Antiparasitic activity of some New Caledonian medicinal plants

Journal of Ethnopharmacology 112 (2007) 7–12

Antiparasitic activity of some New Caledonian medicinal plants Julie Desrivot a,b , Jean Waikedre c , Pierre Cabalion c , Christine Herrenknecht a , Christian Bories b , Reynald Hocquemiller a , Alain Fournet a,d,∗ a

Laboratoire de Pharmacognosie (UMR CNRS 8076-BioCIS), Universit´e Paris-Sud 11, Facult´e de Pharmacie, 92296 Chˆatenay-Malabry, France b Laboratoire de Chimioth´ erapie Antiparasitaire (UMR CNRS 8076-BioCIS), Universit´e Paris-Sud 11, Facult´e de Pharmacie, 92296 Chˆatenay-Malabry, France c Laboratoire Substances Naturelles Terrestres & Savoirs traditionnels (‘SNT&ST’), US 084, IRD, BP A5, 98848 Noum´ ea, New Caledonia d IRD US 084, Laboratoire de Pharmacognosie, Facult´ e de Pharmacie, 92296 Chˆatenay-Malabry, France Received 30 November 2006; received in revised form 10 January 2007; accepted 23 January 2007 Available online 30 January 2007

Abstract Twenty-nine extracts of 18 medicinal plants used in New Caledonia by traditional healers to treat inflammation, fever and in cicatrizing remedies were evaluated in vitro against several parasites (Leishmania donovani, Trypanosoma brucei brucei, Trichomonas vaginalis and Caenorhabditis elegans). Among the selected plants, Scaevola balansae and Premna serratifolia L. were the most active against Leishmania donovani with IC50 values between 5 and 10 ␮g/ml. The almond and aril extracts from Myristica fatua had an IC50 value of 0.5–5 ␮g/ml against Trypanosoma brucei brucei. Only Scaevola balansae extract presented a weak activity against Trichomonas vaginalis. The almond extract from Myristica fatua presented significant activity against Caenorhabditis elegans (IC50 value of 6.6 ± 1.2 ␮g/ml). © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: New Caledonia; Leishmanicidal activity; Trypanocidal activity; Trichomonas vaginalis; Caenorhabditis elegans

1. Introduction The chemotherapy of many parasitic infections remains an unresolved problem. Widespread resistance to drugs is emerging, not only among helminthic diseases (Kaminsky, 2003) but also in protozoan infections, such as Trichomonas vaginalis, the most common parasite causing sexually transmitted diseases (Swygard et al., 2004). The resulting failures of treatment, combined with side effects, make it necessary to search for new drugs by pharmacological screening. Therefore, attempts are being made to identify new candidate antiparasitic agents among naturally occurring compounds with biological properties. New Caledonia possesses a relatively rich flora. The quotient of the number of species of native phanerogams by surface area (density per square kilometer) is 0.157, which is high compared with those obtained for other Pacific islands. Its high ∗

Corresponding author at: IRD US 084, Laboratoire de Pharmacognosie, Facult´e de Pharmacie, 92296 Chˆatenay-Malabry, France. Tel.: +33 1 46 83 55 94; fax: +33 1 46 83 53 99. E-mail addresses: [email protected], [email protected] (A. Fournet). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.01.026

specific endemicity ratio is remarkable (almost 75%) (Jaffr´e et al., 2001). Although parasitic diseases such as leishmaniasis and trypanosomiasis are absent from New Caledonia and Vanuatu, the IRD (Institut de Recherche pour le D´eveloppement) has carried out investigations (Billo et al., 2005a,b) to find new naturally occurring active compounds based on the traditional medicine of these countries. The selection of plants was guided by ethnopharmacological data derived from information collected among traditional healers. These plants are used in traditional medicines of New Caledonia for the treatment of inflammation and fevers. Some were selected for the present study because of their presumed biological activity, particularly that of their essential oils. Crude extracts of 18 plants, including various samples of essential oils, were screened in vitro for activity against leishmaniasis (Leishmania donovani), African trypanosomiasis (Trypanosoma brucei brucei), urogenital trichomoniasis (Trichomonas vaginalis) and nematodes (Caenorhabditis elegans). Among these plants, some: Hernandia cordigera (Richomme et al., 1985), Myoporum crassifolium (Menut et al., 2005), Myoporum tenuifolium (Tomas et al., 1985), Eugenia uniflora (Kanazawa et al., 2000), Citrus macroptera (Gaillard et al., 1995) and Curcuma longa (Singh and Khar,

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J. Desrivot et al. / Journal of Ethnopharmacology 112 (2007) 7–12

Table 1 Ethnobotanical data of studied plants Family name

Scientific name

Voucher

Local name

Part used

Uses

Asteraceae

Wollastonia biflora (L.) DC. (Syn.: Melanthera biflora (L.) Willdenow, Wedelia biflora (L.) DC)

JWHL 83

Leaves

Juice of leaves with water in a complex remedy against children dysentery and diarrhea, also diarrhea caused by exposition to cold when fishing

Cupressaceae

Callitris neocaledonica Dummer

Maudet Caroline

herbe a` cochons, kheejak (nelemwa, nixumwak), khˆagee (Yuanga) thagac (nyelayu de balade), kaket (haveke), ujo (haveke); holaless ne hun¨et¨e (Lenormand), meaning the ‘holaless’, earlier translated as ‘faux topinambour de rocaille’; segue (nengone); hi¨ok, hi¨ek, pace (Iaai) One of the “bois de fer de montagne”

Wood

Cupressaceae

Callitris sulcata (Parlatore) Schlechter

Maudet Caroline

sapin de la comboui, gni`e

Wood

Flacourtiaceae

Homalium deplanchei (Vieillard) Warburg

LIT 0781

chˆene bleu du littoral; dridrem (Drehu)

Leaves

Goodneniaceae Scaevola balansae Guillaumin

JWHL 93

Bark, leaves

Hernandiaceae

Hernandia cordigera Vieillard

JWHL101

Lamiaceae

Premna serratifolia L.

Lit1071

emuet (nemi), mualandio (nyelayu des Belep), jara de veo (aji¨e); darandaveo (one langage near Bourail, orowe or t¨ırˆı) bois bleu, phoap (nyelayu de Balade; same name in nelemwa, nixumwak and nyelayu), epeth (drehu); dr¨ok (Iaa¨ı) hat (nelemwa, nixumwak), khama, khamio (yuanga), alchadscha, al (drehu), ari (Nengone), veek (Iaai)

No traditional uses because of lack of accessibility, as it grows only up in the mountain, near Mt Humboldt, behind the xˆarˆagur`e region Inner wood resistant to decay and termites, traditionally used in the confection of wooden house poles Leaves in a complex remedy against fatigue an/or as a fortifying portion (drehu) A fortifying potion

Moraceae

Ficus prolixa J.G. Forster

SJ2

banian, thuk (nixumwak), hmana (drehu), saa (Drehu), denge, ye-denge (Nengone), b¨ek (Iaai)

Leaves, fruits

Myoporaceae

Myoporum crassifolium J.G. Forster

SereiOuvea MC1

Leaves, wood

Myoporaceae

Myoporum tenuifolium J.G. Forster

C. Bontemps

hanyium ne gej¨e (drehu), hagnyum (Iaii), faux-santal, grande citronelle, kanum(u) faux santal, bois de citronelle, amwat (nelemwa, nixumwak), xabwet (nelemwa, nikumwak), hanyium ne helep (Drehu), hany¨om (iaai)

Bark, leaves

Juvenile forms used in remedies against skin diseases (orowe or tˆırˆı region, near Bourail)

Leaves

Young leaves heated and used in massages to help blood circulation on new cicatrices, juice expressed from leaves against TB (nelemwa); juice from expressed leaves drunk against migraine and general pains (drehu) Dible fruits (nyelay from Belep); crushed leaves are spitted on the chest of a nurse to avoid or treat breast lymphangitis (drehu) Smoke of aerial parts against mosquitoes (nengone)

Leaves, wood

Masticated leaves are projected on skin ulcerations or mycosis, or pressed against mycosis (nelemwa); against odontalgias, eat raw leaves or ingest a remedy containing a decoction of leaves (Drehu); maceration of leaves against epilepsy (drehu); foliar buds masticated against odontalgiae (nengone), masticated with coconut and applicated on head in case of tinea (nengone)

J. Desrivot et al. / Journal of Ethnopharmacology 112 (2007) 7–12

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Table 1 (Continued ) Family name

Scientific name

Voucher

Local name

Part used

Uses

Myristicacae

Myristica fatua Houttuyn

PC1046

Sap, fruit

Yaws, rheumatisms

Myrtaceae

Eugenia uniflora L. (Syn: Eugenia michelii Lamarck)

JW 120 serei

nandai (bislama pidgin, Vanuatu) cerisier kanak, cerisier de Cayenne, cerisier de Mar´e (used in Mar´e), boobo (drehu) No common or vernacular name collected citronnier sauvage, oranger indig`ene (Bril), oranger sauvage, oranger ancien, oranger des morts, buvaang, buvaak (nelemwa, nixumwak), buvaa (yuanga, nyelayu from Balade), dongan (nyelayu from Belep); mid´e, (fwai) puifel¨o (drehu), Ibon (nengone), nig¨ot (iaai) thang (CB), (BHG), walhap (ou) wulang, m`ee` k¨o (xˆarˆac`uu` ), reinette, reinette de la R´eunion chˆene tann, bunier, bugny, buni gingembre sauvage, carry de montagne, carry, safran, kari (nelemwa, nixumwak)

Fruits, sap

Edible fruits (drehu, negone) sap contains skin allergens (drehu) No traditional use collected

Bark, leaves

In remedies against ringworm, complex remedies against sicknesses (yuanga); epilepsy-like symptoms (nelemwa)

Leaves

Sickness (drehu)

Bark, leaves

Ichtyotoxic bark (xˆarˆac`uu` ), anti-inflammatory reputation (drehu)

Podocarpaceae Prumnopytis ferruginoides (Compton) de Laubenfels Rutaceae Citrus macroptera Montrous.

PC/EH 3421A

Rutaceae

Murraya crenulata Oliver

JW3 Serei

Sapindaceae

Dodonea viscosa (L.) N. J. Jacquin

JWHL 97

Sapotaceae

Manilkara dissecta var. pancheri (Baillon) Maas Curcuma longa L.

LIT 0537

Zingiberaceae

JW 78

JW 185 Serei

2006), have been studied previously. This work follows on from our first study (Billo et al., 2005b) of antiparasitic activities in medicinal plants from New Caledonia.

Reputed as a panacea (drehu) Leaves, rhizome

Condiment

Myoporum tenuifolium (leaves), Citrus macroptera (leaves) and Curcuma longa (leaves) were obtained by hydrodistillation. 2.3. Biological assays

2. Materials and methods 2.1. Plant material The study was carried out between March 2000 and November 2005 in the main island of New Caledonia (Grande Terre) and the Loyalties Islands (Mar´e, Lifou and Ouv´ea). Eighteen plants from 13 families, native to New Caledonia, were selected on the basis of ethnopharmacological information based on local medicinal knowledge. They were identified at the SNT&ST and at the Botany Laboratories, Institut de Recherche pour le D´eveloppement (IRD), Noum´ea, New Caledonia. Voucher specimens have been deposited at the Herbarium of IRD Centre, Noum´ea. Plant materials were oven-dried at 40 ◦ C and ground. 2.2. Preparation of plant extracts 2.2.1. Preparation of crude organic extracts Dried powdered material (50 g) was extracted by maceration with 250 ml of methanol or dichloromethane, for 3 h at room temperature with shaking. Total extracts were filtered and concentrated to dryness at reduced pressure. 2.2.2. Preparation of essential oils Extracts of Callitris neocaledonica (heart wood), Callitris sulcata (heart wood), Myoporum crassifolium (heart wood),

The parasites used in this study: Leishmania donovani, Trypanosoma brucei brucei, Trichomonas vaginalis and Caenorhabditis elegans, were obtained from the Antiparasitic Chemotherapy Department of the Faculty of Pharmacy of Chˆatenay-Malabry Univ. Paris-Sud 11, France. Biological tests were performed three times and each concentration was tested in duplicate (n = 6). 2.3.1. Leishmanicidal assay The test was performed as previously described by M’Bongo et al. (1997) and Okpekon et al. (2004). Briefly, promastigotes of Leishmania donovani were grown in 96-well microtiter plates at 27 ◦ C in a 5% CO2 atmosphere in the dark, in M199 medium containing 10% fetal calf serum (FCS) and supplemented with 40 mM HEPES, 100 ␮M adenosine, 0.5 mg hemin/l, and 50 ␮g gentamycin/ml. Two hundred microliters of culture medium were placed in the well containing the maximum concentration of extract (C1 ), and 100 ␮l in the following (C2 –C7 and controls); 2 ␮l of an extract solution of 20 mg/ml in DMSO were added in C1 and a serial dilution in the wells was performed. After 1 h at 27 ◦ C under a 5% CO2 atmosphere, 100 ␮l of culture medium complemented with 1.75 × 106 parasites/ml, from a logarithmic phase culture, were added. The final volume in the well was 200 ␮l. After a 72 h incubation period, the viability of parasites was evaluated by the tetrazolium-dye (MTT)

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Table 2 Antiprotozoal and nematocidal activities of some medicinal plants from New Caledonia Scientific name

Part used

Extract

Leishmania donovani (IC50 ± S.E.M. ␮g/ml)

Trypanosoma brucei brucei (MAC)

Asteraceae

Wollastonia biflora (L.) DC

Leaves

Cupressaceae Cupressaceae Flacourtiaceae

Callitris neocaledonica Dummer Callitris sulcata Schltr. Homalium deplanchei (Vieillard) Warburg

Wood Wood Bark

CH2 CL2 MeOH Hydrodistillate Hydrodistillate CH2 CL2

12.6 ± 1.8 >50 >50 >50 11.5 ± 1.2

>100 >50 >50 >50 >50

69.8 ± 0.8 >100 >100 >100 >100

Goodneniaceae

Scaevola balansae Guillaumin

Bark

Hernandiaceae

Hernandia cordigera Vieillard

Bark

Lamiaceae

Premna serratifolia L.

Bark

Moraceae Myoporaceae

Ficus proxila Forster Myoporum crassifolium Forster & G.Forster

Leaves Wood

MeOH CH2 CL2 MeOH CH2 CL2 MeOH CH2 CL2 MeOH MeOH Hydrodistillate

>50 8.7 ± 1.4 >50 11.5 ± 1.0 >50 4.4 ± 0.4 >50 >50 25.5 ± 2.9

>50 39 >50 48 >50 >50 >50 46 16

>100 29.3 ± 6.2 >100 >100 >100 >100 >100 >100 >100

Myoporaceae

Myoporum tenuifolium Forster & G. Forster Myristica fatua Houttuyn

Resin Leaves

CH2 CL2 CH2 CL2

>50 19.0 ± 1.7

>50 >50

>100 >100

Almonds

26.5 ± 2.7 >50 21.5 ± 1.5 >50 >50 >50

0.5 1.6 0.8 4.7 >50 >50

35.2 ± 4.7 >100 >100 >100 >100 >100

Myristicacae

Trichomonas vaginalis (IC50 ± S.E. ␮g/ml)

Eugenia uniflora L. Prumnopytis ferruginoides (Compton) de Laub. Citrus macroptera Montrouzier Murraya crenulata (Turzaninow) Oliver Dodonea viscosa (L.) N. J. Jacquin

Bark Leaves

CH2 CL2 MeOH CH2 CL2 MeOH Hydrodistillate Hydrodistillate

Leaves Bark

Hydrodistillate Hexane

>50 >50

>50 27.6

>100 >100

Leaves

CH2 CL2

>50

61.4

>100

Sapotaceae

Manilkara dissecta var. pancheri (Baillon) Maas

Leaves

MeOH CH2 CL2

>50 13.4 ± 1.1

>50 >50

>100 >100

Zingiberaceae

Curcuma longa L.

Leaves

MeOH Hydrodistillate

>50 26.0 ± 2.4

>50 >50

>100 >100

Arils Myrtaceae Podocarpaceae Rutaceae Rutaceae Sapindaceae

Reference drugs Amphotericin B Miltefosine Pentamidine Metronidazole Mebendazole

Caenorhabditis elegans (IC50 ± S.E.M. ␮g/ml)

18.9 ± 2.4 6.6 ± 1.2 13.1 ± 2.6 22.1 ± 4.6

0.1 ± 0.02 7.3 ± 0.7 2.6 0.5 ± 0.1

1.0 ± 0.3

J. Desrivot et al. / Journal of Ethnopharmacology 112 (2007) 7–12

Family name

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colorimetric method. The results are expressed as the concentration inhibiting parasite growth by 50% (IC50 ). Amphotericin B and miltefosine were the reference drugs. 2.3.2. Trypanocidal assay The antiparasitic activity was assessed using the method described in Loiseau et al. (2000). Briefly, the bloodstream forms of Trypanosoma brucei brucei were maintained in vitro for 48 h in the dark at 37 ◦ C in a 5% CO2 atmosphere, in minimum essential medium (Gibco-BRL) including 25 mM HEPES and Earle’s salts, and supplemented with 2 mM l-glutamine, 1 g of additional glucose per liter, 10 ml of minimum essential medium nonessential amino acids (100×; Gibco-BRL) per liter, 0.2 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 15% heat-inactivated horse serum (Gibco-BRL), and 50 ␮g gentamycin per milliliter. The 96-well plates were filled as in the leishmanicidal assay, except that the parasites added were 2 × 105 trypomastigotes from the blood of a mouse collected aseptically. The minimum active concentration (MAC) was defined as the minimum concentration at which no viable parasite was observed microscopically. Pentamidine was the reference drug. 2.3.3. Trichomonicidal assay The trichomonicidal activity was assessed by using the method described by Loiseau et al. (1999). Briefly, Trichomonas vaginalis were grown in TYM medium for 48 h in the dark at 35 ◦ C in a 5% CO2 atmosphere. The 96-well plates were filled as in the antileishmanial assay, except that the final volume was 300 ␮l. After 1 h at 35 ◦ C in a 5% CO2 atmosphere, 100 ␮l of culture medium containing 12 × 104 parasites/ml, from a logarithmic phase culture, was added in each well. Trichomonas vaginalis are grown in microaerobic condition (GENbag microaer, Biom´erieux, France). The IC50 value was defined microscopically and/or using bromophenol purple. Metronidazole was the reference drug. 2.3.4. In vitro activity against Caenorhabditis elegans A method described by Ravina et al. (1993) was used. Tests were carried out in 24-well plates (Costar® ) and 4 wells were used for each experimental group. To each well, 1 ml of culture medium (M9 buffer, NG agar, 50 ␮l of Escherichia coli culture in brain heart infusion with 9 mg/ml chloramphenicol instead of ampicillin, Brenner, 1977) was added followed by 7.5 ␮l of the appropriate compound solution or solvent. Finally, 0.5 ml of culture medium containing 10–15 Caenorhabditis elegans (L2 or L3 obtained from synchronous cultures) was added to each well. The effect of compound on the development and reproductive capacity of Caenorhabditis elegans was determined by comparing the population levels attained in the control and test wells after an incubation period of 7 days at 20 ± 1 ◦ C. Mebendazole was used as a reference compound. 3. Results and discussion Twenty-nine extracts (methanol, dichloromethane, hexane, hydrodistillate) of 18 plants belonging to 13 botanical families

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were screened for their antiparasitic activity against Leishmania donovani, Trypanosoma brucei brucei, Trichomonas vaginalis and Caenorhabditis elegans. Five reference drugs: amphotericin B (Fungizon® ), miltefosine (Impavido® ), pentamidine (Pentacarinat® ), metronidazole (Flagyl® ) and mebendazole (Vermox® ) were used as appropriate. The plants studied are presented in Table 1, while the in vitro antiparasitic activities (IC50 values) of their extracts are shown in Table 2. Among the 29 samples investigated in this study, 13 plants showed variable levels of activity against one or more of the test organisms. Extracts from Premna serratifolia and Scaevola balansae showed the strongest activity against Leishmania donovani (IC50 between 4.4 and 8.7 ␮g/ml). For these plants, no information about their phytochemical composition is available. Four extracts showed IC50 between 10 and 15 ␮g/ml: from Wollastonia biflora (Asteraceae), Hernandia cordigera (Hernandiaceae), Homalium deplanchei (Sapindaceae) and Manilkara dissecta var. pancheri (Sapindaceae). For Hernandia cordigera, this activity could be linked to presence of lignans or bisbenzylisoquinoline alkaloids (Richomme et al., 1985; Angerhofer et al., 1999; Jang et al., 2004; Van Miert et al., 2005). For Wollastonia biflora (syn = Wedelia biflora (L.) DC.) no data about its phytochemical composition is available. The most active extracts against the bloodstream forms of Trypanosoma brucei brucei, were those made with dichloromethane from almonds and arils of Myristica fatua, which yielded IC50 values inferior to 1.0 ␮g/ml, while the IC50 value of pentamidine was 2.6 ± 0.4 ␮g/ml. This high activity against Trypanosoma brucei brucei could be due to the high concentration of fatty acids (Shuaibu et al., 2003) or the essential oil (Dorman and Deans, 2000; Cimanga et al., 2002) contained in the fruit. Against Trichomonas vaginalis, only the Myristica fatua almond extract with dichloromethane and the Scaevola balansae bark extract presented IC50 values inferior to 50 ␮g/ml, by respectively of 35.2 ± 4.7 and 29.3 ± 6.2 ␮g/ml. This activity is much lower than that of the reference drug, metronidazole (IC50 value of 0.04 ± 0.01 ␮g/ml). The in vitro activity of the extracts was also tested against the nematode Caenorhabditis elegans. Myristica fatua almonds and arils extracts (IC50 = 6.6 ± 1.2 ␮g/ml) and the resin of Myoporum crassifolium (IC50 = 18.9 ± 2.4 ␮g/ml) were the only samples with any detectable activity, which remained lower than that of the reference drug, mebendazole (IC50 = 1.0 ± 0.3 ␮g/ml) (Table 2). 4. Conclusion In this study, 18 medicinal plants collected in New Caledonia were screened for antiparasitic activity. Some of them presented interesting in vitro activities against visceral leishmaniasis. Against African trypanosomiasis, only Myristica fatua had significant activity against the bloodstream forms of Trypanosoma brucei brucei. These promising results will be followed up by in vivo testing.

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