Antimalarial and cytotoxic activities of ethnopharmacologically selected medicinal plants from South Vietnam

Journal of Ethnopharmacology 109 (2007) 417–427

Antimalarial and cytotoxic activities of ethnopharmacologically selected medicinal plants from South Vietnam Julie Nguyen-Pouplin a,b,∗ , Hop Tran c,d , Hung Tran e , Tuyet Anh Phan f , Christiane Dolecek f , Jeremy Farrar f,g , Tinh Hien Tran h , Philippe Caron c , Bernard Bodo a , Philippe Grellier b a

UMR 5154 CNRS Chimie et Biochimie des Substances Naturelles, Dpt R.D.D.M., Mus´eum National d’Histoire Naturelle, CP54, 63 rue Buffon, 75231 Paris Cedex 05, France b USM 0504 MNHN Biologie Fonctionnelle des Protozoaires, Dpt R.D.D.M., Mus´ eum National d’Histoire Naturelle, CP52, 61 rue Buffon, 75231 Paris Cedex 05, France c Institut de D´ eveloppement Vietnam Pacifique, 42/13 Duong so 4, P.5, Go Vap District, Ho Chi Minh City, Vietnam d Department of Botany, University of Natural Sciences, 225 Nguyen Van Cu, District 5, Ho Chi Minh City, Vietnam e Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy of Ho Chi Minh City, 41 Dinh Tien Hoang, District 1, Ho Chi Minh City, Vietnam f Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietnam g London School of Hygiene and Tropical Medicine, Keppel Street, London, UK h Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietnam Received 31 March 2006; received in revised form 7 July 2006; accepted 11 August 2006 Available online 15 August 2006

Abstract Malaria is a major global public health problem and the alarming spread of drug resistance and limited number of effective drugs now available underline how important it is to discover new antimalarial compounds. An ethnopharmacological investigation was undertaken of medicinal plants traditionally used to treat malaria in the South Vietnam. Forty-nine plants were identified, 228 extracts were prepared and tested for their in vitro activity against Plasmodium falciparum, and assessed for any cytotoxicity against the human cancer cell line HeLa and the embryonic lung MRC5 cell line. In a first screening at a concentration of 10 ␮g/ml, 92 extracts from 46 plants showed antiplasmodial activity (parasite growth inhibition >30%). The IC50 values of the most active extracts were determined as well as their selectivity towards Plasmodium falciparum in comparison to their cytotoxic effects against the human cell lines. Six plants showed interesting antiplasmodial activity (IC50 ranging from 0.4 to 8.6 ␮g/ml) with a good selectivity: two Menispermaceae, Arcangelisia flava (L.) Merr. and Fibraurea tinctoria Lour., and also Harrisonia perforata (Blanco) Merr. (Simaroubaceae), Irvingia malayana Oliv. ex Benn. (Irvingiaceae), Elaeocarpus kontumensis Gagn. (Elaeocarpaceae) and Anneslea fragrans Wall. (Theaceae). © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Antimalarial activity; Cytotoxicity; Medicinal plants; Vietnam; Ethnopharmacological screening

1. Introduction Malaria is a major global public health problem and is responsible for the death of over 1 million people annually, with more than 90% of cases found in sub-Saharan Africa. The increasing global spread of drug resistance to most of the available and ∗

Corresponding author at: UMR 5154 CNRS Chimie et Biochimie des Substances Naturelles, Dpt R.D.D.M., Mus´eum National d’Histoire Naturelle, CP54, 63 rue Buffon, 75231 Paris Cedex 05, France. Tel.: +33 1 40 79 31 19; fax: +33 1 40 79 31 35. E-mail address: [email protected] (J. Nguyen-Pouplin). 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.08.011

affordable antimalarial drugs is a major concern and requires innovative strategies to combat. History shows that plants have been an important source of medicines against malaria with two of the major drugs used in malaria treatment, quinine and more recently artemisinin both having derived from traditional medicine and from plants. These two drugs are now the mainstay of the treatment of severe malaria worldwide and the artemisinin derivatives in combination with a second antimalarial drug are now at the heart of the World Health Organisation strategy to control malaria globally. Traditional medicines are a potential rich source of new drugs against malaria and other infectious diseases and given the remarkable contribution this

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has made to the development of potent antimalarial drugs over the last five hundred years it is clearly an approach that must be continued. Malaria is endemic in Vietnam and remains one of the most important diseases in Asia particularly in the Mekong region home to 300 million people. South-East Asia is also home to the most drug resistant parasites in the world, both Plasmodium falciparum and Plasmodium vivax (World Health Organisation, 2001). Vietnamese traditional medicine is similar in many respects to the traditional Chinese school and has two components: southern medicine “thuoc nam” genuinely Vietnamese and the northern medicine or “thuoc bac”, which is Sino-Vietnamese in origin. In practice, both are performed in parallel and often together. Southern Vietnamese medicine arose under the influence of many ethnic groups including the incorporation of Chinese and Indian teaching, and developed using plants growing in the tropical conditions of southern Vietnam and was primarily concerned with infectious and rheumatic diseases (Hoang et al., 1999; Nghiem, 2002). Considering the great potential of South Vietnam in terms of plant biodiversity, rich traditional knowledge and practice, we were interested to study medicinal plants from this tropical area. In this framework, an ethnopharmacological investigation of South Vietnamese medicinal plants was undertaken through interviews and literature surveys on plants used to treat malaria or malaria-like symptoms. Selected plants were collected and further evaluated for their in vitro antiplasmodial activity and cytotoxicity effect on human cell lines. 2. Methods and materials 2.1. Antimalarial plant inventory An ethnopharmacological investigation of Vietnamese medicinal plants traditionally used against malaria was undertaken starting from a botanist and traditional healer plant selection established by the Institut de D´eveloppement Vietnam Pacifique (IDVP) and enriched by a relevant literature survey and interviews. An inventory of 200 plants was developed. Selection was further optimised to narrow down the identification of potentially new antimalarial compounds. Weighted criteria were applied to this collection using the following: the quantity of documented references, the relevance and redundancy of the information, the specific use of the plant to treat malaria or malaria-like symptoms, the convergence of remedies and practices in different countries, the plant localization, particularly in the south of Vietnam and the chemical composition and biological activity reports. Forty-nine plants were selected. They are listed in Table 1. 2.2. Collection and identification Plant collection was carried out with IDVP in November 2003 in South Vietnam from areas spreading out from Ho Chi Minh City to Dalat through the Ba Ria Vung Tau, Dong Nai and Lam Dong provinces (Fig. 1). Collections were undertaken from areas endemic for malaria, all are secondary forests but with variable

Fig. 1. Map of plant collecting places in Vietnam.

vegetal covers: Binh Chau (dry forest, sea coast), Ma Da (350 m above sea level, slightly wet forest), Bao Loc (800 m above sea level, sempervirent rainforest) and Da Lat (1100 m above sea level, resinous forest). Botanic identification was first conducted in the field. The parts of the plant traditionally used were collected, and allowed to dry. The specimens were then chopped into small pieces at IDVP and put on plates in a drying room for 12 h, at 40–45 ◦ C, until desicated. Plants were crushed into powder using a hammer mill and stored at room temperature in plastic bags under vacuum with a deshumidifying pellet sachet. The specimen identification was confirmed by Prof. Tran Hop (Department of Botany, University of Natural Sciences, Ho Chi Minh City, Vietnam) and by comparison with the collections of the National Museum of Natural History of Paris (MNHN, France). These voucher herbarium specimens were deposited at MNHN (Table 1). Four additional plants used as antimalarial remedies were also investigated: three plants were donated by a traditional medicine monk from the Linh Chieu Pagoda in Dong Nai province (stems of Tinospora crispa Hook.f. Thomson (Menispermaceae), roots of Dichroa febrifuga Lour. (Saxifragaceae), bark of Eurycoma longifolia Jack. (Simaroubaceae)) and one plant was bought in the traditional medicine market of Ho Chi Minh City (leaves of Momordica cochinchinensis (Lour.) Spreng. (Cucurbitaceae)). 2.3. Extraction Crude extractions from the plants were performed in the Pharmacognosy Laboratory of the Faculty of Pharmacy (Ho Chi Minh City, Vietnam) and in the Chemistry Laboratory of Natural Products of MNHN. For each part of plant, 50 g of powdered dried material were sequentially macerated three times in ethanol 80%, for 2 h with constant shaking, at room temperature. The filtrates were pooled and evaporated to dryness and the powdered extract was stored in a dry place in the dark. Crude extract fractionation was performed at the Laboratory of Pharmacognosy, Faculty of Pharmacy (Strasbourg,

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Table 1 Selected plants of South Vietnam used to treat malaria and malaria-like symptoms

a B,

bark; F, fruit; L, leaves; P, pericarp; R, roots; S, stems; Se, seeds; W, whole plant; b V.N., voucher number; n.d., voucher not done.

France), with a semi-automatized Soxhlet extractor (Soxtec Avanti 2055 apparatus, Foss Tecator AB, H¨ogan¨as, Sweden). Each crude extract (2 g) was mixed with sand (15 g) and sequentially extracted with 100 ml of cyclohexane (15 min, 180 ◦ C), methylene chloride (60 min, 180 ◦ C) and methanol (60 min, 270 ◦ C). The filtrates were taken to dryness under vacuum and the powdered residues were stored at 4 ◦ C. The presence of tannins was checked in all methanol extracts. Three of them were positive and their tannins were removed by chromatography on LH20 Sephadex gel; 228 organic extracts were obtained. Extracts were conditioned in 96-tube microplates at a concentration of 10 mg/ml in DMSO and stored at −20 ◦ C. This extract

library is available at the French National Plant Extract Library (http://chimiotheque-nationale.enscm.fr). 2.4. Biological assays 2.4.1. Antiplasmodial activity The antiplasmodial activity of the extracts was determined against the chloroquine-resistant FcB1/Colombia strain of Plasmodium falciparum (IC50 chloroquine = 0.1 ␮M) as previously described (Frappier et al., 1996). Briefly, the extracts at the concentration of 10 mg/ml in DMSO were diluted with culture medium to the expected concentrations in 96-well microplates,

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asynchronous parasite cultures were then added (1% parasitemia and 1.5% final hematocrit) and plates were maintained for 24 h at 37 ◦ C in a candle jar. [3 H] hypoxanthine (0.5 ␮Ci) was subsequently added to each well and parasites were maintained for further 24 h. After freezing and thawing, the cells were harvested from each well onto glass fiber filters and the dried filters were counted in a scintillation spectrometer. The growth inhibition for each well was determined by comparison of the radioactivity incorporated into the treated culture with that in control culture maintained on the same plate. A preliminary screening was performed at the concentration of 10 ␮g/ml in duplicate. Results were averaged to select the active extracts presenting more than 30% of inhibition of parasite growth. The antiplasmodial activity of the most active extracts was further assessed by determining the extract concentration inhibiting 50% of parasite growth (IC50 ) according to Desjardins et al. (1979). Results were expressed as the means ± the standard deviations determined from several independent experiments. Some extracts were also tested on contemporary clinical isolates of Plasmodium falciparum at the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam. The infected blood culture was synchronized at the ring stage by sorbitol treatment (Lambros and Vanderberg, 1979). The extracts were then tested in duplicate at the concentration of 10 ␮g/ml on the infected blood (3% of hematocrit and <1% parasitemia) in 96-well microplates, at 37 ◦ C, under a 5% CO2 atmosphere. After 24–48 h of incubation, when parasites reached the maturation stage controls, Giemsa-stained thick blood films were prepared for each well and the percentage of inhibition of parasite growth was determined under microscope by comparison of the number of schizonts with three or more nuclei out of a total of 200 parasites with that of control wells containing no extract. 2.4.2. Cytotoxicity on mammalian cells and selectivity index The human cervix carcinoma cells HeLa and the human diploid embryonic lung cells MRC5 were seeded into 96-well microplates at 5000 cells per well. After 24 h, cells were washed and maintained with different concentrations of extract for 5 days, at 37 ◦ C, under 5% CO2 atmosphere. The cytotoxicity of the plant extracts was determined using the colorimetric methylthiazoletetrazolium (MTT) assay (Mossman, 1983) and scored as a percentage of absorbance reduction at 540 nm of treated cultures versus untreated control cultures. IC50 values on cell growth were obtained from the drug concentration–response curves. Results were expressed as the means ± the standard deviations determined from several independent experiments. The selectivity index was determined by the ratio of the IC50 value on cancer (HeLa) or normal (MRC5) cells to the IC50 value on Plasmodium falciparum. 3. Results and discussion Two hundred and twenty eight extracts were prepared from the 57 parts of the 49 selected plants and were first tested at the concentration of 10 ␮g/ml on the Plasmodium falciparum

FcB1 strain (Table 2). Ninety-two extracts (40%) showed a significant in vitro antiplasmodial activity (parasite growth inhibition greater than 30%), corresponding to 46 plants among the 49 selected (94%). A similar antiplasmodial activity was also observed for these plants on the clinical Plasmodium falciparum isolates (data not shown). The antiplasmodial activities of the extracts were qualified as “high” when IC50 values were below 1 ␮g/ml, “good” when IC50 values were between 1 and 10 ␮g/ml and “moderate” when IC50 values were above 10 ␮g/ml. The IC50 values of the most active extracts were further determined on Plasmodium falciparum FcB1 strain and on human HeLa and MRC5 cells, and the selectivity indexes (SI) were determined. SI is defined as the ratio of the cytotoxicity on the human cells to the antiplasmodial activity. Those of them that showed high selectivity should offer the potential of safer therapy. All extraction and bioassay results were reported in Table 2. First, our investigation confirmed the antiplasmodial activity of plants already described in the literature by evaluation on different strains or species of Plasmodium. Brucea javanica (Simaroubaceae, IC50 values ranging from 0.1 to 1.1 ␮g/ml) and Eurycoma longifolia (Simaroubaceae, IC50 values from 1.5 to 4.3 ␮g/ml) showed high antiplasmodial activity. This activity is mainly due to quassinoid diterpenoids and some triterpenoids (Kitagawa et al., 1994; Jiwajinda et al., 2002; Kuo et al., 2004). Dichroa febrifuga (Saxifragaceae), the main constituent of the antimalarial chinese remedy, Changshan and containing febrifugine and analogues with high antiplasmodial activity (Kikuchi et al., 2002; Lei and Bodeker, 2004; Jiang et al., 2005) showed a moderate antiparasite activity in our study (IC50 = 13.6 ␮g/ml) as for Sida acuta Malvaceae (Karou et al., 2003), Bixa orellana Bixaceae (Baelmans et al., 2000), Annona squamosa Annonaceae (El Tahir et al., 1999), Annona muricata Annonaceae (Bidla et al., 2004) and Tinospora crispa (Rahman et al., 1999) that have already been identified as antimalarial plants. Among the 38 other plants with antiplasmodial activity, the two Menispermaceae Arcangelisia flava and Fibraurea tinctoria showed the highest activities (IC50 values ranging from 0.4 to 1.1 ␮g/ml) associated with the highest selectivity index (SI up to 325 for the Fibraurea tinctoria stem methanol extract). Arcangelisia flava is a liana with a yellow wood found through out South-East Asia, from China to New Guinea, and broadly used to treat infections. For example, in Vietnam, it is used against malaria and undifferentiated fever. In the Malaysian peninsula, the stem decoction is used against jaundice, worms and intestinal problems and the inhalation of the wood smoke is used to treat mucous membrane infections. In Philippines, this plant is known as antiseptic, the wood and stem decoctions are used to treat wounds, ulcer, skin irritation, mouth diseases and fever and have tonic, emmenagogue and abortive properties (Padua et al., 1999). Fibraurea tinctoria is also a yellow woody climber which looks like Arcangelisia flava. It grows wild in China, Malaysia, Laos, Cambodia and Vietnam. In Vietnam, the stem and the roots are used to treat malaria, fever, diarrhoea, bacillary dysentery, enteritis and dyspepsia in the form of decoction, powder or pills. It is also used in Malaysia against malaria.

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Table 2 Extraction and in vitro bioassays on Plasmodium falciparum and human cells of the selected plants Selection Scientific name

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

JN50

R

E C D M

8.9 1.7 0.5 58.7

20 39 0 10

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN91

B

E C D T

7.3 4.6 0.7 60.5

7 20 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN53

L

E C D M

19.7 9.7 0.7 51.5

28 64 25 38

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN54

L

E C D M

19.1 3.3 0.5 58.5

20 69 33 34

NT 19.9 ± 2.7 NT NT

– 10.0 – –

– NT – –

– 0.5 – –

– – – –

JN60

B

E C D M

11.9 3.8 0.4 82.6

5 56 3 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN60

L

E C D M

12.8 1.7 0.5 88.5

18 75 0 0

NT 12.5 ± 3.9 NT NT

– 7.8 – –

– NT – –

– 0.6 – –

– – – –

JN97

B

E C D M

15.1 4.5 2.8 84.3

46 92 98 0

20.4 ± 2.7 4.6 ± 0.4 3.3 ± 0.4 NT

NT 15.3 10.3 –

NT NT NT –

– 3.3 3.1 –

– – – –

JN92

B

E C D M

22.2 2.2 5.0 84.6

24 22 12 9

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN96

L

E C D M

21.6 12.3 2.9 57.9

25 55 51 0

62.0 ± 0.8 NT 17.3 ± 1.1 NT

40.0 – 17.5 –

NT – 32.2 –

0.7 – 1.0 –

– – 1.9 –

JN88

B

E C D M

16.0 3.1 11.7 78.6

3 44 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN59

R

E C D M

18.1 3.5 0.8 92.3

17 40 33 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

Amaranthaceae Achyranthes aspera L.

Anacardiaceae Spondias pinnata (Koenig et L.f.) Kurtz

Annonaceae Annona muricata L.

Annona squamosa L.

Cananga odorata (Lam.) Hook.f. et Thoms Cananga odorata (Lam.) Hook.f. et Thoms

Xylopia vielana Pierre ex Fin. et Gagnep.

Apocynaceae Thevetia peruviana (Pers.) K. Schum.

Wrightia dubia (Sims.) Spreng. syn.: Wrightia rubiflora Pit.

Plumeria rubra L.

Arecaceae Calamus salicifolius Becc.

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Table 2 (Continued ) Selection Scientific name

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

JN56

L

E C D M

22.1 3.1 0.7 76.9

29 40 14 71

NT NT NT 13.9 ± 0.6

– – – 34.2

– – – NT

– – – 2.5

– – – –

JN62

L

E C D M

9.5 16.3 10.7 78.3

15 35 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN80

L

E C D M

17.0 6.0 2.2 68.1

6 56 0 7

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

n.d.

L

E C D M

17.6 8.8 3.7 68.4

20 35 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN68

L

E C D M

13.9 7.0 1.6 85.1

4 53 6 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN69

B

E C D M

10.2 1.8 0.4 88.1

70 48 7 97

5.1 ± 0.2 NT NT 4.3 ± 0.4

18.0 – – 20.3

NT – – 25.6

3.5 – – 4.7

– – – 5.9

JN79

S

E C D M

7.6 4.8 1.3 72.7

11 50 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN87

W

E C D M

11.4 14.9 2.5 73.6

19 39 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN58

L

E C D M

16.4 11.9 9.3 36.2

21 62 19 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN63

W

E C D M

11.8 2.2 0.3 77.6

1 69 0 0

NT 10.8 ± 1.1 NT NT

– 9.9 – –

– 12.9 – –

– 0.9 – –

– 1.2 – –

JN89

L

E C D M

16.7 14.5 29.0 36.5

5 35 3 18

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

Bixaceae Bixa orellana L.

Bombacaceae Ceiba pentandra (L.) Gaertn.

Convolvulaceae Ipomoea pes-caprae (L.) Sweet

Cucurbitaceae Momordica cochinchinensis (Lour.) Spreng Dilleniaceae Dillenia indica L.

Elaeocarpaceae Elaeocarpus kontumensis Gagn.

Euphorbiaceae Homonoia riparia Lour.

Phyllanthus amarus L.

Fabaceae caesalpinoideae Caesalpinia pulcherrima (L.) Swartz.

Christia vespertilionis (L.f.) Bekh.f.

Fabaceae papilionoideae Pterocarpus macrocarpus Kurz.

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Table 2 (Continued ) Selection Scientific name

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

JN82

B

E C D M

10.2 19.1 1.5 60.9

25 93 60 0

NT 5.5 ± 0.5 6.2 ± 1.1 NT

– 8.5 18.5 –

– NT NT –

– 1.5 3.0 –

– – – –

JN82

P

E C D M

7.0 4.1 1.1 51.4

19 63 35 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN82

Se

E C D M

25.3 49.7 0.2 26.7

10 29 7 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN71

L

E C D M

19.6 8.3 1.1 79.7

3 52 18 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN75

S

E C D M

9.4 1.0 0.4 65.5

16 48 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN76

S

E C D M

3.9 7.2 1.9 69.4

17 61 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN81

L

E C D M

24.5 4.6 3.5 78.5

40 40 0 95

10.5 ± 1.0 NT NT 5.0 ± 0.7

11.7 – – 14.8

NT – – 50.5

1.1 – – 2.9

– – – 10.0

JN64

L

E C D M

7.1 24.0 16.8 43.3

23 39 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN55

B

E C D M

16.8 1.6 1.0 78.1

0 29 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN90

W

E C D M

5.1 11.0 0.9 78.9

26 42 76 3

NT NT 7.5 ± 1.1 NT

– – 29.7 –

– – 19.4 –

– – 3.9 –

– – 2.6 –

JN94

W

E C D M

11.4 14.4 2.1 41.9

18 78 42 0

NT 14.1 ± 5.9 NT NT

– 6.5 – –

– NT – –

– 0.5 – –

– – – –

Milletia diptera Gagn.

Milletia diptera Gagn.

Milletia diptera Gagn.

Gentianaceae Fagraea fragrans Roxb.

Gnetaceae Gnetum sp.

Gnetum sp.

Irvingiaceae Irvingia malayana Oliv. ex Benn.

Lauraceae Cinnamomum iners Reinn.

Lecythidaceae Barringtonia acutangula (L.) Gaertn.

Malvaceae Sida acuta Burm.f.

Urena lobata L.

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Table 2 (Continued ) Selection Scientific name

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

JN66

S

JN72

R

E C D M E C D M

8.4 3.6 2.2 73.3 13.0 1.2 18.6 74.5

94 95 98 98 94 37 96 97

0.7 ± 0.1 1.1 ± 0.1 0.4 ± 0.0 0.9 ± 0.1 1.0 ± 0.2 NT 0.7 ± 0.1 1.1 ± 0.1

21.3 NT 8.8 40.7 NT – NT NT

155.0 NT 56.5 147.7 NT – NT NT

29.6 – 25.0 44.7 – – – –

215.3 – 161.4 162.3 – – – –

JN72

S

E C D M

5.2 7.6 14.9 59.7

94 54 97 97

1.1 ± 0.2 NT 0.5 ± 0.1 1.0 ± 0.3

70.6 – 53.4 99.9

99.8 – 29.0 335.3

63.0 – 100.8 97.0

89.1 – 54.6 325.5

n.d.

S

E C D M

5.7 17.9 10.0 63.6

30 55 26 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN73

L

E C D M

4.7 17.5 3.9 58.8

35 75 51 13

NT 15.9 ± 0.9 5.8 ± 1.2 NT

– NT 14.9 –

– NT NT –

– 2.6 –

– – – –

JN74

L

E C D M

3.8 21.3 0.6 29.8

10 34 0 16

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN84

L

E C D M

18.1 5.3 2.4 71.4

26 47 0 14

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN61

L

E C D M

15.4 3.8 1.1 66.0

0 47 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN85

B

E C D T

37.4 0.2 0.1 95.0

13 54 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN86

B

E C D M

12.9 4.9 1.5 83.2

2 29 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN86

L

E C D M

17.3 8.5 1.8 83.6

2 43 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN51

L

E C D M

18.7 14.1 1.0 70.2

8 61 18 13

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

Menispermaceae Arcangelisia flava (L.) Merr. Fibraurea tinctoria Lour. syn. Fibraurea chloroleuca Miers. Fibraurea tinctoria Lour. syn. Fibraurea chloroleuca Miers.

Tinospora crispa Hook.f. Thomson

Moraceae Ficus hispida L.f.

Ficus racemosa L.

Morus alba L.

Rhizophoraceae Carallia brachiata (Lour.) Merr.

Rubiaceae Nauclea officinalis (Pierre ex Pit.) Merr.

Neolamarckia cadamba (Roxb.) Bosser

Neolamarckia cadamba (Roxb.) Bosser

Rutaceae Feroniella lucida (Schaff.) Sw.

J. Nguyen-Pouplin et al. / Journal of Ethnopharmacology 109 (2007) 417–427

425

Table 2 (Continued ) Selection Scientific name

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

n.d.

R

E C D M

4.5 6.0 9.2 75.8

18 61 74 0

NT NT 13.6 ± 1.7 NT

– 34.2 33.3 –

– – NT –

– – 2.5 –

– – – –

JN57

R

E C D M

7.2 6.0 9.5 73.6

95 98 99 94

0.6 ± 0.1 1.1 ± 0.2 0.1 ± 0.0 1.1 ± 0.2

NT NT NT NT

NT NT NT NT

– – – –

– – – –

n.d.

B

E C D M

5.1 18.8 3.2 75.2

80 76 96 0

7.9 ± 1.3 10.0 ± 2.9 1.5 ± 0.4 37.5 ± 3.9

17.4 3.5 2.1 NT

21.6 7.4 2.5 NT

2.2 0.4 1.5 –

2.7 0.8 1.7 –

JN70

L

E C D M

19.2 11.8 6.4 37.5

86 65 93 99

4.3 ± 0.5 NT 4.1 ± 0.4 3.9 ± 0.4

NT – 11.2 31.4

NT – 27.8 43.1

– – 2.7 8.1

– – 6.7 11.1

JN78

L

E C D M

18.7 7.3 1.3 72.5

45 94 0 94

9.4 ± 1.1 6.7 ± 0.8 NT 5.1 ± 0.1

8.7 3.9 – 14.3

NT 8.4 – 56.0

0.9 0.6 – 2.8

– 1.2 – 12.5

JN98

R

E C D M

15.9 1.5 2.3 88.7

13 32 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN52

L

E C D T

26.4 8.2 9.3 46.9

23 68 50 21

NT 15.3 ± 0.8 8.6 ± 1.4 NT

– NT 23.4 –

– NT 69.5 –

– 2.7 –

– – 8.0 –

Saxifragaceae Dichroa febrifuga Lour.

Simaroubaceae Brucea javanica (L.) Merr.

Eurycoma longifolia Jack syn. Eurycoma apiculata A.W. Bennett Eurycoma longifolia Jack syn. Eurycoma apiculata A.W. Bennett

Harrisonia perforata (Blanco) Merr.

Taccaceae Tacca chantrieri Andr´e

Theaceae Anneslea fragrans Wall.

Tiliaceae JN77

L

E C D M

13.2 8.3 10.5 51.5

5 61 0 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN65

L

E C D M

27.0 3.2 0.6 91.5

10 23 0 14

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

JN65

R

E C D M

12.0 6.7 5.4 72.4

20 75 38 0

NT 10.0 ± 1.5 NT NT

– 21.0 – –

– NT – –

– 2.1 – –

– – – –

JN65

S

E C D M

8.2 4.7 1.8 76.9

19 29 34 0

NT NT NT NT

– – – –

– – – –

– – – –

– – – –

Grewia paniculata Roxb.

Verbenaceae Clerodendrum inerme (L.) Gaertn.

Clerodendrum inerme (L.) Gaertn.

Clerodendrum inerme (L.) Gaertn.

426

J. Nguyen-Pouplin et al. / Journal of Ethnopharmacology 109 (2007) 417–427

Table 2 (Continued ) Selection Scientific name

Vitex negundo L.

a b c d e f

Extraction

Antiplasmodial activitya , Plasmodium falciparum FcB1

Cytotoxicitya , IC50 (␮g/ml)

Selectivity index (SI)b

V.N.c

Partd

Extractione

Yield (%)f

Inhibition % 10 ␮g/ml

IC50 (␮g/ml)

Hela cells

MRC5 cells

Hela cells

MRC5 cells

JN95

L

E C D M

21.2 1.8 0.4 94.5

9 67 35 0

NT 17.3 ± 3.2 NT NT

– NT – –

– NT – –

– – – –

– – – –

NT, not tested. Selectivity index (SI), ratio of cytotoxicity on Hela or MRC5 cells to antiplasmodial activity against FcB1 strain of Plasmodium falciparum. V.N., voucher number; n.d., voucher not done. B, bark; L, leaves; R, roots; S, stem; Se, seeds; W, whole plant. C, cyclohexane extract; D, methylene chloride extract; E, crude extract; M, methanol extract; T, methanol extract after tannins removal. Extract yield %: E extract yield percentage is regarding the dry powdered plant; C, D and M extract percentages are regarding E extract.

These two plants from the Menispermaceae family were reported to contain protoberberine alkaloids as berberine, palmatine, jatrorrhizine and columbamine (Barbosa-Filho et al., 2000). The biological properties of protoberberine-like components have been abundantly studied. Particularly, they possess strong antiplasmodial activity (Iwasa et al., 1998) and inhibit Plasmodium falciparum telomerase activity (Sriwilaijareon et al., 2002) that could explain the antiplasmodial activity we observed from these two plants. Good antiplasmodial activity (IC50 ranging from 3.3 to 8.6 ␮g/ml) was also observed for Xylopia vielana (Annonaceae), Elaeocarpus kontumensis (Elaeocarpaceae), Irvingia malayana (Irvingiaceae), Harrisonia perforata (Simaroubaceae), Ficus hispida (Moraceae), Milletia diptera (Fabaceae) and Anneslea fragrans (Theaceae), however, with lower SI values (up to 12.5 for the Harrisonia perforata methanol leaf extract). The cyclohexane extracts of Clerodendrum inerme (Verbenaceae), Christia vespertilionis (Fabaceae), Cananga odorata (Annonaceae), Urena lobata (Malvaceae), Wrightia dubia (Apocynaceae) and Vitex negundo (Verbenaceae), also exhibited antiplasmodial activity with IC50 values ranging from 10 to 20 ␮g/ml. However, they generally showed high cytotoxicity upon mammalian cells (SI < 2). Four plants which have not be previously studied for their antimalarial properties, showed interesting activity. Irvingia malayana (IC50 = 5 ␮g/ml on Plasmodium falciparum and SI = 10 on MRC5 cells for the leaf methanol extract) is a high tree (15–20 m) widespread in South-East Asia. It is a hardwood widely used in the construction industry and the nut which is rich in oil is used in cooking. We believe this is the first time that antimalarial activity was reported for this plant. Another specie, Irvingia gabonensis (Aubry-Lecomte ex O’rorke) Baill., also showed definite but weaker antiplasmodial activity (IC50 of 21.6 ␮g/ml) against the same Plasmodium falciparum FcB1 strain (Zihiri et al., 2005). Irvingia malayana has been recently referenced in the International Union for the Conservation of Nature red list of threatened species (World Conservation Monitoring Centre, 1998), and this plant clearly requires further investigation to evaluate its medical activity and to ensure its preservation.

Harrisonia perforata, a small tree, was promising with an IC50 value of 5.1 ␮g/ml on Plasmodium falciparum and SI = 12.5 on the MRC5 cells for the leaf methanol extract. Limonoids, quassinoids and chromones were isolated from its leaves and its bark (Tanaka et al., 1995; Kamiuchi et al., 1996; Khuong-Huu et al., 2001). These classes of molecules possess high antimalarial activity (Murakami et al., 2003; Krief et al., 2004). Elaeocarpus kontumensis (IC50 of 4.3 ␮g/ml on Plasmodium falciparum and SI = 5.9 on MRC5 cells for the leaf methanol extract) is a shrub or small tree common in secondary forest. No biological properties were reported in the literature for this species although activities were described for other species of Elaeocarpus, e.g. antimicrobial, antiinflammatory properties (asthma, arthritis, liver diseases). Chemically, this genus showed the presence of glycosides, alkaloids, flavonoids and steroids (Singh et al., 2000). Anneslea fragrans (IC50 of 8.6 ␮g/ml on Plasmodium falciparum and SI = 8 on MRC5 cells for the leaf methylene chloride extract) is a big tree and the leaves have been used to treat fever in Cambodia and the bark used in dysentery and diarrhoea in China and Cambodia, as well as in liver inflammation in Vietnam (Lemmens and Bunyapraphatsara, 2003). We believe that this is the first time that an antimalarial activity of this plant has been reported. In conclusion, our ethnopharmacological investigation of plants from southern Vietnam allowed the identification of several plants with antimalarial activity. Some such as Brucea javanica and Eurycoma longifolia have already been described in the litterature and our study confirmed these observations. Furthermore, our study identified six plants with possible novel antimalarial compounds: two Menispermaceae, Arcangelisia flava (L.) Merr. and Fibraurea tinctoria Lour., Harrisonia perforata (Blanco) Merr., Irvingia malayana Oliv. ex Benn., Elaeocarpus kontumensis Gagn. and Anneslea fragrans Wall. These plants have not been previously studied for their antiplasmodial activity and yet showed selectivity for Plasmodium falciparum when tested on mammalian cells. Their investigation by bioguided fractionation is in progess to identify the active antimalarial compounds.

J. Nguyen-Pouplin et al. / Journal of Ethnopharmacology 109 (2007) 417–427

Acknowledgments The authors would like to thank the members of the Laboratory of Pharmacognosy in Strasbourg for their warm support and help in the crude extract fractionation, in particular R. Anton, A. Lobstein and B. Weniger. Special thanks to Marcel Hibert, the director of the French Chemical and Plant Extract National Library. Warm thanks also to S. Hul and F. Jacques of MNHN to have shared their plant knowledge and to have kindly performed and confirmed the botanical determinations. Plant collection was kindly supported by IDVP. J. Nguyen-Pouplin was supported by the French Ministry of Education and Research. Some of this work was funded by the Wellcome Trust UK. JF is a Senior Wellcome Trust Research Fellow. References Baelmans, R., Deharo, E., Bourdy, G., Mu˜noz, V., Quenevo, C., Sauvain, M., Ginsburg, H., 2000. A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part IV. Is a new haem polymerisation inhibition test pertinent for the detection of antimalarial natural products? Journal of Ethnopharmacology 73, 271–275. Barbosa-Filho, J.M., Leit˜ao da-Cunha, E.V., Gray, A.I., 2000. Alkaloids of Menispermaceae. The Alkaloids: Chemistry and Biology 54, 1–190. Bidla, G., Titanji, V., Joko, B., Ghazali, G., Bolad, A., Berzins, K., 2004. Antiplasmodial activity of seven plants used in African folk medicine. Indian Journal of Pharmacology 36, 245–246. Desjardins, R.E., Candfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity by a semi-automated microdilution technique. Antimicrobial Agent and Chemotherapy 16, 710–718. El Tahir, A., Satti, G.M.H., Khalid, S.A., 1999. Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Journal of Ethnopharmacology 64, 227–233. Frappier, F., Jossang, A., Soudon, J., Calvo, F., Rasoanaivo, P., RastimamangaUrveg, S., Saez, J., Schrevel, J., Grellier, P., 1996. Bisbenzylisoquinoline as modulators of chloroquine resistance in Plasmodium falciparum and multidrug resistance in tumor cells. Antimicrobial Agent and Chemotherapy 6, 1476–1481. Hoang, B.C., Pho, D.T., Huu, N., 1999. Overview of Vietnamese traditional medicine. In: Mai, Q.L., Tran, D.L., Ngo, K.X., Tran, H.M. (Eds.), Vietnamese Traditional Medicine. The Gioi Publishers, Hanoi, pp. 1–28. Iwasa, K., Kim, H.-S., Wataya, Y., Dong-Ung, L., 1998. Antimalarial activity and structure–activity relationships of protoberberine alkaloids. European Journal of Medicinal Chemistry 33, 65–69. Jiang, S., Zeng, Q., Gettayacamin, M., Tungtaeng, A., Wannaying, S., Lim, A., Hansukjariya, P., Okunji, C.O., Zhu, S., Fang, D., 2005. Antimalarial activities and therapeutic properties of febrifugine analogs. Antimicrobial Agents and Chemotherapy 49, 1169–1176. Jiwajinda, S., Santisopasri, V., Murakami, A., Kawanaka, M., Kawanaka, H., Gasquet, M., Elias, R., Balansard, G., Ohigashi, H., 2002. In vitro anti-tumor promoting and anti-parasitic activities of the quassinoids from Eurycoma longifolia, a medicinal plant in Southeast Asia. Journal of Ethnopharmacology 82, 55–58. Kamiuchi, K., Mitsunaga, K., Koike, K., Ouyang, Y., Ohmoto, T., Nikaido, T., 1996. Quassinoids and limonoids from Harrisonia perforata. Heterocycles 43, 653–664. Karou, D., Dicko, M.H., Sanon, S., Simpore, J., Traore, A.S., 2003. Antimalarial activity of Sida acuta Burm.f. (Malvaceae) and Pterocarpus erinaceus Poir. (Fabaceae). Journal of Ethnopharmacology 89, 291–294.

427

Khuong-Huu, Q., Chiaroni, A., Riche, C., Nguyen-Ngoc, H., Nguyen-Viet, K., Khuong-Huu, F., 2001. New rearranged limonoids from Harrisonia perforata III. Journal of Natural Products 64, 634–637. Kikuchi, H., Tasaka, H., Hirai, S., Takaya, Y., Iwabuchi, Y., Ooi, H., Hatakeyama, S., Kim, H.S., Wataya, Y., Oshima, Y., 2002. Potent antimalarial febrifugine analogues against the Plasmodium malaria parasite. Journal of Medicinal Chemistry 45, 2563–2570. Kitagawa, I., Mahmud, T., Simanjuntak, P., Hori, K., Uji, T., Shibuya, H., 1994. Indonesian medicinal plants. VIII. Chemical structures of three new triterpenoids, bruceajavanin A, dihydrobruceajavanin A, and bruceajavanin B, and a new alkaloidal glycoside, bruceacanthinoside, from the stems of Brucea javanica (Simaroubaceae). Chemical and Pharmaceutical Bulletin 42, 1416–1421. Krief, S., Martin, M.T., Grellier, P., Kasenene, J., Sevenet, T., 2004. Novel antimalarial compounds isolated in a survey of self-medicative behavior of wild chimpanzees in Uganda. Antimicrobial Agents and Chemotherapy 48, 3196–3199. Kuo, P.-C., Damu, A.G., Lee, K.-H., Wu, T.-S., 2004. Cytotoxic and antimalarial constituents from the roots of Eurycoma longifolia. Bioorganic & Medicinal Chemistry 12, 537–544. Lambros, C., Vanderberg, I., 1979. Synchronisation of P. falciparum erythrocytic stages in culture. The Journal of Parasitology 65, 418–420. Lei, S.H.-L., Bodeker, G., 2004. Changshan (Dichroa febrifuga)—ancient febrifuge and modern antimalarial: lessons for research from a forgotten tale. In: Willcox, M., Bodeker, G., Rasoanaivo, P. (Eds.), Traditional Medicinal Plants and Malaria. CRC Press LLC, Boca Raton, pp. 61–82. Lemmens, R.H.M.J., Bunyapraphatsara, N., 2003. Plant Resources of SouthEast Asia. No. 12(3). Medicinal and Poisonous Plants 3. Backhuys Publishers, Leiden, p. 664. Mossman, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. Journal of Immunological Methods 65, 55–63. Murakami, N., Sugimoto, M., Kawanishi, M., Tamura, S., Kim, H.S., Begum, K., Wataya, Y., Kobayashi, M., 2003. New semisynthetic quassinoids with in vivo antimalarial activity. Journal of Medicinal Chemistry 46, 638–641. Nghiem, V.D., 2002. Indigenous peoples in Vietnam and their knowledge of biodiversity in traditional health care. Biodiversity 3, 22–23. Padua, L.S., Bunyapraphatsara, N., Lemmens, R.H.M.J., 1999. Plant Resources of South-East Asia. No. 12(1). Medicinal and Poisonous Plants 1. Backuys Publishers, Leiden, p. 711. Rahman, N.N.N.A., Furuta, T., Kojima, S., Takane, k., Mohd, M.A., 1999. Antimalarial activity of extracts of Malaysian medicinal plants. Journal of Ethnopharmacology 64, 249–254. Singh, R.K., Acharya, S.B., Bhattacharya, S.K., 2000. Pharmacological activity of Elaeocarpus sphaericus. Phytotherapy Research 14, 36–39. Sriwilaijareon, N., Petmitr, S., Mutirangura, A., Ponglikitmongkol, M., Wilairat, P., 2002. Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine. Parasitology International 51, 99–103. Tanaka, T., Koike, K., Mitsunaga, K., Narita, K., Takano, S., Kamioka, A., Sase, E., Ouyang, Y., Ohmoto, T., 1995. Chromones from Harrisonia perforata. Phytochemistry 40, 1787–1790. World Conservation Monitoring Centre, 1998. Irvingia malayana. In: IUCN 2004. 2004 IUCN Red List of Threatened Species. Available at: http://www.redlist.org (downloaded on 28 November 2005). World Health Organisation, 2001. Informal Consultation on Monitoring Resistance to Antimalarial Drugs in the Mekong Region (Cambodia, China, Lao People’s Democratic Republic, Myanmar, Thailand, Vietnam). World Health Organization Regional Office for the Western Pacific, Phnom Penh, p. 52. Zihiri, G.N., Mambu, L., Gu´ed´e-Guina, F., Bodo, B., Grellier, P., 2005. In vitro antiplasmodial activity and cytotoxicity of 33 west African plants used for treatment of malaria. Journal of Ethnopharmacology 98, 281–285.