/ml, < T 0.31n.r. MBC = 0.63 mg/ml n.r. GIZ > V, M GIZ (2000 ≥ n.r. GIZ (5 0.63n.r. MBC = 1.25 mg/ml GIZ (2000 n.r. GIZ (5 n.r.n.r. GI “ 0.63 GI “ MBC = 1.25mg/ml -guaiene, 6% -guaiene, 6% -guaiene, 6% ␤ ␤ ␤ limonene) Hexane leaf extract (8 days)Methanol leaf extract (8 days)Acetone and ethanol root extracts (8Chloroform days) root extract (8 days)EO (40% carvone, 10% 0.125 EO (37% citral, 15% myrcene, 5%5% geraniol, germacrene D) 0.5 0.25 EO (22% citral, 10% nerol, 0.0312 6%90% geraniol) ethanol leaf extract (48 h)Hexane, ethanol and methanol leaf extracts (8 days) n.r. >1 GIZ Acetone root extract (8 days)Chloroform root extract (8 days)Ethanol root extract (8 days)Ethyl acetate root extract (48 h, r.t.)Methanol root extract (48 h, r.t.) 0.5 Aqueous 0.25 root extract (48 h, r.t.) 0.5; 1 1 GIZ n.r. GI “ EO (37% citral, 15% myrcene, 5%5% geraniol, germacrene D) limonene) EO (40% carvone, 10% 90% ethanol leaf extract (48 h)Hexane, ethanol and methanol leaf extracts (8 days) >1 Acetone and ethanol root extracts (8Chloroform days) root extract (8 days)EO (37% citral, 15% myrcene, 5%5% geraniol, germacrene 0.25 D) 0.0312 limonene) EO (22% citral, 10% nerol, 6% geraniol)Ethyl acetate, methanol and water root extracts (48 h, r.t.) Hexane, ethanol n.r. and methanol leaf extracts, chloroform, acetone and ethanol root extracts (8 days) EO (40% carvone, 10% Ethanol leaf extract (8 days) 0.0625 GIZ (2000 EO (22% citral, 10% nerol, 6% geraniol) n.r. GIZ (5 Lippia alba MRSA Staphylococcus aureus Staphylococcus aureus (BMB9393) Bacillus subtilis Lactobacillus casei Enterococcus faecalis Micrococcus luteus Bacilli Gram + Cocci Gram + Table 2 Antibacterial and antifungal activities of essential oils and extractsClassification of Bacteria Species Tested material MIC (mg/ml) Other results References T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 217 ıs et al. (2005) ıs et al. (2005) ıs et al. (2005) ıs et al. (2005) ´ ´ ´ ´ Alan Antoun et al. (2001) Pino Alea et al. (1996) Holetz et al. (2002) Alan Sena Filho et al. (2006) Pino Alea et al. (1996) Pino Alea et al. (1996) Holetz et al. (2002) Aguiar (2006) Sena Filho et al. (2006) Pino Alea et al. (1996) Sena Filho et al. (2006) Aguiar (2006) Pino Alea et al. (1996) Sena Filho et al. (2006) Aguiar (2006) Alan Alan Aguiar (2006) Oliveira et al. (2006) Pino Alea et al., 1996 g/ml ”) ␮ − g/mlRi g/disc) g/disc) g/disc) ␮ ”) ”) ”) ␮ ␮ ␮ g/disc) < Ka − − − ␮ L/disc) > V, M ␮ ” (50 and 100 mg/ml) ”(2000 ” (50 and 100 mg/ml) ” (50 and 100 mg/ml) ”(2000 ” (50 and 100 mg/ml) ”(2000 g/disc) g/disc) − − − − − − − ”), PC = 1 ␮ ␮ − acetate extract, < T 1 mg/ml (“ (30 (10 n.r. GI < 50% (“ 2.5n.r. 2.5 mg/ml GI 8 mg/ml < 50% (“ n.r. GI “ 2.5 2.5 mg/ml n.r. GI “ R n.r.2.5 GI “ >2 MBC = 2.5 mg/ml GI + at 100 mg/ml ethyl n.r.0.63 GI “ n.r. MBC = 1.25 mg/ml GI “ n.r. GI < 50% (“ n.r. GI < 50% (“ n.r. GIZ (2000 0.63n.r. MBC = 1.25 mg/ml GIZ (5 -guaiene, 6% -guaiene, 6% -guaiene, 6% -guaiene, 6% -guaiene, 6% -guaiene, 6% ␤ ␤ ␤ ␤ ␤ ␤ Aqueous (decoction, 3 h) and methanolunknown) (time leaf extracts 90% ethanol leaf extract (48 h)Aqueous (decoction, 3 h) and methanolunknown) (time leaf extracts >1 Hexane, ethanol, methanol leaf extracts, chloroform, acetone, ethanol root extracts (8 days) limonene) EO (40% carvone, 10% Ethyl acetate, methanol and water root extracts (48 h, r.t.) 90% ethanol leaf extract (48 h)Aqueous root extract (48 h, r.t.) >1 n.r. GI “ limonene) EO (40% carvone, 10% limonene) EO (40% carvone, 10% Hexane, ethanol and methanol leaf extracts, chloroform, acetone and ethanol root extracts (8 days) Ethyl acetate and methanol root(48 extracts h, r.t.) Aqueous root extract (48 h, r.t.) n.r. GI “ limonene) EO (40% carvone, 10% Ethyl acetate, methanol and water root extracts (48 h, r.t.) Hexane, ethanol, methanol leaf extracts, chloroform, acetone, ethanol root extracts (8 days) limonene) EO (40% carvone, 10% Aqueous (decoction, 3 h) and methanolunknown) (time leaf extracts Aqueous (decoction, 3 h) and methanolunknown) (time leaf extracts Hexane, ethanol, methanol leaf extracts, chloroform, acetone, ethanol root extracts (8 days) 95% ethanol leaf extract n.r. 82% inhibition at 100 EO (22% citral, 10% nerol, 6% geraniol) n.r. EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) EO (40% carvone, 10% limonene) . sp Escherichia coli Salmonella Pseudomonas aeruginosa Enterobacter aerogenes Klebsiella pneumoniae Serratia marcescens Shigella flexneri Shigella sonnei Mycobacterium smegmatis Mycobacterium tuberculosis Streptococcus mutans Staphylococcus epidermidis Bacilli Gram - Mycobacteria 218 T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 Oliveira et al. (2006) Holetz et al. (2002) Aguiar (2006) Oliveira et al. (2006) Holetz et al. (2002) Aguiar (2006) Oliveira et al. (2006) Holetz et al. (2002) Oliveira et al. (2006) Oliveira et al. (2006) Duarte et al. (2005) Oliveira et al. (2006) g/ml ␮ AB AB ≤ ≤ g/mlRi g/disc) g/disc) ␮ ␮ ␮ g/disc) < Ke g/disc) < Ke ␮ ␮ L/disc) L/disc) > AB L/disc) > AB L/disc) L/disc) > AB L/disc) > AB L/disc) > AB L/disc) > AB ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ g/disc) g/disc) ”(2000 ”(2000 inimal bactericidal concentration; n.r.: not recorded; PC: g/disc) g/disc) g/disc) g/disc) g/disc) g/disc) g/disc) g/disc) ␮ ␮ − − ”), PC = 1 ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ − (10 (10 (300 (10 (10 (300 (10 (10 (10 (“ (10 , the positive control was trimethoprim (8 mg/ml), which inhibited 100% of ıs et al. (2005) ´ n.r. GI “ n.r. n.r. GIZ (5 n.r.n.r. GI “ GIZ (5 n.r. GIZ (5 n.r. GIZ (5 n.r. GIZ (5 n.r. GIZ (5 Alan 3 h, agitation) R × EO (22% citral, 10% nerol, 6% geraniol)90% ethanol leaf extract (48 h)Chloroform root n.r. extract (8 days) >1 n.r. GIZ (5 GIZ (2000 90% ethanol leaf extract (48 h)Hexane, ethanol, methanol leaf extracts, acetone, ethanol root extracts (8 days) >1 EO (22% citral, 10% nerol,EO 6% (37% geraniol) citral, 15% myrcene, 5% geraniol, germacrene D) EO (22% citral, 10% nerol, 6%90% geraniol)ethanol leaf extract (48 h) n.r. n.r. 0.125 EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) Acetone and ethanol root extracts (8 days) n.r. GIZ (2000 EO (22% citral, 10% nerol, 6%90% geraniol)ethanol leaf extract (48 h)Hexane, ethanol and methanol leaf extracts (8 days) Chloroform root n.r. extract (8 days)EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) >1 2 EO (22% citral, 10% nerol, 6% geraniol) n.r. GIZ (5 EO (22% citral, 10% nerol, 6% geraniol) n.r. EO (76% linalool)70% ethanol leaf extract (2 EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) 0.60 (nistatin: 0.05) EO (22% citral, 10% nerol,EO 6% (37% geraniol) citral, 15% myrcene, 5% geraniol, germacrene D) n.r. EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) 95% ethanol stem extract n.r. 19% inhibition at 100 EO (37% citral, 15% myrcene, 5% geraniol, germacrene D) . T544 5VPL serotype B Shigella sonnei ) -444 Serotype A 1 Candida tropicalis Chrysonilia sitophila Cryptococcus neoformans T Candida guilliermondii Candida krusei Candida parapsilosis Candida albicans Candida albicans Fonsecaea pedrosoi Trichophyton rubrum Continued Fungi Table 2 ( Classification Species Tested material MIC (mg/ml) Other results References AB: amphothericin B; EO: essentialpositive oil; control; GI: R: growth resistant; inhibition; Ri:bacterial GIZ: rifampicin; growth, growth except r.t.: against room inhibition temperature; zone; T: Ka: tetracycline; kanamycin; V: Ke: vancomycin. ketoconazole; In M: the methicillin; case MBC: of m T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 219

(MIC = 600–630 ␮g/ml) were observed against Staphylococcus hardly be linked to any traditional mode of administration and epidermidis, Enterococcus faecalis, Serratia marcescens, Can- the positive control, diazepam (0.75 mg/kg i.p.), was much more dida albicans (Pino Alea et al., 1996; Duarte et al., 2005). active. The relevance of any conclusion concerning the sedative Moreover, the growth inhibition zone of citral essential oils properties of the essential oil of Lippia alba in the context of a (5 ␮L/disc) was superior to that of vancomycin (10 ␮g/disc) traditional use is doubtful at the moment. in methicillin-resistant Staphylococcus aureus (Oliveira et al., Tests concerning the sedative activity of Lippia alba were 2006). also performed with polar extracts of Lippia alba. Several hydroethanolic extracts (40, 60 and 80% ethanol; 200 mg/kg, 5.1.2. Antiviral activity p.o.), which contained no detectable amounts of volatile com- Only extracts of Lippia alba were used to evaluate antivi- pounds were tested on mice. The 80% ethanolic extract did ral activities. An ethanolic extract exhibited anti-HSV-1 effect reduce latency to induce sleep and potentiate pentobarbital- at 12.5 to 50 ␮g/ml, obtained by assessing cytopathic effect induced sleeping time. The results were similar to those of and protein synthesis ([35S]methionine incorporation), but also diazepam (1 mg/kg, i.p.) on sleep latency, but much weaker on toxicity against HeLa cells at the higher tested concentrations. its duration. In the rota-rod test, it also significantly increased The same extract was ineffective against poliovirus type 1 and the number of falls and reduced the time spent on the rotatory VSV (Abad et al., 1997). A methanolic extract made from bar, when compared with the control group. The rectal tem- leaves and twigs did not show any activity against BVDV-1 (a perature was not affected in this case and the extracts failed to model of anti-HCV activity), HSV-1, HSV-2, HIV and influenza reduce significantly the number of convulsions and mortality virus type A, whereas an infusion of the ground material had after pentylenetetrazole injection. The sedative effect seemed to a selectivity index (cytotoxic concentration/antiviral concen- be related to flavonoid content (Zetola´ et al., 2002). Recently, tration ratio) of 19.2 against influenza virus type A (H3N2) after isolating pure polar compounds from a Lippia alba sam- (EC50 = 122.7 ␮g/ml) (Ruffa et al., 2004). Of the three fractions ple from Guadeloupe (France) (Hennebelle et al., 2006b), it (dichloromethane, ethyl acetate and butanol) obtained from an was established that its major metabolites had only weak or ethanolic extract, the butanolic fraction showed activity against moderate activities on benzodiazepine and GABAA receptors, HSV-1, acyclovir-resistant strain 29R (EC50 =2␮g/ml, selec- the most actively tested product being luteolin-7-diglucuronide tivity index = 8), and the ethyl acetate fraction had a moderate (IC50 = 101 and 40 ␮M, respectively) (the IC50 of diazepam and effect on poliovirus type 2. None of the three fractions had muscimol were 10.0 and 24.5 nM, respectively) (Hennebelle et any effect on HSV-1, strain KOS (Andrighetti-Frohner¨ et al., al., 2007). 2005). 5.2.2. Analgesic and anti-inflammatory activities 5.1.3. Antiprotozoal activity Essential oils and an extract of Lippia alba showed anal- A methanolic extract of aerial parts showed IC50 val- gesic properties. As far as the essential oil is concerned, the two ues of 58.1 ␮g/ml on Entamoeba histolytica (moderate) and chemotypes used for the tests are citral-limonene and carvone- 109.4 ␮g/ml on Giardia lamblia (weak). The positive controls limonene. Both essential oils (1–2 mg/kg p.o.) inhibited up to were emetin (1.05 and 0.42 ␮g/ml) and metronidazole (0.04 80.5% acetic acid-induced writhing in mice (100% inhibition and 0.21 ␮g/ml) (Calzada et al., 2006). The activity against was obtained for 10 mg/kg meperidine, i.p.). The effect was Trichomonas vaginalis was low (IC50 = 227.9 ␮g/ml; metron- weaker in the formalin test, as comparable inhibition values idazole: 0.037 ␮g/ml) (Calzada et al., 2007). A 95% ethanol were reached only with 10 mg/kg p.o. Moreover, the effect leaf extract was one of the five most active (50–100% parasite was observed mainly during the second (inflammatory) phase, suppression on Plasmodium falciparum D-6 clone), out of 37 the first (neurogenic) phase being little affected. The citral- tested species (Antoun et al., 2001). limonene chemotype (50 mg/kg p.o.) was the only essential oil that increased latency time to the stimulus in the hot plate 5.2. Effects on the nervous system test to the same extent (51.5%) as meperidine (10 mg/kg, i.p.; 55.7%). The inhibitory effect of the citral-limonene essential oil 5.2.1. Neurosedative activity was reversed by the opioid antagonist naloxone, suggesting a Another widely investigated aspect was neurosedative activ- morphinic action mechanism, whereas the effect of the carvone- ity. Three essential oils of Lippia alba (55% citral, 10% limonene chemotype was not reversed by naloxone. (Viana et ␤-myrcene = EO I; 63% citral, 23% limonene = EO II; 55% al., 1998). carvone, 12% limonene = EO III) and some of their major An ethanolic leaf extract (150 mg/kg i.p.) and a chloroformic constituents (citral, myrcene, limonene) were tested for their root extract (367 mg/kg i.p.) had significant anti-inflammatory behavioural effects on mice. They all exhibited anxiolytic activities (35.1 and 58.7% decrease of granulocytes counted effects in the elevated plus maze test (100–200 mg/kg, i.p.), and in exudate, respectively) in the carrageenan-induced air-pouch decreased rectal temperature. EO II and EO III and the pure com- model in mice. In the same test, phenylbutazone (100 mg/kg, i.p.) pounds exhibited sedative activities at the same dose range (open provoked a 70.5% decrease in the granulocyte number (Aguiar, field test, rota-rod test). Citral, myrcene and limonene poten- 2006). Use of the intraperitoneal route does not make it possi- tiated pentobarbital-induced sleeping time (Vale et al., 1999, ble to suppose any anti-inflammatory properties of traditional 2002). These results, obtained by intraperitoneal injection, can preparations of Lippia alba. 220 T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222

5.3. Cardiovascular activity An ethanolic leaf extract and a chloroformic root extract exhibited no cytotoxic activity against HeLa cells in the MTT Traditional use against hypertension was not conﬁrmed by method for counting living cells. IC50 were both >30 ␮g/ml. pharmacological experimentation, as a 95% ethanolic extract Antitumoral activity against Sarcoma 180 cells in mice was also failed to inhibit KCl- and phenylephrine-induced contractions determined and the inhibition capacities of both extracts on the in isolated rat aorta rings (Guerrero et al., 2002). An aqueous tumor (47.4 and 29.3%, respectively) were below the 58% level, extract diminished the cardiac rate without affecting the con- considered by the NCI to be the signiﬁcance threshold. The tractile force of isolated rat hearts at a dose of 77 ␮g/ml (Gazola positive control was actinomycin D, which caused 87% tumor et al., 2004). inhibition at 0.3 mg/kg (Aguiar, 2006).

5.4. Miscellaneous 6. Toxicity

Several papers report activities that are unrelated to the tra- Aguiar (2006) determined the oral LD50 of Lippia alba ditional use of the plant. For the most part, they were assessed extracts, the values being 460 mg/kg for an ethanolic leaf extract during screenings for speciﬁc properties. and 1466 mg/kg for a chloroformic root extract in mice.

5.4.1. Antioxidant activity 7. Ethnopharmacological appraisal Columbian essential oil samples were used to establish antioxidant activities. One, which contained 61% carvone, was Many traditional uses of Lippia alba (Mill.) N.E. Brown tested by the DPPH assay (EC50 = 0.28 kg oil/mmol DPPH) have been reported on. A review of well-conducted surveys con- and TEAC (TAA = 14.4 mmol Trolox/kg oil), with very low firms that this species has four main uses: gastrointestinal and activities (Puertas-Mejia et al., 2002). Another (51% carvone, respiratory troubles, sleep or anxiety disorders and cardiovas- 33% limonene) inhibited FeSO4-induced linoleic acid per- cular complaints (Table 1). Only one study was dedicated to oxydation. Its activity was higher than or similar to those digestive (antiulcerogenic) properties. It has not been reviewed of positive controls between 5 and 20 g/L, e.g. at 5 g/L, here because the dose was very high (12.5 g dry plant/kg in rat) 72.0, 55.0 and 55.3% linoleic acid remain, for the essen- (Pascual et al., 2001). Only one moderate activity on Entamoeba tial oil, vitamin E and BHA, respectively (Stashenko et al., histolytica, which can cause diarrhea and dysentery, could jus- 2004). The presence of limonene, with conjugated double tify the use of this species in some severe digestive troubles bonds, may explain this good activity, compared to the pre- in immunocompetent adults. The traditional use against flu may vious paper. Pure products from polar leaf extracts from find an explanation in an antiviral activity against influenza virus. Lippia alba have recently been tested for antioxidant activi- Potential anti-inflammatory properties might play a part in res- ties. Phenylethanoids proved to be potent inhibitors of DPPH piratory ailments (Viana et al., 1998; Aguiar, 2006), but existing (IC50 = 8.2–15.8 ␮M). Among the tested flavonoids, luteolin-7- works lack positive controls or the mode of administration does diglucuronide was the only one to reach an IC50 (38.2 ␮M). The not correspond to traditional practice. As regards neuroseda- reference compounds were Trolox (12.5 ␮M) and chlorogenic tive activity, existing data on sedative properties of this essential acid (20.8 ␮M). Among the major compounds, luteolin-7- oil, or its major constituents citral, limonene, myrcene, and car- diglucuronide (19), verbascoside (10) and calceolarioside E vone (Vale et al., 1999, 2002; De Sousa et al., 2007) have been (13) inhibited superoxide radical with higher activities in obtained with high doses (100–200 mg/kg) and by intraperi- PMA-stimulated neutrophil granulocytes than in a cell-free toneal injection, so they are of little interest in an assessment system (hypoxanthine–xanthine oxidase), suggesting an anti- of the plant. The sedative activity of a hydroethanolic extract on inflammatory effect via the regulation of respiratory burst mice and the effect of luteolin-7-diglucuronide (18) on BZD and (Hennebelle et al., 2007). GABA receptors (Zetola´ et al., 2002; Hennebelle et al., 2007) are more convincing. Activities are weak, but some traditional 5.4.2. Cytotoxic activity modes of consumption use considerable amounts of plant. For Eighteen extracts made from three parts of the plant (roots, instance, the Creole name twa tass for Lippia alba means that twigs and leaves) with six different solvents (hexane, chlo- three cups are drunk before sleep. Usage as an antihypertensive roform, acetone, ethanol, methanol, and water) were tested remedy was not confirmed by experimentation, but verbascoside against Hep-2 and NCI-H292 cells. The positive control was (10) and isoverbascoside (12), potent vasorelaxant compounds vincristine (IC50 = 2.8 and 43.8 ng/ml, respectively). According (Yoshikawa et al., 2006), and glycosides of apigenin and luteolin, to the threshold of significance determined by the NCI for crude both of which possess vasorelaxing properties (Woodman and extracts (IC50 <30␮g/ml), three extracts were cytotoxic to Hep- Chan, 2004), were isolated. This highlights the problem of chem- 2 cells: chloroformic root extract (11.5 ␮g/ml), ethanolic and ical variability, which is common in plants, but is particularly methanolic leaf extracts (8.2 and 14.4 ␮g/ml, respectively). Five important in Lippia alba (Hennebelle et al., 2006a). Variationsin extracts were toxic to NCI-H292 cells: chloroformic, acetonic essential oil composition and yield (0.1–2.2%) make it necessary and ethanolic root extracts (4.6, 16.3 and 11.4 ␮g/ml, respec- to link biological results to a precise and well-defined sam- tively), chloroformic twig extract (19.7 ␮g/ml) and hexanic leaf ple. This has generally been done. Less investigation has been extract (25.1 ␮g/ml) (Costa et al., 2004). made of any variation in secondary non-volatile compounds, but T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222 221 differences exist in HPLC profiles and isolated compounds, fol- Albuquerque, U.P.,Medeiros, P.M.,Almeida, A.L.S., Monteiro, J.M., Lins Neto, lowing the geographical origin of samples (Barbosa et al., 2006a; E.M.F., Melo, J.G., Santos, J.P., 2007b. Medicinal plants of the caatinga Hennebelle et al., 2006b; Hennebelle, 2006). Thus, differences (semi-arid) vegetation of NE Brazil: a quantitative approach. Journal of Ethnopharmacology 114, 325–354. in the amount and composition of metabolites may explain sur- Andrighetti-Frohner,¨ C.R., Sincero, T.C.M., da Silva, A.C., Savi, L.A., Gaido, prising biological results. This leads us to think that future works C.M., Bettega, J.M.R., Mancini, M., de Almeida, M.T.R., Barbosa, R.A., should provide accurate information on the major compounds Farias, M.R., Barardi, C.R.M., Simoes,˜ C.M.O., 2005. Antiviral evalua- that are present in the extract or, at least, on the amount of com- tion of plants from Brazilian atlantic tropical forest. Fitoterapia 76, 374– pounds such as phenylethanoids or flavonoids, which are quite 378. Antoun, M.D., Ramos, Z., Vazques, J., Oquendo, I., Proctor, G.R., Gerena, L., easy to determine. Franzblau, S.G., 2001. Evaluation of the flora of Puerto Rico for in vitro Additional, precisely and quantitatively ascertained, uses antiplasmodial and antimycobacterial activities. Phytotherapy Research 15, consist mainly of local applications against wounds, skin dis- 638–642. eases or pain and may be explained by inhibitory activities of Arango Caro, S., 2004. Guia de plantas medicinales de uso comun en Salento, the leaf essential oil and extracts on skin pathogens such as Colombia. Missouri Botanical Garden Press, Saint-Louis, pp. 49–50. Barbosa, F.G., Lima, M.A.S., Silveira, E.R., 2005. Total NMR assignments of Staphylococcus aureus, Staphylococcus epidermidis, Candida new [C7-OC7]-biflavones from leaves of the limonene-carvone chemotype albicans or HSV (Aguiar, 2006; Pino Alea et al., 1996; Duarte of Lippia alba (Mill) N.E. Brown. Magnetic Resonance in Chemistry 43, et al., 2005; Andrighetti-Frohner¨ et al., 2005). 334–338. A convincing analgesic activity of the essential oil (Viana Barbosa, F.G., Lima, M.A.S., Braz-Filho, R., Silveira, E.R., 2006a. Iridoid and et al., 1998) may not only play a part in several traditional phenylethanoid glycosides from Lippia alba. Biochemical Systematics and Ecology 34, 819–821. uses: digestive troubles and respiratory infections, but also in Barbosa, F.F., Barbosa, L.C.A., Melho, E.C., Botelho, F.M., Santos, R.H.S., other indications that have been mentioned, such as headaches 2006b. Influenciaˆ da temperature do ar de secagem sobre o teor e a or gynecological troubles (Scarpa, 2004; Leonti et al., 2003; composiçao˜ qu´ımica do oleo essencial de Lippia alba (Mill) N.E. Brown. Arango Caro, 2004). It has been reported (although not quanti- Quimica Nova 29, 1221–1225. tatively) that the plant was used to prepare antimalarial remedies Borges-Argaez,´ R., Canche-Chay, C.I., Pena-Rodr˜ ´ıguez, L.M., Said-Fernandez,´ S., Molina-Salinas, G.M., 2007. Antimicrobial activity of Diospyros anisan- in French Guiana (Vigneron et al., 2005). An interesting activity dra. Fitoterapia 78, 370–372. against a chloroquine-resistant clone of Plasmodium falciparum Calzada, F., Yepez-Mulia,´ L., Aguilar, A., 2006. In vitro susceptibility of (Antoun et al., 2001) should encourage further studies. Entamoeba histolytica and Giardia lamblia to plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology 108, 367–370. 8. Conclusion Calzada, F., Yepez-Mulia,´ L., Tapia-Contreras, A., 2007. Effect of Mexican medicinal plant used to treat trichomoniasis on Trichomonas vaginalis Lippia alba is a popular medicinal plant that is considered trophozoites. Journal of Ethnopharmacology 113, 248–251. useful in numerous ailments, mainly digestive, respiratory, car- Costa, M.C.C.D., Aguiar, J.S., do Nascimiento, S.C., 2004. Atividade citotoxica´ diovascular troubles and anxiety. Variousbiological studies have de extratos brutos de Lippia alba (Mill.) N.E. Brown (Verbenaceae). Acta Farmaceutica Bonaerense 23, 349–352. been dedicated to this species, but a small number of them are Day, M.D., Mc Andrew, T.D., 2003. The biology and host range of Falco- useful in evaluating its traditional uses. The sedative activity nia intermedia (Hemiptera: Miridae), a potential biological control agent deserves further investigation, for example clinical trials. Some for Lantana camara (Verbenaceae) in Australia. Biocontrol Science and anti-infectious properties have also been revealed, but the effects Technology 13, 13–22. on respiratory pathogens have not been well studied. Digestive De Sousa, D.P., De Farias Nobrega,´ F.F., De Almeida, R.N., 2007. Influence of the chirality of (R)-(−)- and (S)-(+)-carvone in the central nervous system: and respiratory complaints are important indications and should a comparative study. Chirality 19, 264–268. be studied more thoroughly at relevant doses. Special atten- Di Stasi, L.C., Oliveira, G.P., Carvalhaes, M.A., Queiroz-Junior, M., Tien, O.S., tion should be given to the characterisation of assayed extracts, Kakinami, S.H., Reis, M.S., 2002. Medicinal plants popularly used in the because of their chemical variability. Brazilian Tropical Atlantic Forest. Fitoterapia 73, 69–91. Duarte, M.C.T., Figueira, G.M., Sartoratto, A., Rehder, V.L.G., Delarmelina, C., 2005. Anti-Candida activity of Brazilian medicinal plants. Journal of References Ethnopharmacology 97, 305–311. Fournet, J., 2002. Flore Illustree´ des Phanerogames´ de Guadeloupe et de Mar- Abad, M.J., Bermejo, P., Villar, A., Sanchez Palomino, S., Carrasco, L., 1997. tinique (tome 2). CIRAD, Gondwana Editions, Tartane. Antiviral activity of medicinal plant extracts. Phytotherapy Research 11, Franco, E.A.P., Barros, R.F.M., 2006. Uso e diversidade de plantas medicinais 198–202. no Quilombo Olho D’agua´ dos Pires, Esperantina Piau´ı. Revista Brasileira Adams, C.D., 1972. Flowering Plants of Jamaica. University of the West Indies, de Plantas Medicinais 8, 68–88. Glasgow University Press, Glasgow. Gazola, R., Machado, D., Ruggiero, C., Singi, G., Macedo Alexandre, M., 2004. Aguiar, J.S., 2006. Atividades antimicrobiana, citotoxica,´ antitumoral e antiin- Lippia alba, Melissa officinalis and Cymbopogon citratus: effects of the flamatoria´ de extratos brutos de Lippia alba (Mill.) N.E. Brown. Mestrado aqueous extracts on the isolated hearts of rats. Pharmacological Research em cienciasˆ biologicas,´ Universidade de Pernambuco. 50, 477–480. Alan´ıs, A.D., Calzada, F., Cervantes, J.A., Torres, J., Ceballos, G.M., 2005. Gazzaneo, L.R.S., de Lucena, R.F.P., de Albuquerque, U.P., 2005. Knowledge Antibacterial properties of some plants used in Mexican traditional medicine and use of medicinal plants by local specialists in a region of Atlantic Forest for the treatment of gastrointestinal disorders. Journal of Ethnopharmacol- in the state of Pernambuco (Northeastern Brazil). Journal of Ethnobiology ogy 100, 153–157. and Ethnomedicine 1, 1–8, doi:10.1186/1746-4269-1-9. Albuquerque, U.P., Monteiro, J.M., Ramosa, M.A., Amorim, E.L.C., 2007a. Giron,´ L.M., Freire, V., Alonzo, A., Caceres,´ A., 1991. Ethnobotanical sur- Medicinal and magic plants from a public market in northeastern Brazil. vey of the medicinal flora used by the Caribs of Guatemala. Journal of Journal of Ethnopharmacology 110, 76–91. Ethnopharmacology 34, 173–187. 222 T. Hennebelle et al. / Journal of Ethnopharmacology 116 (2008) 211–222

Guerrero, M.F., Puebla, P., Martin, M.L., Arteaga, L., San Roman, L., 2002. Scarpa, G.F., 2004. Medicinal plants used by the Criollos of Northwestern Assessment of the antihypertensive and vasodilator effects of ethanolic Argentine Chaco. Journal of Ethnopharmacology 91, 115–135. extracts of some Colombian medicinal plants. Journal of Ethnopharmacol- Sena Filho, J.G., Melo, J.G.S., Saraiva, A.M., Gonçalves, A.M., Psiottano, ogy 80, 37–42. M.N.C., Xavier, H.S., 2006. Antimicrobial activity and phytochemical pro- Heinrich, M., Rimpler, H., Barrera, N.A., 1992. Indigenous phytotherapy of file from the roots of Lippia alba (Mill.) N.E. Brown. Revista Brasileira de gastrointestinal disorders in a lowland Mixe community (Oaxaca, Mexico): Farmacognosia 16, 506–509. ethnopharmacologic evaluation. Journal of Ethnopharmacology 36, 63–80. Sena Filho, J.G., Duringer, J.M., Uchoa, D.E.A., Xavier, H.S., Barbosa Filho, Hennebelle, T., 2006. Investigation chimique, chimiotaxonomique et pharma- J.M., Braz Filho, R., 2007. Distribution of iridoid glycosides in plants from cologique de Lamiales productrices d’antioxydants. Memoire´ de doctorat, the genus Lippia (Verbenaceae): An investigation of Lippia alba (Mill.) N.E. Universite´ des Sciences et Technologies de Lille. Brown. Natural Product Communications 2, 715–716. Hennebelle, T., Sahpaz, S., Dermont, C., Joseph, H., Bailleul, F., 2006a. The Senatore, F., Rigano, D., 2001. Essential oil of two Lippia spp. (Verbenaceae) essential oil of Lippia alba: analysis of samples from French overseas growing wild in Guatemala. Flavour and Fragrance Journal 16, 169– departments and review of previous works. Chemistry and Biodiversity 3, 171. 1116–1125. Silva, N.A., Oliveira, F.F., Costa, L.C.B., Bizzo, H.R., Oliveira, R.A., 2006. Hennebelle, T., Sahpaz, S., Joseph, H., Bailleul, F., 2006b. Phenolics and iridoids Caracterizaçao˜ qu´ımica do oleo´ essencial da erva cidreira (Lippia alba of Lippia alba. Natural Product Communications 1, 727–730. (Mill.) N.E. Br.) cultivada em Ilheus´ na Bahia. Revista Brasileira de Plantas Hennebelle, T., Sahpaz, S., Gressier, B., Joseph, H., Bailleul, F., 2007. Antiox- Medicinais Botucatu 8, 52–55. idant and neurosedative properties of polyphenols and iridoids from Lippia Singh, G., Rao, G.P., Kapoor, P.S., Singh, O.P., 2000. Chemical constituents and alba. Phytotherapy Research 22, 256–258. antifungal activity of Lippia alba Mill. leaf essential oil. Journal of Medicinal Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.G., Nakamura, C.V., and Aromatic Plant Sciences 22, 701–703. Filho, B.P.D., 2002. Screening of some plants used in the brazilian folk Stashenko, E.E., Jaramillo, B.E., Mart´ınez, J.R., 2003. Comparacion´ de la com- medicine for the treatment of infectious diseases. Memorias´ do Instituto posicion´ qu´ımica y de la actividad antioxidante in vitro de los metabolitos Oswaldo Cruz 97, 1027–1031. secundarios volatiles´ de plantas de la familia Verbenaceae. Revista de la Howard, R.A., 1989. Flora of the Lesser Antilles. Arnold Arboretum, Harvard Academia Colombiana de Ciencias 27, 579–597. University, Jamaica Plain, Massachussetts. Stashenko, E.E., Jaramillo, B.E., Martinez, J.R., 2004. Comparison of differ- IPNI (International Plant Names Index). Available at: http://www.ipni.org ent extraction methods for the analysis of volatile secondary metabolites (accessed in 12 November, 2007). of Lippia alba (Mill.) N.E. Brown, grown in Colombia, and evaluation of ISB (Institute for Systematic Botany): Atlas of Florida Vascularplants. Available its in vitro antioxidant activity. Journal of Chromatography A 1025, 93– at: http://www.plantatlas.usf.edu/synonyms.asp?plantID=1589 (accessed in 103. 26 August, 2007). Toscano-Gonzalez,´ J.Y., 2006. Uso tradicional de plantas medicinales en la Leonti, M., Sticher, O., Heinrich, M., 2003. Antiquity of medicinal plant usage in vereda San Isidro, municipio de San Jose´ de Pare-Boyaca:´ un estudio pre- two Macro-Mayan ethnic groups (Mexico).´ Journal of Ethnopharmacology liminar usando tecnicas´ cuantitativas. Acta Biologica Colombiana 11, 137– 88, 119–124. 146. Matos, F.J.A., Machado, M.I.L., Craveiro, A.A., Alencar, J.W., 1996. Essential Vale, T.G., Matos, F.J.A., Lima, T.C.M., Viana, G.S.B., 1999. Behavioral effects oil composition of two chemotypes of Lippia alba grown in northeast Brazil. of essential oils from Lippia alba (Mill.) N.E. Brown chemotypes. Journal Journal of Essential Oil Research 8, 695–698. of Ethnopharmacology 167, 127–133. Oliveira, D.R., Leitao,˜ G.G., Santos, S.S., Bizzo, H.R., Lopes, D., Alviano, Vale, T.G., Furtado, E.C., Santos Jr., J.G., Viana, G.S.B., 2002. Central C.S., Alviano, D.S., Leitao,˜ S.G., 2006. Ethnopharmacological study of two effects of citral, myrcene and limonene, constituents of essential oil Lippia species from Oriximina,´ Brazil. Journal of Ethnopharmacology 108, chemotypes from Lippia alba (Mill.) N.E. Brown. Phytomedicine 9, 709– 103–108. 714. Pascual, M.E., Slowing, K., Carretero, E., Villar, A., 2001. Antiulcerogenic Vendruscolo, G.S., Mentz, L.A., 2006. Estudo da concordanciaˆ das citaçoes˜ de activity of Lippia alba (Mill.) N.E. Brown (Verbenaceae). Il Farmaco 56, uso e importanciaˆ das especies´ e fam´ılias utilizadas como medicinais pela 501–504. comunidade do bairro Ponta Grossa, Porto Alegre, RS, Brasil. Acta Botanica Pino Alea, J.A., Ortega Luis, A.G., Rosado Perez,´ A., Rodr´ıguez Jorge, M., Brasilica 20, 367–382. Baluja, R., 1996. Composicion´ y propiedades antibacterianas del aceite esen- Verpoorte, R., Houghton, P.J., Heinrich, M., Mukherjee, P.K., Schmeda cial de Lippia alba (Mill.) N.E. Brown. Revista Cubana de Farmacia 30, Hirschmann, G., van Staden, J., Yesilada, E., 2006. Editorial. Journal of 0–10. Ethnopharmacology 103, 309–310. Pinto, E.P.P., Amorozo, M.C.M., Furlan, A., 2006. Conhecimento popular sobre Viana, G.S.B., do Vale, T.G., Rao, V.S.N., Matos, F.J.A., 1998. Analgesic and plantas medicinais em comunidades rurais de mata atlantica—Itacare,´ BA, antiinflammatory effects of two chemotypes of Lippia alba: a comparative Brasil. Acta Botanica Brasilica 20, 751–762. study. Pharmaceutical Biology 36, 347–351. Puertas-Mejia, M., Hillebrand, S., Stashenko, E., Winterhalter, P., 2002. In vitro Vigneron, M., Deparis, X., Deharo, E., Bourdy, G., 2005. Antimalarial remedies radical scavenging activity of essential oils from Columbian plants and in French Guiana: a knowledge attitudes and practices study. Journal of fractions from oregano (Origanum vulgare L.) essential oil. Flavour and Ethnopharmacology 98, 351–360. Fragrance Journal 17, 380–384. Von Poser, G.L., Toffoli, M.E., Sobral, M., Henriques, A.T., 1997. Iridoid gluco- Robard, I., 2003. Plantes medicinales´ d’outre-mer et pharmacopees:´ aspects sides substitution patterns in Verbenaceae and their taxonomic implication. juridiques, economiques´ et culturels. Phytotherapie—De´ la Recherche ala` Plant Systematics and Evolution 205, 265–287. Pratique 1, 16–21. Woodman, O.L., Chan, E.C.H., 2004. Vascular and anti-oxidant actions of Rodrigues, A.C.C., Guedes, M.L.S., 2006. Utilizaçao˜ de plantas medicinais no flavonols and flavones. Clinical and Experimental Pharmacology and Phys- Povoado Sapucaia, Cruz das Almas—Bahia. Revista Brasileira de Plantas iology 31, 786–790. Medicinais 8, 1–7. Yoshikawa, M., Matsuda, H., Morikawa, T., Xie, H., Nakamura, S., Muraoka, Ruffa, M.J., Wagner, M.L., Suriano, M., Vicente, C., Nadinic, J., Pampuro, O., 2006. Phenylethanoid oligoglycosides and acylated oligosugars with S., Salomon,´ H., Campos, R.H., Cavallaro, L., 2004. Inhibitory effect of vasorelaxant activity from Cistanche tubulosa. Bioorganic and Medicinal medicinal herbs against RNA and DNA viruses. Antiviral Chemistry and Chemistry 15, 7468–7475. Chemotherapy 15, 153–159. Zetola,´ M., Lima, T.C.M., Sonaglio, D., Gonzalez-Ortega, G., Limberger, R.P., Sastre, C., Breuil, A., 2007. Plantes, milieu et paysages des Antilles françaises. Petrovick, P.R., Bassani, P.R., 2002. CNS activities of liquid and spray-dried Ecologie, biologie, identification, protection et usages. Biotope, collection extracts from Lippia alba—Verbenaceae (Brazilian false melissa). Journal Parthenope,´ Meze.` of Ethnopharmacology 82, 207–215.