Journal of Ethnopharmacology Medicinal plants from ...

Journal of Ethnopharmacology 123 (2009) 413–422

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Medicinal plants from the Yanesha (Peru): Evaluation of the leishmanicidal and antimalarial activity of selected extracts Valadeau Céline a,b , Pabon Adriana c , Deharo Eric b,d , Albán–Castillo Joaquina e , Estevez Yannick b , Lores Fransis Augusto f , Rojas Rosario g , Gamboa Dionicia g , Sauvain Michel b,d , Castillo Denis g , Bourdy Geneviève b,d,∗ a

IFEA UMIFRE 17 CNRS/MAEE, Casilla 18-1217, Lima, Peru Université de Toulouse, UPS, UMR 152 (Laboratoire de pharmacochimie des substances naturelles et pharmacophores redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France c Grupo Malaria, Grupo Malaria-Unversidad de Antioquia, Calle 62 # 52-59, Torre 1, Lab. 610 (SIU) Universidad de Antioquia, Medellín, Colombia d IRD, UMR-152, Mission IRD Casilla, 18-1209 Lima, Peru e Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Jesus Maria, Lima, Peru f Comunidad Yanesha de San Pedro de Pichanaz, Sector Azulis, Distrito Palcazú, Prov. Oxapampa, Región Pasco, Peru g Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Avenida Honorio Delgado 430, San Martin de Porres, Lima, Peru b

a r t i c l e

i n f o

Article history: Received 17 December 2008 Received in revised form 3 March 2009 Accepted 19 March 2009 Available online 5 April 2009 Keywords: Yanesha Plasmodium Leishmania Leishmanicidal agents Traditional medicine Antimalarial Peru

a b s t r a c t Aim of the study: Ninety-four ethanolic extracts of plants used medicinally by the Yanesha, an Amazonian Peruvian ethnic group, for affections related to leishmaniasis and malaria were screened in vitro against Leishmania amazonensis amastigotes and against a Plasmodium falciparum chloroquine resistant strain. Materials and methods: The viability of Leishmania amazonensis amastigote stages was assessed by the reduction of tetrazolium salt (MTT) while the impact on Plasmodium falciparum was determined by measuring the incorporation of radio-labelled hypoxanthine. Results and conclusions: Six plant species displayed good activity against Plasmodium falciparum chloroquine resistant strain (IC50 < 10 ␮g/ml): a Monimiaceae, Siparuna aspera (Ruiz & Pavon), A. DC., two Zingiberaceae, Renealmia thyrsoidea (Ruiz & Pavon) Poepp. & Endl. and Renealmia alpinia (Rottb.), two Piperaceae (Piper aduncum L. and Piper sp.) and the leaves of Jacaranda copaia (Aubl.) D. Don (Bignoniaceae). Eight species displayed interesting leishmanicidal activities (IC50 < 10 ␮g/ml): Carica papaya L. (Caricaceae), Piper dennisii Trel (Piperaceae), Hedychium coronarium J. König (Zingiberaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Renealmia alpinia (Rottb.) Zingiberaceae, Lantana sp. (Verbenaceae), Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae) and Calea montana Klat. (Asteraceae). Most of them are used against skin affections by Yanesha people. Results are discussed herein, according to the traditional use of the plants and compared with data obtained from the literature. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Yanesha is a group of Amerindian people living in the upper Amazon, in the department of Oxapampa, Pasco Province (Peru). The actual overall population is estimated at 8540. They belong to the Arawak linguistic family, and through successive migrations during centuries, they moved from the Atlantic coast up to the ultimate oriental foothill of the Andes, where they settled down.

∗ Corresponding author at: IRD, UMR-152, Mission IRD casilla, 18-1209 Lima, Peru. Tel.: +51 1 441 32 23; fax: +51 1 441 32 23. E-mail address: [email protected] (B. Geneviève). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.041

Since then, they shared a vast territory in the central area of Peru, known as “selva central” together with other ethnic groups such as Ashanika, Matsiguengua, and migrants (Smith, 2006). Because the Yanesha pharmacopoeia has not been studied, and also because they live in an endemic place for malaria and leishmaniasis, we undergo a study aiming to document the traditional use of plants and to determine the activity of their crude extracts against Plasmodium and Leishmania parasites. Results are discussed herein in relation with traditional use in Yanesha medicine. 2. Materials and methods All chemicals were from Sigma–Aldrich (USA).

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V. Céline et al. / Journal of Ethnopharmacology 123 (2009) 413–422

Fig. 1. Place of study.

2.1. Ethnobotanical survey Ethnobotanical survey took place in the frame of a more general ethnomedical study and was performed in three communities settled in different ecological sites in Pasco province, department of Oxapampa (see Fig. 1): Tsachopen (Miraflores sector, 250 inhabitants, altitude of 1770 m), Loma Linda (Loma Linda Laguna sector, 290 inhabitants, altitude of 300 m), and San Pedro de Pichanaz (Azulis sector, 122 inhabitants, altitude of 800 m). This survey was undertaken with the formal agreement of the three Yanesha communities. Informant consensus forms were completed and signed. An authorization of collect was delivered by INRENA (Instituto Nacional de Recursos Naturales, Ministerio de Agricultura, Lima, Peru, 122-2007-INRENA-IFFS-DCB). Ethnobotanical and ethnomedicinal data were collected following two methodologies. Species were collected in different ecotopes with the help of 30 informants willing to share with us their knowledge. For each collected plant species, its names, uses, and other related indications were recorded. Through structured and semi-structured interviews, data regarding concept of diseases, treatments, diagnostics, and other topics of interest were noted. All data were checked again during a 4-day workshop with 8 members of the Yanesha communities. All collected plant species were deposited at the National Herbarium of the San Marcos University in Lima, Peru (USM) and in HOXA (Herbario Selva Central, Jardín Botánico de MissouriOxapampa, Peru). Specialists identified all collected material.

2.2. Selection of plant species for evaluation When possible, plants were selected according to their Yanesha use. For malaria, we retained species used against malaria, or Yonna˜ nets (see below) but also species that are used to cure symptoms that can be possibly related with malaria crisis such as fever, strong headache, chills, vomits, associated or not with diarrhoea and liver pain. For cutaneous leishmaniasis, we tried to select plants used against Uta or Mare˜ nets (see below) and also plants indicated as useful to treat various skin ailments in general, because

in the absence of diagnosis, cutaneous leishmaniasis looks very much alike other ulcerative skin pathologies, including bacterial or fungal affections. We selected all together a total of 94 species corresponding to 94 plant parts (see Table 1 ). 2.3. Treatment of plant samples Fifty grams of air-dried powdered plant material were macerated for a week at room temperature in 95% ethanol (500 ml). After filtration, the solvent was evaporated to dryness under reduced pressure at a temperature lower than 40 ◦ C. 2.4. Biological tests 2.4.1. Antimalarial tests Assays on Plasmodium falciparum in vitro were conducted according to Bertani et al. (2005). Plasmodium falciparum FCR3 chloroquine resistant strain-infected erythrocytes were plated at 1% parasitaemia (5% hematocrit) with 3H-hypoxanthine (ICN Biochemicals) (1 ␮Ci/ml), in 96-well microtiter plates and exposed to different concentrations of the extracts (Desjardins et al., 1979). After 48 h of incubation, at 37 ◦ C and 5% CO2 , 5% O2 and balanced N2 atmosphere, plates were frozen–defrosted and each well was harvested onto a glass fiber filter. The incorporated 3H-hypoxanthine was determined with a beta-counter. Growth curves were obtained. Concentration of the extract required to inhibit 50% of parasite growth (IC50 ) was determined by linear interpolation, and standard deviations were calculated using Excel software. All experiments were conduced in triplicate. Reference compound was chloroquine. 2.4.2. Leishmanicidal tests Experiments were conducted on axenic amastigotes of Leishmania amazonensis (strain MHOM/BR/76/LTB-012) according to Estevez et al. (2007). Axenically grown amastigotes were maintained by weekly subpassages in MAA/20 medium (Sereno and Lemesre, 1997a) at 32 ± 1 ◦ C with 5% CO2 in 25 cm2 tissue culture flasks.


415

Table 1 Yanesha traditional indication of plants, and activity of selected extracts. Species family (voucher number)

Medicinal use

Remedy, mode of administration

Part of plant tested

IC50 Leishmania (␮g/ml)

IC50 malaria (␮g/ml)

Acalypha macrostachya Jacq. Euphorbiaceae (CV627) Acalypha mapirensis Pax Euphorbiaceae (JAC16414) Acrocarpus fraxinifolius Wight & Arn. Fabaceae (CV188) Anthurium croatii Madison Araceae (JAC16435) Anthurium ernestii Engl. Araceae (JAC16441) Anthurium ernestii Engl. Araceae (JAC16441) Begonia glabra Aublet Begoniaceae (JAC16388) Begonia parviflora Poepp. & Endl. Begoniaceae (CV516) Calea montana Klatt Asteraceae (CV109)

Uta de agua

See Table 2

Leaves

32.9 ± 4.7

24.7 ± 9.5

Dermatosis

Latex applied on skin

Aerial part

32.7 ± 9.8

NA

Internal bruises

Bark boiled. Preparation drunk

Bark

93.2 ± 8.8

49.8 ± 16.3

Gastro-intestinal disorders with fever Snake or spider bites

Stem

NA

NA

Leaves

31.7 ± 0.3

22.9 ± 11.5

Same as above

Root and stem boiled. Preparation drunk Boil roots, drink and apply mashed root as poultice Same as above

Roots

23.1 ± 4.5

27 ± 11.5

Stomach acidity

Eat raw leaves

Leaves

>100

22.1 ± 7.8

Malaria, fever

See Table 2

Leaves

18.1 ± 8.2

NA

Infected wounds (a) and Panacea (b)

Leaves

10 ± 5.6

NA

Malaria (a) and Uta de agua (b) Infected wound, bruise

(a) Mashed fresh leaves in poultice and (b) drink a tea made out from the leaves. See Table 2 Applied juice from crushed leaf buds

Leaves Leaves

11 ± 1.2 >100

19.8 ± 5.8 45.2 ± 13.7

Infected wounds (a) and malaria (b) Muscular sprains

(a) Heated leaves applied as a dressing and (b) see Table 2 Heated leaves applied as a dressing

Leaves

9.8 ± 2.6

NA

Leaves

9.8 ± 2.6

NA

Feeling of weakness

Bath with the leaves

Leaves

50.8 ± 8.8

NA

Skin care

Leaves

>100

30.4 ± 14.4

Vomits, nauseas

Maceration of leaves in water, as a wash Tea with leaves to drink

Leaves

28.8 ± 4.3

NA

To induce abortion

Roots in the form of decoction to drink

Roots

50.8 ± 7.4

28.6 ± 11.0

Kidney, liver, back pain

Hot leaves applied as a poultice

Entire plant

33.1 ± 2.7

NT

Vomits, stomach trouble, diarrhoea Acute respiratory infection (a), snake or spider bites (b), teeth pain (c) and Uta de agua (d) Uta de agua

Entire plant boiled. Preparation drunk

Entire plant

33.1 ± 2.7

NT

(a) Stem boiled, preparation drunk, (b) grate stem and apply directly, (c) apply sap directly and (d) see Table 2 See Table 2

Stem

>100

NA

Leaves

25.6 ± 7.7

NT

Leaves

>100

22.9 ± 1.5

To strengthen new born

Bath with leaves, drink some tea from leaves Bath with the leaves

Leaves

>100

23.5 ± 8.6

Malaria

See Table 2

Leaves

>100

NA

Rheumatic pain, arthritis

Grate rhizome, put to boil. To drink

Rhizome

10 ± 2.6

26.3 ± 9.5

Rheumatic pain, arthritis

Poultice of mashed hot boiled leaves

Leaves

17.9 ± 5.1

25.7 ± 9.6

Fever, stomach pain (a), kidney pain (b), and to induce labour (c) Uta de agua

(a) Poultice with hot leaves, (b) trunk bark boiled to drink and (c) trunk bark boiled to drink See Table 2

Bark

>100

NA

Leaves

10 ± 7.2

NA

Malaria

See Table 2

Leaves

63.3 ± 9.7

NA

Malaria (a) and Uta de agua (b)

(a and b) See Table 2

Leaves

37.4 ± 6

NA

Malaria (a) and Uta de agua (b)

(a and b) See Table 2

Leaves

16.5 ± 4.5

8.1 ± 1.5

Uta de agua Fever with sprains

See Table 2 Tea with leaves and bath

Leaves Leaves

10 ± 2.1 >100

17.5 ± 6.0 NA

Fever, rheumatic pain, flu

Bark

21.8 ± 9

24.5 ± 9.5

Uta de agua

Steam bath with leaves, leaves in poultice See Table 2

Bark

14.1 ± 0.5

26.3 ± 10.8

Stomach pain and diarrhoea


Bark

>100

NA

Carica papaya L. Caricaceae (CV438) Cecropia latiloba Miq.Cecropiaceae (CV106) Cestrum racemosum Ruiz & Pav. Solanaceae (JAC16378) Cestrum racemosum Ruiz & Pav. Solanaceae (JAC16378) Clusia hammeliana Pipoly Clusiaceae (JAC16384) Clusia trochiformis Vesque Clusiaceae (CV96) Columnea guttata Poepp. Gesneriaceae (JAC16385) Daucus montanus Humb. & Bonpl. ex Spreng. Apiaceae (CV198) Desmodium adscendens (Sw.) DC. Fabaceae (CV759) Desmodium adscendens (Sw.) DC. Fabaceae (CV759) Dieffenbachia williamsii Croat Araceae (CV315) Euphorbia heterophylla L. Euphorbiaceae (CV551) Gloxinia sylvatica (HBK.) Hanst. Gesneraiceae (GB3224) Gurania lobata (L.) Pruski Cucurbitaceae (JAC16377) Hamelia patens Jacq. Rubiaceae (JAC16361) Hedychium coronarium J. König Zingiberaceae (JAC16412) Hedyosmum lechleri Solms Chloranthaceae (CV84) Heliocarpus americanus L. Tliaceae (CV41) Hyptis lacustris A. St.-Hil. ex Benth. Lamiaceae (CV418) Iresine diffusa Humb. & Bonpl. ex Willd. Amaranthaceae (JAC16379) Irlbachia alata (Aubl.) Maas Gentianaceae (CV24) Jacaranda copaia (Aubl.) D. Don Bignoniaceae (CV451) Lantana sp. Verbenaceae (CV68) Liabum eriocaulon Poepp. Lamiaceae (GB3257) Mansoa alliacea (Lam.) A.H. Gentry Bignoniaceae CV364 Munnozia hastifolia (Poepp.) H. Rob. & Brettell Asteraceae (JAC16374) Nectandra cuspidata Nees & Mart. Lauraceae (CV82)

For long walks

416


Table 1 (Continued ) Species family (voucher number)

Medicinal use





Oreocallis grandiflora (Lam.) R. Br. Proteaceae (CV81) Oxalis boliviana Britton Oxalidaceae (CV181)

Panacea

Leaf tea to drink

Leaves

23.7 ± 4.2

NA

Infected wounds (a) and nauseas, stomach troubles (b)

Leaves

>100

34.9 ± 10.7

Peperomia distachya (L.) A. Dietr .Piperaceae (CV72) Persea peruviana Nees. Lauraceae(CV107) Philodendron ernestii Engl. Araceae (CV529) Phoradendron crassifolium (Pohl ex DC.) Eichler Loranthaceae (CV31) Phthirusa robusta Kunth Viscaceae (CV38)

Stomach parasites

(a) Leaves to cook in water, applied as poultice and (b) leaves to cook in water. Eat leaves Leaf tea to drink

Leaves

25.9 ± 8.1

22.1± 5.0

Broken bones

Bark in powder, as a plaster

Bark

>100

NA

Stomach parasites

Stem is toasted, and eaten

Stem

>100

28.5 ± 12.4

Broken bones; stomach ulcers (alcohol) Broken bones (a) and stomach ulcers (alcohol) (b)

Leaves

14.2 ± 4.1

NA

Leaves

37.7 ± 6.4

23.9 ± 10.4

Phthirusa stelis (L.) Kuijt Viscaceae (JAC16367) Phytolacca rivinoides Kunth & C.D. Bouché Phyotlacaceae (CV294) Piper aduncum L. Piperaceae (CV887) Piper crassinervium Kunth Piperaceae (CV52) Piper dennisii Trel. Piperaceae (CV515) Piper politaereum Trel. Piperaceae (CV612) Piper sp. Piperaceae (CV880) Piper sp1. Piperaceae (CV791) Piper umbellatum L. Piperaceae (JAC16362) Piper umbellatum L. Piperaceae (JAC16362) Pityrogramma calomelanos L. Adiantaceae (CV762) Pseudolmedia laevis (Ruiz & Pav.) J.F. Macbr. Moraceae (CV714) Psychotria poeppigiana Müll. Arg. Rubiaceae (CV400) Renealmia alpinia (Rottb.) Maas Zingiberaceae (JAC16436) Renealmia thyrsoidea (Ruiz &Pavon) Poepp. & Endl. Zingiberaceae (CV622) Rollinia mucosa (Jacq.) Baill. Annonaceae (CV63) Ruellia ruiziana (Nees) Lindau Acanthaceae (JAC16421) Sanchezia oblonga Ruiz & Pav. Acanthaceae (JAC16382) Satyria panurensis (Benth. ex Meisn.) Hook. f. Ex Ericaceae (JAC16402) Selaginella conduplicata Spring Selaginellaceae (CV137) Senna ruiziana (G. Don) H.S. Irwin & Barneby Fabaceae (CV441) Siparuna aspera (Ruiz & Pav.) A. DC. Monimiaceae (C060) Siparuna radiata (Poepp. & Endl.) A. DC.Monimiaceae (CV149) Solanum peruvianum L. Solanceae (CV182) Stachytarpheta cayennensis (Rich.) Vahl. Verbnaceae (CV903) Tabernaemontana sananho Ruiz & Pav. Apocynaceae (CV272) Tetragastris panamensis (Engl.) Kuntze Burseraceae (JAC16444) Tripogandra serrulata (Vahl) Handlos Commelinaceae (JAC16376) Urera baccifera (L.) Gaudich. ex Wedd. Urticaceae (CV131)

Broken bones

Leaves

28.5 ± 2.4

NA

Heart pain

Leaf tea to drink and mashed leaves in poultice; leaf tea to drink (a) Leaf tea to drink and mashed leaves in poultice and (b) leaf tea to drink and mashed leaves in poultice on stomach Leaf tea to drink and mashed leaves in poultice Fruits mashed in poultice, on thorax

Fruit

26.3 ± 7.2

26.4 ± 10.7

Fever, general infection Traumas, bruises

Steam bath, and some tea drunk Mashed fresh leaves in poultice

Leaves Leaves

NT 25.8 ± 3.2

9.6 ± 1.7 33.5 ± 15.9

Rheumatic pain, arthritis Lack of appetite (a) and leg oedema (b) High fever, diarrhoea, asthenia High fever, diarrhoea, asthenia Boils, dermatosis

Mashed fresh leaves in poultice (a) Leaf eaten raw and (b) steam bath with leaves Steam bath and some tea drunk Steam bath and some tea drunk Previously heated leaves in poultice

Leaves Leaves

10 ± 1.5 50.3 ± 6.7

21.4 ± 7.0 NA

Leaves Leaves Leaves

29.5 ± 12.4 19.9 ± 5.9 39.5 ± 3.2

7.3 ± 0.8 NA 19.2 ± 5.1

Miscarriage

Tea from leaves to drink

Leaves

39.5 ± 3.2

19.2 ± 5.1

Uta de agua, dermatosis

See Table 2

Leaves

88 ± 2.8

49.9 ± 16.5

Infected wounds

Latex applied on skin

Leaves

32 ± 8.2

22.3 ± 7.7

High fever, diarrhoea, asthenia

Steam bath with leaves

Leaves

>100

NA

Gynaecological haemorrhages

Rhizome crushed and boiled in water. To drink Rhizome crushed and boiled in water. To drink Leaf juice to drink

Rhizome

9 ± 0.6

10 ± 1.4

Rhizome

10 ± 0.8

6.8 ± 1.5

Leaves

25.2 ± 0.4

12.8 ± 1.5

To give a sensation of well being Strong headache

Leaves in the form of a bath

Leaves

>100

33.7 ± 5.3


Leaves

>100

26.8 ± 11.1

Muscular sprains

Leaves

>100

NA

Fever and asthenia

Squash leaves, drink juice. In the form of a poultice Steam bath with leaves

Leaves

86.1 ± 6.2

NA

Uta seca, dermatosis

Cut green pod, apply exuding sap

Leaves

>100

25.4 ± 10.1

Fever and asthenia


Leaves

NT

6.4 ± 2.3

Fever and asthenia


Leaves

>100

21.7 ± 7.2

Muscular sprains

Poultice of mashed hot boiled leaves

Aerial part

14.2 ± 4.2

26.9 ± 11.2

Panacea

Whole plant as a tea

Aerial part

>100

NA

Leg oedema, anaemia


Bark

30.9 ± 2.5

NA

Gynaecological haemorrhages

Grated bark to boil. Preparation drunk

Bark

>100

NA

Dark urine with fever

Leaves squashed, juice drunk

Leaves

>100

NA

Diarrhoea, stomach trouble (a), cold with fever and (b) rheumatic pain, arthritis (c) Malaria

(a and b) Tea with leaves to drink and (c) tea with leaves to drink, and poultice of leaves See Table 2

Leaves

48.6 ± 7.2

NA

Aerial part

33.3 ± 4.1

23.5 ± 8.6

Athlete foot

Mash leaves, apply as poultice

Leaves

>100

38.7 ± 10.1

Verbena littoralis Kunth Verbanaceae (CV376) Vernonanthura patens (Kunth) H. Rob Asteraceae (JAC16366)

To give a sensation of well being Throat “cancer”


417

Table 1 (Continued ) Species family (voucher number)

Medicinal use





Virola sp. Myristicaceae (JAC16397) Vismia baccifera (L.) Triana & PlanchClusiaceae (JAC16368) Vismia pozuzoensis Engl. Clusiaceae (JAC16405) Vismia sp. Clusiaceae (GB3202) Warszewiczia coccinea (Vahl) Klotzsch Rubiaceae (JAC16391) Xiphidium caeruleum Aubl. Haemodoraceae (JAC16360) Amphotericine B (reference drug for Leishmania) Chloroquine (reference drug for malaria)

Internal bruises Infected wounds

Bark boiled. Preparation drunk Latex applied over the wound

Bark Bark

>100 >100

23.5 ± 6.2 NA

Cold with fever

Steam bath with leaves, drink some tea

Leaves

>100

22.9 ± 8.9

Uta de agua Convulsions, epilepsy

See Table 2 Grated bark in cold water to drink

Stem Bark

54.3 ± 10.4 >100

NA NA

Infected wounds

Grated inside of stem, applied as poultice

Stem

>100

NA

Cultures were initiated with 5 × 105 amastigotes in 25 cm2 tissue culture flasks with 5 ml of medium. To determine the activity of the extracts, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) micromethod was used as previously described (Sereno and Lemesre, 1997b). Briefly, 100 ␮l of axenically grown amastigotes were seeded in 96-well flat bottom microtiter plates. Extracts, dissolved in DMSO, were added at final concentrations ranging from 100 to 1 ␮g/ml. After 72 h of incubation, 10 ␮l of MTT (10 mg/ml) was added to each well and plates were further incubated for 4 h. The enzymatic reaction was then stopped with 100 ␮l of 50% isopropanol 10% sodium dodecyl sulfate and incubated for an additional 30 min under agitation at room temperature. Finally, the optical density (OD) was read at 570 nm with a 96-well scanner (Bio-Rad). All experiments were performed in triplicate, and standard deviations were calculated using Excel software. Percent growth inhibition of the parasite was calculated by the following formula: % of inhibition =

(OD control − OD drugs) × 100 OD control

The concentration inhibiting 50% of the parasite growth (IC50 ) was calculated after evaluating percent growth inhibition at different concentrations. Reference compound was Amphotericin B. 3. Results and discussion Ethnopharmacological data on selected species together with results in both tests are given in Table 1. (In order to make it easier to read, we just give a brief description of the ethnopharmacological uses and remedies of selected species, but complete data can be provided on simple request to corresponding author.) Table 2 summarizes complete ethnopharmacological information on plants specifically used by the Yanesha against malaria or cutaneous leishmaniasis. 3.1. Ethnobotanical results Both malaria and leishmaniasis are well known by the Yanesha, and because they live with these diseases for centuries, they built up a cultural vision of them. 3.1.1. Malaria According to the Yanesha cosmovision, in ancient times, illnesses were human beings. The myth of origin of malaria (Valadeau et al., 2008) tells that Yonna˜ nets (malaria) came to earth with three other illnesses: Errasanãts, Shornan˜ets and Puetsarnãts (haemorrhagic fever, smallpox and pox) in order to kill Yaneshas. They started their trip from a mountain, in Cacazu valley, and travelled to

0.14 ± 0.03 0.3 ± 0.02

the lower parts of the valley to a place called Iscozacin. They walked slowly, killing and eating Yanesha people all along the way, moving from one place to another when they had nothing more to eat in a village. Finally, when they reach the end of the valley, they decided to return to their departure point. At that time, they were in great state of starvation, because the whole population had vanished, from fear or death. Two of them died, Shornan˜ets and Puetsarnãts. Yonna˜ nets and Errasanãts set apart, and Yonna˜ nets decided to stay on a rock near the Azulis river, since then called “malaria table”, keeping up eating and killing people, and finally disappeared suddenly. Errasanãts was desperate to go back to its place of origin, and completely lost, asked the only Yanesha left in a village to show him the way back to the mountain where he came from. After getting the guarantee he will not be killed, the man accepted. While walking, Errasanãts showed him all plants that could be used as remedy to cure malaria, haemorrhagic fever, smallpox and pox. Once in the mountain, he vanished in the air. As demonstrated by many studies (Desowitz, 1991), malaria was introduced into South America from Africa by slave trading during colonial times. According to colonial sources (Santos Granero, 2004), between 1711 and 1739, when the missionary control of this area took place, due to the introduction of many European epidemic diseases (flu, chicken pox, measles) the overall indigenous population drop down dramatically leading to a child mortality rate of 50% in 1739. Without doubt, the travel of Yonna˜ nets and its companions as described by the Yanesha is a metaphoric description of the sudden apparition of these introduced diseases, their mode of propagation and their disappearance. According to Yanesha, Yonna˜ nets is characterized by a strong fever that makes the body shake, together with a strong feeling of tiredness when the fever is over. They also recognize that this disease is rather to be encountered in the lower part of the valley, in the Amazonian lowlands (where warmer and humid climates prevail) and that higher in the mountains, there are very few cases of Yonna˜ nets. Two kinds of remedies are used by the Yanesha against malaria, preventive ones and curative ones (see Table 2). Preventive remedies, are used at a family or a community level, when people have good reasons to suspect that there are cases of malaria in the community or in the neighbouring villages. These remedies are based on the use of three species: Capsicum frutescens L. (Solanaceae), Jacaranda copaia (Aubl.) D. Don (Bignoniaceae) and Irlbachia alata (Aubl.) Maas (Gentianaceae). The first one is administered in the form of a “smoke bath”: fruits and leaves of Capsicum frutescens are mixed up with chopped tobacco leaves (Nicotiana sp., Solanaceae), placed over a small fire, and people have to stand for a while in the profuse prickly smoke. The other plants are administered in the form of a steam bath. Leaves are put into water in an aluminium pot, over a fire. When the water steams, the person place the pot

418


Table 2 Species and mode of preparation of remedies used by the yaneshas for the treatment of malaria (Yonna˜ nets) or cutaneous leishmaniasis (Mare˜ nets). Disease as described by the yaneshas

Species determination, yanesha name, herbarium number

Remedy and mode of administration

Yonna˜ nets

Begonia parviflora Poepp. & Endl (Begoniaceae), ˜ Yonnantsopar, CV516, GB3201

Yonna˜ nets

Bidens pilosa L. (Asteraceae), Poconcpan, CV617, GB3236

Yonna˜ nets

Carica papaya L. (Caricaceae), Papaya, CV976

Yonna˜ nets

Cestrum racemosum Ruiz & Pav. (Solanaceae), t˜epeshech, CV600, JAC16378 Hamelia patens Jacq. (Rubiaceae), Arotapan, CV247, GB3209

Prepare a tea with some leaves. Drink two or three small glasses (50 ml) during the day (it is a strong remedy). At the same time, prepare a steam bath with the leaves. It is also recommended to rub all over the body the inside part of the stem, finely grated. This helps the fever to low down quickly. A handful of leaves is mashed, and placed in half liter of boiling water. The water is let to cool down. Drink half a cup (150 ml) three times a day. Prepare a steam bath. Two yellow leaves (the one ready to fall on the ground) are placed in boiling water for some minutes. Drink half a cup (150 ml) three times a day. Collect from 10 to 15 leaves, and prepare a steam bath. Then after the steam bath, apply the cooked leaves all over the body. Prepare a tea with some leaves. Drink three cups a day. At the same time, make a steam bath with leaves and stems. After the steam bath use the water in the form of a bath. Repeat the treatment until feeling better and complete disparition of fever. 250 g of aerial part is mashed, and covered by some cold water. After a while, when the preparation has turned very bitter, all are drunk at once. For the children it is better to prepare a tea with 4–5 leaves in a cup of boiling water, and give by spoonful. At the same time, make a stem bath with a bunch of whole plant or a bath. Mash 1–2 leaves in water or in masato (manioc beer). Drink half a cup (150 ml) three times a day. At the same time prepare a steam bath with the leaves or a bath. Prepare some leaves in the form of a tea, which is drunk. For preventive and curative purposes, prepare a steam bath (or a bath). Prepare a tea with some mashed leaves and drink 3 cups a day, until feeling better. At the same time, prepare a steam bath, or a bath, and give to all members of the family. Prepare a tea with some leaves and drink 3 cups a day, until feeling better. Apply the white latex directly on the affected part. During three day. It is said that cicatrization starts at day 3. Apply the latex directly on the skin.

Yonna˜ nets

Yonna˜ nets

Iresine diffusa Humb. & Bonpl. ex Willd. (Amaranthaceae), Charrtapan, JAC16379

Yonna˜ nets

Irlbachia alata (Aubl.) Maas (Gentianaceae), Puepa’t˜pan, CV24*, GB3200 Jacaranda copaia (Aubl.) D. Don (Bignoniaceae), Charapachpan, CV559 Urera laciniata Goudot ex Wedd. (Urticaceae), Rrenaj, CV436

Yonna˜ nets Yonna˜ nets

Yonna˜ nets Mare˜ nets Mare˜ nets

Verbena littoralis Kunth (Verbenaceae), Pesherr Acalypha macrostachya Jacq. (Euphorbiaceae), ˜ Marentsopar, CV250* Carica papaya L. (Caricaceae) CARICACEAE, Papaypan, CV438 ˜ nt, ˜ Euphorbia heterophylla L. (Euphorbiaceae), tãte’ne CV551 Dieffenbachia williamsii Croat (Araceae), Corech CV315, JAC16423 Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae), Ollamepan, CV418 and Hyptis obtusiflora C. Presl ex Benth. (Lamiaceae), Ollamepan, CV333 Munnozia hastifolia (Poepp.) H. Rob. & Brettell (Asteraceae), Huallapnarren, JAC16374

Cut the top of the stem, or a leaf, and apply exuding latex to the affected part, few times a day until cure. Put a handful of plant in boiling water for a while, and wash the affected part once a day until cure. At the same time, use the “boiled leaves” in the form of a dressing on the ulcer. Cut the top of the stem, or a leaf, and apply exuding sap to the affected part, once a day until cure. Apply previously heated leaves, mashed over the skin in the form of a dressing. Aplicar directamente el látex blanco que sale del tallo cortado sobre la zona afectada. Se aplica también para cicatrizar heridas.

Mare˜ nets

Irlbachia alata (Aubl.) Maas (Gentianaceae), Puepa’t˜pan, CV644 Jacaranda copaia (Aubl.) D. Don (Bignoniaceae) Charapachpan, CV559

Machacar bien las hojas y aplicarlas en la parte afectada. Cambiar este emplasto todos los días. Leaves are previously boiled in water. When soft, they are applied on the affected part like a poultice.

Mare˜ nets

Lantana sp. (Verbenaceae), t˜epeshpan, CV68, CV76

Mare˜ nets

Pityrogramma calomelanos (L.) Link (Pteridaceae), Seseronapan, CV424 ˜ Vismia sp. (Clusiaceae), Marentsorech, GB3203

Prepare a concentrated infusion with few leaves. Clean the affected part with this, and then, apply the hot leaves as a dressing. Repeat everyday, until cure. Prepare a concentrated infusion with few leaves. Clean the affected part with this, and then, apply the hot mashed leaves as a dressing. Repeat everyday until cure. Cut a piece of the stem of this liana, and apply the thick orange latex directly on the affected part at night. Leave as a plaster all night long. Repeat this treatment until cicatrization occurs.

Mare˜ nets Mare˜ nets

Mare˜ nets

Mare˜ nets

Mare˜ nets

between his feet, and cover his entire body by large traditional clothes, and stay there, sweating, for some minutes. In some cases, previously heated stones in an open fire are added to this preparation, increasing steam and sweating. For small children or for weak people, these plants would preferably be given in the form of a bath, avoiding the steam, judged too strong. According to Yanesha, in case of malaria or in case of any strong fever, the intense heat of the steam can damage kidney and liver: hence one must be very cautious in their use. Ten species are used for curative purposes: Begonia parviflora Poepp. & Endl (Begoniaceae), Bidens pilosa L. (Asteraceae), Carica papaya L. (Caricaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Hamelia patens Jacq. (Rubiaceae), Iresine diffusa Humb. & Bonpl. ex Willd. (Amaranthaceae), Irlbachia alata, Jacaranda copaia, Urera laciniata Goudot ex Wedd. (Urticaceae) and Verbena littoralis Kunth (Verbenaceae). According to Yanesha people, together with the administration of remedies, a special diet must be respected,

including food restrictions together with special social behaviour rules. For people who suffer of Yonnan˜ets it is highly recommended to avoid sexual relations, spicy food (for example Capsicum frutescens fruits), canned food, fermented drinks (like the traditional manioc beer), alcoholic beverages, salt and sugar. Cestrum racemosum is the only plant that has to be administered in the form of a bath or as a steam bath, and cannot be taken orally. The other species are administered as tea, followed by a steam bath (or a bath) with the same plant species. Most of the remedies were single plant remedies, but it was said to us, that depending on the severity of the crisis and also the skill and the knowledge of people, some plants can be mixed together to enhance the activity of the remedy. Of the above-mentioned plants, yellow leaves of Carica papaya are a common remedy for malaria, but generally in association with other plants (Milliken and Bruce, 1997; Morton, 1981; Vigneron et al., 2005). In our model, these leaves did not display any activ-


ity, thus confirming results from previous studies (Bertani et al., 2005). Irlbachia alata use against malaria is also reported in different countries (Brandao et al., 1992; Johnston and Colquhoun, 1996; Vigneron et al., 2005), but was never found to be active in the intra-erythrocytic test. Nevertheless, xanthones isolated from this plant was shown to inhibit the biocrystallization of heme, a crucial process for Plasmodium growth (Hnawia et al., 2003). Unfortunately, none of the Yanesha plants claimed to be used directly against malaria was active in our in vitro model. Nevertheless, among all of the selected extracts, six plants showed very good activity (IC50 < 10 ␮g/ml): a Monimiaceae Siparuna aspera (Ruiz & Pavon), A. DC. and two Piperaceae (Piper sp., and Piper aduncum), all of them also used in steam bath against high fever and general infectious disease, the leaves of Jacaranda copaia used against leishmaniasis and the rhizomes of two Zingiberaceae, Renealmia thyrsoidea (Ruiz & Pavon) Poepp. & Endl. and Renealmia alpinia (Rottb.). The genus Siparuna is widely used in traditional pharmacopoeias all over central and South America. In French Guiana, teas made out of leaves of Siparuna guianensis Aubl. and Siparuna pachyantha A.C. Sm. are used by Creoles and Wayãpi Indians against malaria and fevers while Palikur Indians used them as cataplasm for antiinflammatory purposes (Grenand et al., 2004). Some years ago, we tried to isolate compounds responsible for the antimalarial activity of Siparuna guianensis, but the activity was lost during bioguided fractionation (unpublished data). Other Siparuna (Siparuna andina (Tul.) A. DC., Siparuna pauciflora (Beurl.) A. DC. and Siparuna tonduzian Perkins) from Central America were also shown to have antiplasmodial activity (Jenett-Siems et al., 1999). Isoquinoline and sesquiterpene derivatives were isolated from Siparuna pauciflora. The nor-boldine was the only one with some antimalarial properties, IC50 : 3.1 ␮g/ml (Jenett-Siems et al., 2003). A flavanone was also shown to be active (Jenett-Siems et al., 2000). As far as we know, it is the first time that the antimalarial activity of Siparuna aspera has been reported. At the best of our knowledge, it is also the first time that antimalarial activity was described for Piper aduncum. Unfortunately, it was impossible to have a complete identification of the other active Piper, but generally speaking, this genus is known to produce chalcones, amides and prenylated aromatic acid derivatives with antiprotozoal properties. Some of them also showed interesting antimalarial properties, and much work has been undertaken related with this large genus (Rukachaisirikul et al., 2002, 2004; Portet et al., 2007; Flores et al., 2008). Regarding the good activity displayed in our model we think these plants worth a bioguided fractionation in order to isolate compounds responsible for the displayed activity. Jacaranda copaia has been shown to have some leishmanicidal properties (see below) based on quinone derivatives jacaranone and triterpene. Nevertheless these products were found to be toxic for ˜ macrophages (Chan-Bacab and Pena-Rodríguez, 2001). The antimalarial activity detected herein could be due to similar toxic effect, but this should be checked. Finally, rhizomes of both Renealmia species are active on both tests: we discuss these results below. 3.1.2. Leishmaniasis All over Peru, and also in some other Latin American countries suffering from this disease (Bolivia), cutaneous leishmaniasis is designated by the Quechua name of Uta (Herrer and Battistini, 1951). In the IMT-AvH (Instituto de Medicina Tropical Alexander von Humboldt) from Lima (Peru), the reference centre for the treatment of leishmaniasis in this country, it has been estimated that 80% of cases called Uta by the patients were indeed due to a Leishmania infection. Yanesha people differentiate two types of Uta: Uta de agua and Uta seca (“Watery Uta” and “Dry Uta” in local spanish). According to the

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Yanesha, Uta de agua is a skin ulcer, starting small and getting bigger slowly, “like if it was eating from deep inside” with a lot of material giving a watery effect, and painful. We also observed directly some cases of Uta de agua and some specific scars (round, with no sharp angles, with the skin a little bit depressed and coloration lines in a concentric pattern), so we concluded that, for Yanesha people also the term Uta de agua refers to cutaneous leishmaniasis, and its Yanesha name is Mare˜ nets. For the Yanesha, Uta seca is a dermatosis, starting by small vesicles, extending rapidly, becoming very itchy with a whitish and squeamish aspect, the centre drying and the sides being red, and bleeding when scratched. In our opinion, this could be much more linked with an infection due to Microsporum sp. or Trichophyton sp., or maybe another fungal infection, and certainly not cutaneous leishmaniasis. Oppositely to malaria, leishmaniasis is a South American endemic disease, occurring since pre-Incaic times (Urteaga-Ballon, 1991). According to the Yanesha cosmovision, Mare˜ nets is represented as a very short legged man with a heavy backpack on his shoulder. In a mythic time, when part of the Yanesha humanity became animals, plants, or diseases, Mare˜ nets transformed in a small fly. Since then Mare˜ nets spirit, inside the fly, introduces himself under the skin, through the bite of the fly, in order to make his victim die, and rapt his spirit forever. Mare˜ nets will preferably attack a person from the opposite sex. If the affected person does not cure himself (herself) this means that he/she accepts its deadly love. This is why it is said that the treatment against Mare˜ nets has to be administered by a single man, for a female patient (or vice versa). This is also why the patient must not show any emotion of regret (like sobbing, crying) during the length of the painful treatment. The associated diet is to avoid sexual relations, eating salt, salted fish or spicy food. Twelve species were designated as useful against Mare˜ nets (see Table 2). Most widely used treatment consists on the application of the white latex of Acalypha macrostachya Jacq. (Euphorbiaceae), a common shrub growing in disturbed places. Stem apex is cut, and the freshly dripping whitish latex is directly applied all over the affected part. This application has to be repeated for 3 days in a row: it is said that it is very painful, “like if the skin was burning”, but very efficient: at the third day the cicatrization process has started. For some Yanesha people, it is so efficient that it can also be used in case of muco-cutaneous leihmaniasis, dripping the latex right in the cavity. During our fieldwork, we were able to observe a treatment with this latex, and can hereby confirm its potent activity. Among the species used by the Yanesha to cure cutaneous leishmaniasis, some are well known species or genus that also have been recorded in other places with similar uses, such as Jacaranda copaia (Aublet, 1775; Devez, 1932; Fleury, 1986; Bourdy, 1999; Grenand et al., 2004), Irlbachia alata (Bourdy, 1999), Munnozia hastifolia (Poepp.) H. Rob. & Brettell (Asteraceae) (Fournet et al., 1993), Dieffenbachia spp. (Araceae) (Grenand et al., 2004; Kvist et al., 2006), Lantana sp. (Verbenaceae), Pityrogramma calomelanos L. (Adiantaceae) (Van Andel, 2000). Surprisingly, even if Tabernaemontana sananho Ruiz & Pav. (Apocynaceae) is easy to find in the Yanesha area, it does not seem to be used against leishmaniasis, as ˜ it is the case in many other lowland Amazonian places (Munoz et al., 1994; Kvist et al., 2006; Estevez et al., 2007). Over the 12 species designated as useful for Mare˜ nets we were able to collect 11 of them. Nine of them displayed activity, ranging from very good to moderate in our model, thus validating their use. Best activity (IC50 < 10) was observed for Carica papaya, Hyptis lacustris, and Lantana sp. The fresh flowing papaw latex applied directly on skin (against sloughing or infected burns and wounds, remove damage tissues in anthrax, and for other dermatosis such as eczema and psoriasis) is a quite common household remedy, almost everywhere in the world where this tree grows (Watt and Breyer-

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Brandwijk, 1962; Pétard, 1986). These traditional uses are explained by the presence of the protein-digestive enzymes such as papain, which has the ability to digest mammalian tissues and reduce bacteria adherences (Watt and Breyer-Brandwijk, 1962). Still, it is the first time, to the best of our knowledge, that was recorded the use of the papaw latex against leishmaniasis, and maybe the application helps in reducing the over-infection generally associated with these open wounds. In our test, we worked with the ethanolic extract of leaves, in which carpain was isolated. This compound has been extensively studied for its cardiac, diuretic, and amoebicidal effect (Hornick et al., 1978; Phillipson and O’Neill, 1987) and its action has been compared with emetine. It is not sure that this very same product is responsible for the leishmanicidal activity of papaw leaf, hence further biological pharmacology should go on with this extract. Hyptis is a wide genus, with many reported medicinal species. Some Hyptis are traditionally used for skin problems due to fungal affections or other dermatosis (Bourdy, 1999; Grenand et al., 2004; Robineau et al., 2007). Some abietan diterpenes isolated from Hyptis suaveolens (L.) Poit. have been shown to have anti-inflammatory (Grassi et al., 2006) and antiplasmodial properties (Chukwujekwu et al., 2005). Essential oils isolated from the genus Hyptis, have also been shown to have analgesic and regenerative properties (Melo et al., 2006; Santos et al., 2007; Menezes et al., 2007; Arrigoni-Blank et al., 2008). According to the wide range of activity of the genus, related to skin diseases, and the lack of references on Hyptis lacustris specifically, this plant should be considered for activity-guided fractionation studies. Munnozia hastifolia was not as active as the plant described above but its activity correlated with traditional use and leishmanicidal activity of another Munnozia, Munnozia maronii (André) H. Rob. growing in sub-Andean tropical region of Bolivia. A bioguided fractionation process leads to an active sesquiterpene lactone, Dehydrozaluzanin-C, with very good activity against promastigotes of many Leishmania strains (Fournet et al., 1993). Indeed, Munnozia hastifolia should be also considered for further analysis. In earlier studies one of us showed that jacaranone, a benzoquinole isolated from the leaves of Jacaranda copaia in French Guiana, was active against promastigotes of Leishmania amazonensis but was toxic for murine peritoneal macrophages. It was also showed that Jacaranone was weakly active in vivo against Leishmania amazonensis when administered subcutaneously and was toxic when applied inside the lesion (Sauvain et al., 1993). The toxicity and the already isolated compounds are not pleading for deeper studies of this plant. Moderate to poor activity was observed with lactiferous species such as Acalypha macrostachya Wild. (Euphorbiaceae), Euphorbia heterophylla L. (Euphorbiaceae), Vismia sp. (Clusiaceae). In our point of view, this can be explained because we could not reproduce in vitro, the condition of administration of the remedy, using leaves or stem instead of latex. In our model, Irlbalchia alata (Aubl.) Maas (Gentianaceae) also displayed a weak activity. Finally, Pityrogramma calomelanos and Dieffenbachia williamsii Croat (Araceae) did not display activity in our model. Precedent work was undertaken in French Guiana on another Dieffenbachia species, Dieffenbachia seguine (Jacq.) Schott, also used against cutaneous leishmaniasis, but no direct activity was demonstrated against Leishmania. The authors concluded that it was the necrotic effect of the plant that was involved in its use (Grenand et al., 2004). The same was said for Dieffenbachia aff. costata H. Karst.ex Schott used in the Chayahuita ethnic group (Estevez et al., 2007). In our survey some other species not directly used by the Yanesha against Uta de agua also displayed very good activity. They are: Calea montana Klatt, Cestrum racemosum, Piper dennisii Trel., Hedychium coronarium J. König, Renealmia alpinia, Renealmia thyrsoidea.

Calea montana is used by the Yanesha as a plaster on infected wounds. From another species, Calea uniflora Less. actives chromanones against promastigotes of Leishmania major and acetophenones with weak trypanocidal activity have been isolated (do Nascimento et al., 2004, 2007). Therefore, it should be interesting to verify if the same products are also present in Calea montana, and also to check if these products are also active on Leishmania. Leaves of Cestrum racemosum, used against malaria, are also used against infected wounds or parasitic sores (scabies) in the form of a plaster. The same use has been reported for other Cestrum South American i.e.: Cestrum parqui L’Hér., Cestrum strigillatum Ruiz & Pavon (Bourdy et al., 2004). The good antifungal activity of Cestrum auriculatum L’Hér., another Peruvian plant, was reported by Rojas et al. (2003), but as far as we know, no leishmanicidal or antiplasmodial activity of Cestrum was ever reported in the literature. This genus is known for its contents in saponins (Fouad et al., 2008) and this class of compounds showed a strong in vivo activity against leishmaniasis (Germonprez et al., 2005). This is why, we consider this plant worthy for further investigations. Piper dennisii Trel. (Piperaceae) is traditionally used by the Yanesha in the form of a poultice applied on sore joins due to rheumatic pain, or arthritis. In a previous study on Chayahuitas etnia we showed that Piper hispidum Kunth and Piper strigosum Trel. were also active against Leishmania. Chalcones are known to support the leishmanicidal activity of the genus (Chen et al., 1993; Torres-Santos et al., 1999; Hermoso et al., 2003). No antiplasmodial or leishmanicidal properties of Hedychium coronarium J. König (Zingiberaceae) rhizome have been reported in the literature. Nevertheless, the aromatic oil extracted from the rhizome, was shown to have antifungal and antibacterial properties (Joy et al., 2007). A labdane diterpene, coronarin D, isolated from the rhizome of this plant was shown to induce apoptosis in various cellular types (Kunnumakkara et al., 2008). Other labdane diterpenes isolated from the same species were also found to inhibit nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages (Matsuda et al., 2002). Several authors (see Vincendeau et al., 2003 for review) consider that Leishmania parasite controls the macrophage by preventing the production of NO. Thus the inhibition of the production of NO by diterpenes of Hedychium should reinforce this mechanism. We showed herein that rhizomes of Renealmia alpinia and Renealmia thyrsoidea were active against Leishmania and Plasmodium. Renealmia alpinia has been reported as febrifuge in Suriname and cytotoxic labadanes terpenes were isolated from its leaves (Zhou et al., 1997; Yang et al., 1999). Labdane diterpenes isolated from African Zingiberaceae have been showed to inhibit plasmodial growth at ␮M concentrations (Duker-Eshun et al., 2002; Kenmogne et al., 2006). But the genus Renealmia is known also for arylheptanoids contents (Sekiguchi et al., 2002). It must be highlighted that aryl-heptanoids, like curcumin isolated from Curcuma longa L. (another Zingiberaceae), showed leishmanicidal properties (Araujo et al., 1999), this activity being related with an apoptotic effect (Alves et al., 2003; Das et al., 2008). Nevertheless, the presence of curcumin derivatives, known to be able to neutralize the NO production by the macrophages (Chan et al., 2005), is also in contradiction with the antiparasitic putative role of the plant as for the diterpenes isolated from Hedychium coronarium. This aspect is planned to be studied by our group for both Zingiberaceae. 4. Conclusion The purpose of this work was to screen plant extract, selected from the traditional Yanesha pharmacopoeia, against cutaneous leishmaniasis and malaria. From the ethnopharmacological survey, we discovered that both diseases were integrated in the Yanesha cosmovision. Regarding leishmaniasis, the description provided by


the Yanesha, coupled with our visual observations, which gave the warranty that there was a good correlation between the Yanesha names of Mare˜ nets and cutaneous leishmaniasis. Over the 11 species used by Yanesha people against Mare˜ nets, the high majority of them displayed activity in our model. Most used species (Acalypha macrostachya) displayed a weak activity, in contradiction with its traditional reputation and positive therapeutic effect, but this is certainly due to the impossibility to repeat the exact conditions of administration of this plant. It is also important to stress that a large proportion of plants used by the Yanesha against leishmaniasis are also used for the same purposes in other part of the Amazon, hence pleading for a common pot of pan-Amazonian species with the same uses. Regarding malaria, the situation is a little bit more complex. No species directly indicated by the Yanesha to be useful in case of Yonna˜ nets was active in our model. For Yanesha people more salient symptoms of malaria are high fever with chills. Even if these symptoms indeed are characteristic of a malaria crisis, they can also occur in other pathologies, and one might suspect that in places with low endemicity, there are more doubts about the match between these symptoms and a true malaria crisis. This point was mentioned by the Yanesha themselves and confirmed by the nearest hospital (Alarcon, 2004). Also indeed, the way of administration of these remedies, as steam bath, makes difficult to set up experimental conditions to assert their activity, even if it has been demonstrated that this way of administration displayed effective antimicrobial or antimalarial activity (Lopes et al., 1999; Braithwaite et al., 2008). Still, we do consider that it could be very interesting to work on some of these plants, belonging to Siparuna and Piper genus, administered through steam bath against high fever and displaying good activity in the Plasmodium model. Because of the originality of the way of administration, maybe some very new and original findings in the treatment of malaria could be done.

Acknowledgments The authors gratefully acknowledge the financial assistance of CONCYTEC from Peru and DSF-IRD (JEAI) from France. We express our thanks to members of the Yanesha community who were willing to share with us their knowledge about medicinal plants. The authors are grateful to Candy Ruiz from LID-UPCH, for technical assistance in plants extractions. Céline Valadeau, Adriana Pabon and Denis Castillo received grants from the Institut Français des Etudes Andines, Colciencias and the Belgian technical cooperation respectively. Part of this work was supported by a grant from the International Foundation for Sciences attributed to Dionicia Gamboa.

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