23. Nematocidal Effects of Mexican-Medicinal Plant Extracts (Research note)

Jun Maki'. Fumiko A b e \o Yamauchi". Hikaru Okabe', Ricardo Reyes-Chilpa , Manuel Jiménez Estrada \

Miguel A. Martínez Alfaro', Abigail Aguüar C \& Hiroshige Akahane^

1) Kitasato university School of Medicine 2 ) Faculty of Pharmaceutical Sciences. Fukuoka University 3 ) Institute of Chemistry, National University of México 4) Department of Botany, National University of México 5) I. M. S. S. 6 ) Faculty of Medicine. Fukuoka University

ABSTRACT In vitro effects of the Mexican-medicinal plant ext rac ts on a nematode ,

Angiostrongylus cantonensis iAc) were tested and discussed from the viewpoints of local public health and social pharmacy. Immobilizing efficacy of the extracts on the fírst-stage lar\'ae (Ll) of Ac was studied in the test tubes. As a result, some of them were found effective, suggesting that the local plants such as Hura polyandra míght be utilized for the sterilization of nematodes in or on the soil of the local área with parasitic endemicity.

IP4TRODUCTION The present authors have been interested in the discipline of ¡niernational

medical cooperation programs for underdeveloped áreas on the earth. Such cooperation programs are supported and carried out by governmental (for instance. so-called JICA or Japan International Cooperation Agency), nongovern mental or other international (for example WHO) inslitutions. Or sometimes it wili be practised based on a cooperative work between universities like this project study.

We have visited some local áreas with endemic diseases in tropical and subtropical countries. Staying there, we have put plans into practice for the control of obstínate

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— m — parasitic diseases.

Human on earth are mostly afflicted with parasitic infections stiü today. Among scientists specializing in tropical medicine, the parasitoses of malaria, trypanosomiasis, leishmaniasis, filariasis and schistosomiasis are usuaily recognized as the 5 important parasitic diseases in the global sense. The important parasitoses above mentioned are all rampant in Latin American áreas such as México, Central American and Caribbean countries. They are transmitted via insects or snails (schistosomiasis).

Of no less importance of public health are the parasitic nematodes in or on the soil, and on vegetables, being mostly of ubiquitous distribution in tropical, subtropical and températe zones. People have been infected with them and afflicted in their daily life. The daily sterilization of their infective forms has been hard in the local áreas. This has been so, partly because effective chemicals are expensive for the local standard. Other factors making the sterilization virtually impossible are difficulties in the transportaticm of the sterilizer and the risk of the pollution of the environment with the chemicals even if they can afford to purchase them. This seems to be where scientific utilization of local medicinal plants would hopefully come in.

The project study has been carried out on the test of medicinal plant extracts on Trypanosoma cruii, causative agent of American trypanosomiasis. Incidental to this, the present cooperative work was undertaken in an attempt to clarify the possible efficacy of the extracts and preparations from Mexican plants against nematodes irrespect ive of their effects on protozoas. For the model sys tem, a nematode, Angiostrongylus cantonensis was chosen in this study.

MATERIALS AND METHODS All the plants used were collected or purchased in México. The extractions

were prepared from the plants in National University of México (UNAM). Their activity against T.cruzi was tested in Japan (Fukuoka University) in the project study (Abe et a l . , in press) . Detailed preparation method for the extractions is described in the report on T.cruzi by Abe et al. (in p ress ) . These extracts were sent to Kitasato University School of Medicine for the test in their activity against a parasitic nematode, A.cantonensis. The extracts that could be solubilized in water were tested in their activity.

In the laboratory in Kitasato University School of Medicine, the life cycle of the nematode has been maintained using rats as a final host and an experimental intermedíate host, Biomphalaria glabrata. The snail was exposed to 1,200 Ist-stage larvae/snail, ovemight at room temperature (about 25c). They were then maintained in an aquarium about 2 months, fed on iettuce, to raise the 3rd-stage larvae. These larvae were isolated from the infected snails by peptic digestión [j%(w/v)peps¡n in 1 %(v/v)HCl] and were given orally by the aid of a stomach tube to a rat weighing about 250 g. About 2 months after the oral inoculation, the Ist-stage larvae were collected from the faeces of the infected rats.

One mi of the suspensión of the fírst-stage larvae (1000 iarvae/ml) and 1 mi of the extract (1 rag/mi) were mixed in a lOml tube and put into an incubator at 28*0.

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Nematocidal Effects of Mexicao-Medicinal Plant E x t r a c t s (Research note) (Jun Maki, e t a l . ) — 1 9 5 —

After the incubation for 4 hrs-3 days, the numbers of moving and immobUized larvae were counted under a light microscope. Controls free from the extracts were run simultaneously.

Five mi of saline containing one adult worm and 5ml of the extract (0.1 mg/ mi saline) were put into a tube. The control was free from the extract. The tubes were kept at 37C for 18hrs.

RESULTS AND DISCUSSION No complete efficacy was observed except a small number of the extracts, for

instance that from Hura polyandra (the family ñame of Euphorbiaceae) as shown in TABLE 1. This was expected to have component(s) with somewhat effectiveness. The possible activity might also be attributable to the extracts from other plants, C.argentea, H.palens, P.aureum and Senna s p . .

In addition to the test against Ll , the possible effect of the drugs against adult A.cantonensis was also examined (TABLE 2). No efficacy was seen, however.

In the nature, the life cycle of A.cantonensis is usuaily maintained with rats as a final host and snails as an intermedíate host. Humans are infected directly with the infective-stage larvae (the 3rd-stage larvae) harboured in the intermedíate hosts. Needless to say, we have to avoid the contact with such snails, which should be eliminated or killed safely. At the same time it is advisable to disinfect the Ll on the earth and in the fresh water successfully because with Ll being killed, the cycle is interrupted leading to the control of Axantonensis that has been maintained in the nature.

Larval A.cantoensis in mammalians are successfully eliminated with benzimidazoles (Maki & Yanagisawa, 1986; Hwang & Chen. 1988). These are the 2nd-5th stage larvae. No study has been done on drug effects against the Ll to the present athors knowledge, The present study cíearly demonstrated that at least a small number of the plant extracts are effective in immobilizing the L l . In spite of the preliminary data, the present result appears to be encouraging for the possible sterilization of the Ll in local endemic áreas.

The crude extracts with the efficacy are hopefully used as nematocidal agents. Cmde dmgs are readily available in such an área even with inconvieniCTces in tran^rtat ion. It does not seem that the inhabitants have any problems in the pnces and transportation of their locally harvested herbs so far as they are sold at markets. So, utilization of crude drugs for killing Ll would be practicable in local endemic áreas.

Thus, we are now interested in the efficacy from a viewpoint of public health and social pharmacy, hoping that the plant extracts in rural áreas might possibly kill other kinds of nematodes in the soil and water. A sound conclusión on this point must await further studies based on the data in laboratory.

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ACKNOWLEDGEMENTS This research is fínancially supported mainiy by Fukuoka University the fund

(No. 001001) from the Central Research Institute, 2000-2001 and partly by Kitasato University. Dr. Masahiro Kuwada, Kitasato University School of Medicine is gratefully acknowledged for the cooperation for the present study.

7'-̂ í* TABLE 1. Effect of the extracts on the motility of {.-.y, : the Ist-stage larvae (A.cantonensis 1000 larvae/ml)

Extract Moving larvae (%) days after the exposure

1 3 days

Urtica dioica 17 4 Hamelia patens 4 0 Cecropia obtusifolia 23 4 Glirícidin sepium 38 56 Phleboidum aureum 10 0 Senna sp 12 0 Zomia sp 32 11 Hura polyandra 0 0 Eysenhartia polystachia 82 88 Pouteria zapata 28 25 Celosia argéntea 9 0 Mirabais jalap^^ 18 5 Mirabilis jalapa" 42 5 Lippia dulcís 47 12 Portulacca oleraceae 28 33 Control 100 55

l)ieaves 2 ) F o o t s

Guareque (60%MeOH)' 3 GuarequefMeOH-J)** 21 Control SO

3) & 4) are additional data. For the preparation method, see the report by Abe et al. (in press).

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Nematocidal Effects of Mexican-Medic ina l Plant E x t r a c t s (Fesearcb note) (Jun Maki . e t a l . J — 1 9 7 —

TABLE 2. Effect of methanol extract ÍTMII some Mexican-ptant extracts on the adult Angiostrongylus cantonensis

RREFERENCES Abe, F. el al, (2002) in press. Maki & Yanagisawa (1986) Studies on anthemintic effects of flubendazole and mebendazole on the rat lungworm Angiostrongylus cantonensis in mice and rats . Journal of Parasitology, 72(2) 512-516. Kao-Pin Hwang & Eng-Rin Chen (1988) Larvicidal effect of albendazole against Angiostrongylus cantonensis in mice. American Journal of Tropical Medicine and Hygiene, 39(2)191-195.

Extract Immobilizing effect of the extract compared to the control

Sicana odorífera Uppia dulcis Haemaioxylom brasileío Eysenhartia polystachia

negative negative negative negative

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