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Des sources du savoir aux médicaments du futur

 | 
Jacques Fleurentin
, 
Jean-Marie Pelt
, 
Guy Mazars

2. Élaboration des pharmacopées

From ethnopharmacology to laboratory results: does discrepancy mean irrelevancy?

Michel Sauvain

Résumé

Abstract
Since 1992, our research team (a combination of bolivian and french researchers) is working on the problematic of the evaluation of the biological activity of plants used traditionally in different Bolivian ethnias (Chacobo, Mosetene, Alteños, Tacana). Species retained have been screened against malaria, in vitro and on the animal. More than 200 different species have been evaluated, and only a weak percentage did show a strong correlation between uses and biological results. These discrepancies can be explained by different considerations:
Firstly, the difficulties related with the interpretation of the traditional use of plants, generally presented as usefull for aleviate different symptoms, more or less related with a malarial attack. The criterium to select the plant has to be discussed. Also, different successive treatments are administered to the patient until he cures. Synergism effects could be responsible for the antimalarial activity and the method to study this type of effect has to be invented.
Then, the traditional way of using medicinal plant (preparation, administration) cannot always be taken into account in the screening test performed in the laboratory: therefore the in vivo inactivity of extracts may be correlated with the way of administration, having a strong perturbating effect on the absorption rate, delivery process, metabolisation process. The laboratory tests have difficulties to mesure the part of acquired human immunity, etc. The validity and accuracy of the antimalarial screening test aimed to evidenciate antimalarial agents from plant extract will be discussed.

Texte intégral

1Malaria ravages the tropical countries. This disease is the third cause of world mortality with two in three million victims a year essentially in Africa. Healing plants are generally the only weapons which possess the populations touched to fight against it, plant remedies being proposed mostly to reduce the dimension of the symptoms.

2From the works that IRD realize in Bolivia with his partners, we suggest the methodological tracks allowing to validate the efficiency of antimalarial remedies. This demonstration will lean on the pharmacological and clinical data of two natural antimalarial compounds, quinine and artemisinin.

Malaria in Bolivia

3Bolivia is situated in the center of the American south continent between 10 °and 23 ° of south latitude. The surface is 1.098.581 km2 (8.000.000 of inhabitants). The low regions of Bolivia are affected by malaria, the touched population having increased by 700% between 1981 and 1999. The number of cases listed a year is at present more than 70000 cases. This statistics covers only partially the reality in touched zones which remain difficult of access for health services.

4The three Amerindian groups with whom we worked live in representative zones of the three ecosystems where develops the malaria:

5Chacobo lives in Amazonian zone with a transmission of the malaria all year long, two species are involved: Plasmodium falciparum and P. vivax;

6Alteños lives in zones of dry valley (Cochabamba's Department), the transmission is made only during rainy season from December till March, 2 500 cases of malaria are put back a year in this region, all due to Plasmodium vivax;

7Mosetene lives on the oriental Andes characterized by a wet tropical vegetation of height, the transmission of the malaria is here intermediate among those met in Amazonia and in dry valleys.

Type of criteria to select supposed antimalarial medicinal plants

8We established three type of criteria to select plants. They are classified in descending order by importance:

9Malaria

10The notion of malaria was integrated recently into the traditional medicine of the groups studied. Health centers educate the populations at the risks of the malaria since about fifty years

11Fevers

12The fevers can be of different origins, the fact that healers are conscious of the regularity of the accesses and their seasonal appearance is an additional criterion of choice of plants for testing

13Infringements of the digestive system (vomits, hepatomegalia, etc. ) The chronic malaria pulls particular infringements of the digestive system among which the inflation of the liver or hepatomegalia which notes especially at the child's after an repeated exposure to the disease.

Pharmacological criteria of sorting active plants

14Two methods were used in these in vitro studies, the one economic is based on the use of two doses allowing to place the interest of the noticed activity, the other one requiring at least four increasing doses spaced out by a log of concentration allows to establish a IC50, finer criterion of observation of the activity. The choice of the concentrations of test is based on the in vitro activity of the alcoholic extracts of Cinchona (IC50 lower than one µg / ml) and of those of Artemisia annua (CI50 of the order of some µg / ml) and of that in vivo of the alcoholic extract of Cinchona (100 mg / kg / D during 4 Days). The activity is appreciated according to a scale described in the table 1.

Antimalarial activities of the plants selected from the pharmacopoeias of three Bolivian ethnic groups

15Chacobo (Muñoz and col., 2000)

16Four plants were found as antimalarial, two of them are active in vivo. Twelve plants are used against the symptoms of the malaria, 50% turned out active

17Mosetene (Muñoz and col., 2000)

18Two plants are presumed antimalarial, any is active in vivo. Fifteen plants are used against the symptoms of the malaria, 50% are active.

19Alteños (Muñoz et col., 2000)

20An antimalarial plant is inactive on the animal. On twenty plants used against the symptoms of the malaria, 50% are active.

21Healing plants for which we propose a follow-up study are active on the experimental malaria of the mouse. One can notice that about half of the plants used in three ethnic groups are active. But it's necessary to qualify the comment. Indeed if the choice limits itself to plants possessing activities comparable to those of reference extracts, only four Chacobo plants and a Mosetene plant can be selected for follow-up studies. Thoses plants represent 10% of the presumed antimalarial healing plants. The correlation between the activities measured into the animal and on the cellular models is indispensable for the pursuit of the works in particular for the isolation of one or several active principles. It is also an element allowing to distinguish a parasiticide activity the other activities as the immunomodulatory or febrifugal action.

Factors which can have an influence on the results

22The translation in modern medical terms of the traditional medicine is a preliminary stage indispensable to the selection of plants. The attentive study of the traditional medical use can give a supplementary chance to a plant activity which is not confirmed pharmacologicaly by current assays. For example, the high frequency of the report of a use can encourage to deepen the study of the antimalarial activity of a medicinal plant with adapted methods. Indeed, in our studies as in many of the others, extracts were standardized to allow a comparison of the activities among them. The other method would consist in reproducing the exact conditions of every traditional preparation to estimate the activity of it.

23In Bolivia, importance of the malaria is different from a region in the other one, the type of transmission varies also continuous or seasonal. The acquired immunity is consequently different from a population in the other one, from an age to the other one and the efficiency of plant remedies can so be variable. The pharmacological tests most usually used are not necessarily relevant to measure this modulation of the activity. Two examples will allow to illustrate the interest to follow the methodology of the traditional preparations:

Associations of plants and synergy of activity

24In the Mosetene pharmacopoeia , two plants are used in association against malaria: Tesseria integrifolia Ruiz and Pavon and Hymenachane donacifolia Beauv. In the preparation some termit eggs are added. Both plants tested separately in our assays present an antimalarial activity in different degrees. In what measure can this «combinatorial chemistry» improve the result of plants tested independently? What could be the effective experimental method which would allow to estimate these combinations?

Convergence of manners and modes of use

25Ceissospermum spp is a genus very used in Brazil Amazonian (Brandao and col., 1992), in the three Guyana (Grenand and col., 1987) and in Bolivia against malaria. It is so about a convergence of uses. The species of the genus Geissospermum live in the same ecosystem (trees of the primary forest) and are often confused by healers. We found the use as antimalarial to Chacobos (Geissospermum leave (Vell.) Miers) and we tested it. It turned out active in vivo but also toxic. This genus was studied chemically.

26Two types of compounds were described:

  • dimeric indol alkaloids of geissospermine type which are known for their toxicity to which one can attribute in any hypothesis to the mortality of animals;
  • indol monomeric alkaloids like flavopereirin among whom the reduced by-product of which was shown active in vitro on Plasmodium falciparum (Wright and col., 1996). The main chemical characteristic of this last compound is the presence of a quaternary ammonium which assures its preferential extraction in aqueous medium. One could so resume the study of the antimalarial activity of the genus by testing the activity of the decoction or the alcoholic extract taken by oral way, traditional preparations described in pharmacopoeias. If these preparations turn out more active than toxic, it will be also interesting to measure flavopereirin content in these last ones.

The raw extract of Cinchona bark is pharmacologically and clinically bioactive

27The quinine is always the major antimalarial compound prescribed in the most grave cases of malaria resisting to other antimalarials. The ethanolic extract of Cinchona where from is extracted quinine, has a IC50 of 0,2 μg / ml on in vitro Plasmodium falciparum model. The effective dose 50 of the raw extract on mice infected by Plasmodium berghei is 100 mg/kg/J for 4 days (test of Peters).

28The concentration of quinine is 5% in the barks of wild Cinchona (Cinchona calisaya Wedd.). The extraction by warm water of the Cinchona barks gives a return in quinine of 3% and the extraction by ethanol has a return on 5%.

29The classic treatment of the malaria attack is the grip of 1,5 grams of quinine a day for 7 days. The absorption of the decoction of 50 grams of Cinchona bark in a half-liter of water during a day corresponds to the necessary dose to cure the disease.

30All the ethnopharmacological data are so gathered to assert that the oral administration of a medicinal preparation of barks of Cinchona should be able to cure an malarial access in the same way as a treatment with quinine sulfate.

Artemisia annua, medicinal Chinese plant is really an antimalarial plant?

31The active principle of Qinghao (Artemisia annua L.), artemisinin is more active than the reference molecule chloroquine (Meshnik and col., 1996).

32In the traditional preparation (Klayman, 1985) one boils about 20 grams of leaves in a liter of water, this extraction corresponds in theory to the extraction of 100 mg of artemisinin, the maximal concentration of the active principle in the richest variety being 0.5%. The crystallized artemisinin is insoluble in water.

33To cure 50% of the patients, it is necessary to absorb by oral way, 5 grams of artemisinin for three days (Chinese Cooperative Research Group, 1982), measures that it seems impossible to achieve with the absorption of the traditional preparation.

Discussion

34The contradictions which conceals the case of Artemisia annua L. are fascinating. It is about a medicinal plant used since two thousands years against malaria. This plant contains one of the most powerful antimalarial of the current pharmacopoeia, the artemisinin with an in vitro activity among the strongest of the order of a nanomole. The chemical structure of this compound, a sesquiterpen lactone with a bridge endoperoxyde is unique in chemistry of natural substances. The mechanism of action seems itself very different from those proposed for the other classes of antimalarials. On the contrary, the bioavailability as soon as it is extracted from the plant is very poor due to its insolubility in water, the necessary doses to treat a patient are very important without common measures with what one can hope to extract from the small amount of leaves which are of use to the preparation of the traditional herbal tea. These contradictory facts have already made the object of studies trying to advance the role of synergic substances (Elford and col., 1987; Chen Liu and col., 1992; Agtmael and col., 1999) or statements of scientists of good name on the fate which would have led to the discovery of the compound artemisinin (Meshnik, 1998) and the antimlarial activity of which would be without report with the medicinal use of the plant. We bend very gladly over the clinical observation led by a non-governmental organisation, ANAMED supported scientifically by university of Tübingen in Germany which tried recently to demonstrate the activity on the human malaria of an Artemisia annua L. traditional preparation in zone of strong endemic malaria in Democratic Republic of Congo (Mueller and col., 2001). This study demonstrates that the warm water extracts 40% of artemisinin contained in the leaves of a rich variety of Artemisia annua L. The clinical observation show important decline of para- sitaemia of the treated patients (48 treated subjects) and a decrease of the symptoms of malaria. This study considered by these authors as preliminary would deserve to be resumed by following the good practices of the antimalarial clinical assays (Rogier and coll., 2001). One can notice that most of the clinical trials led with healing plants suffer strong methodological deficiencies. However this way of clinical research on antimalarial healing plants (AHP) deserves to be encouraged. WHO already support the forming of scientists' network on this question (RITAM or "Research Initiative one Traditionnal Antimalarial Methods"). This network suggests answering the essential questions that we have to try to land through our experience in Bolivia: do treatments with the AHP work? What research type should to one do to develop the use of the AHP as actual and acceptable treatments? What ethical and legal procedures should one carry out and how?

Table 1. Scale of activity allowing to estimate the activities of plant extracts on experimental malarial models, in vitro on Plasmodium falciparum erythrocytic schizogonia and in vivo on the murin model of malaria (test of Peters)

Table 1. Scale of activity allowing to estimate the activities of plant extracts on experimental malarial models, in vitro on Plasmodium falciparum erythrocytic schizogonia and in vivo on the murin model of malaria (test of Peters)

Bibliographie

References

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Table des illustrations

Titre Table 1. Scale of activity allowing to estimate the activities of plant extracts on experimental malarial models, in vitro on Plasmodium falciparum erythrocytic schizogonia and in vivo on the murin model of malaria (test of Peters)
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