Pharmacological value of plants of Rwandese traditional medicine: chemotherapeutic value of some Rwandese plants

PIERRE CLAVER RWANGABO

Institut de Resherche
Scientifique et Technologique
Butare, Rwanda

Introduction

A detailed study of Rwandese plants was done in order to enhance the knowledge of traditional Rwandese therapy and that of biological activity in medical flora. This study, like many others done in Rwanda and elsewhere, aims at discovering new or better medicines from plants. It also justifies the therapeutic use of certain plants by traditional Rwandese healers. This paper summarises the methodology used and describes the major results of the study.

The study involves some plants widely used in traditional Rwandese medicine. These are Rubus rigidus Sm. ("Umukeri") from the Rosaceae family; Lantana trifolia L. ("Mugengeri") of the Verbenaceae family and Vernonia amygdalina Del. (UMUBILIZI) which belongs to the Asteraceae family.

The paper presents in a condensed form the results obtained up to now from a systematic study seeking to show the pharmacological value and/or chemotherapeutic value of these plants. In order to prove the therapeutic use of these plants, we started with the following general hypothesis: in addition to its psycho-socio-cultural value acknowledged by everybody, traditional medicine also uses plants which have a biological effect that can be demonstrated by using scientific models employed in biomedical research. For one plant species studied, results of which indicated the possibility of its clinical use, toxicological aspects were also examined. The results are promising.

While pursuing research on the therapeutic activity of plants, the isolation and identification of chemical molecules make it possible to have a more complete phytochemical knowledge of the plants being studied for the first time and also to obtain toxicological information which is often inaccessible when working with raw extracts,. The isolation of chemical molecules is important in writing pharmacopoeia of these plants and the identification of chemical molecules is very useful not only in the production of medicines, but also allows the researchers to give useful advice to traditional practitioners who use these species.

A description of the general methodology is given, followed by a detailed study of every species. This study focuses on the chemical molecules that have been identified and their biological activity. Since a tentative conclusion is given during the study of each species, a brief discussion is provided to summarise the importance of the plants studied in the development of a national medico-pharmaceutical sector.

General methodology

The plants studied were selected from the whole range of Rwandese medicinal flora, using in particular the information provided by the traditional practitioners on the ethnopharmacological activity of the species.

For each species, research was done using a methodology that can be summarised by the following seven major elements:

(a) a botanical description and the geographical distribution of selected plants;
(b) an inventory of the uses of plants in Rwandese and central African traditional therapy;
(c) preliminary biological screening of the whole extract of the plant;
(d) phytochemical screening and detailed bibliographical study of the whole extract of the plants;
(e) chromatographic fractionation of extracts as well as investigating the previously identified activity;
(f) isolation, purification and identification of the products responsible for the activity;
(g) detailed study of the therapeutic effect and possible toxicity of active products compared with products already known in therapy.

The botanical study was done at CURPHAMETRA where specimens of the plants can be found. The study of the geographical distribution of the plants was done using specimens at CURPHAMETRA and the Herbarium of the National Botanical Garden of Belgium.

The phytochemical methods of extraction, isolation and identification of plant products have been described in many books. Some particular aspects of this research have been presented in detail in the cited references. A summary of some techniques used in the research on biological activity is presented in this paper, giving the specific character of some of them. Dilution and diffusion were used to demonstrate the antibacterial and antifungal chemotherapeutical activity. Each time we tested the microorganisms that represent major groups recognised as the main pathogenic agents. For purified active products, we investigated the minimum inhibitory concentration (MIC) according to the standard method. Whenever possible, the activity of a product was compared to a known control product used in therapy.

The antiviral activity of plant extracts and of pure products was studied using a more complex technique which necessitates culture and maintenance of cellular tissues, growth of virus on those cells and testing of the antiviral activity by observing the absence or persistence of the cytopathogenic effect of virus according to whether the tested product has any antiviral effect or not. We mainly used dilution of virus in plates of microtitration. The selection of the virus was done in such a way as to include representatives of different classes. Thus the Adenovirus was selected to represent the ADV virus without coating, the Poliovirus and the Coxsachievirus represent the ARN virus without coating, while the measles virus at the Semliki Forest represent the ARN with a coating. Cardiovascular activity was demonstrated using the following experiment models: blood platelets of rabbits, the right and left auricles of guinea - pigs, the central artery of the rabbit's ear, and microsomes of the sheep's seminal vesicle. Some of these experiments were done at the University of Anvers (UIA), in Belgium.

Toxicological studies on 3-methoxyquercetine (3-MQ) isolated from Vernonia amygdalina were done at Butare, at the CURPHAMETRA on experimental models described below.

1. Toxicity of 3-MQ in internal usage (9)

Preparation and administration of the product

The 3-MQ was extracted from the flowers of Vernonia amygdalina (omubilizi). An aqueous suspension was prepared by grinding the powder in a mortar; the suspension was added to a concentration of 15 mg to 20 ml of distilled water and was administered to mice in ratios of 30 mg per kg of the animal's body weight. This translates into 0.8 ml of the suspension for a mouse weighing 20 g. The suspension was administered using a plastic syringe with an unoxidable tip and unlikely to cause trauma in the animal.

Handling and observation of mice

Ten white mice (colony OFI) of both sexes, of more or less identical age and of average weight of 22.4 g were divided into two groups of 5. The general condition and temperature of each animal were observed and noted the day before the administration of the product. The following day, one of the two groups was given a suspension of 3-MQ proportional to the weight of the animals while the other group received an equal amount of distilled water. The weight and temperature of the animals were noted everyday at the same hour and focused on:

· the general condition of every animal
· nervousness and any sign of drowsiness
· temperature
· the condition and form of coat/fur
· the body weight of every animal

The animals were kept in groups of 2 in rectangular plastic cages.

The product was administered for 10 consecutive days and the above- mentioned aspects were initially observed for 44 days.

The animals were given food, water, and libitum. On same dates, the 1st, 9th, 11th, 22nd, 29th, 36th and 44th days, every mice was weighed from the 2 groups and the average weight on those dates was calculated. The comparison of these average weights and initial weight allowed us to evaluate the effect of the treatment on weight evolution and consequently on growth of animals in the experiment.

We used the following procedure to investigate the influence of the product on reproduction: a group of 10 female mice of the same colony were given an aqueous suspension for 10 days. Another group of female mice was given distilled water in the same way like in the previous experiment. On the 11th day, the mice from the 2 groups were mated and kept in pairs in groups of different cages for each lot where they were given normal food, drink and libitum.

The number of new-born mice in each lot was counted and compared.

2. Toxic manifestation of V. amygdalina in UE (10)

The experiment involved 10 adult rabbits with weights between 2.8 and 4 kg; they were divided into 2 groups of 5. After weighing them and observing carefully the general condition of the animals, every rabbit was shaved over an area of 4x4 cm on the back. They were kept in individual cages, fed and given libitum. An ointment of 5% of aqueous-methanolic fraction made from the fruits of the plant was applied in the shaved area of the first lot of rabbits. The ointment had a vaseline base.

The second control group was treated with vaseline only. A small amount of ointment or the vaseline were accordingly applied once a day in the same way by rubbing lightly in order to cover uniformly the bare area.

The experiment lasted a month (from 4th December 1987 to 4th January 1988). On the 18th day the administering of the medicine was stopped. The two groups were observed daily and compared on the following aspects: the general condition of the animals, weight, growth of hairs and especially any manifestation of irritation on the treated surface.

Detailed study of the plants and results

1. Rubus rigidus

The species is widely found in Rwanda and neighbouring countries. The Rwandese traditional practitioners use it mainly to treat bacterial and fungal diseases but it is also used in other areas of pathology such as poisoning, snakebites, etc.

A preliminary study showed an antibacterial and antifungal activity in the whole extract of the plant. Phytochemical studies made possible the isolation and identification of pygallic acid, commonly known as pyrogallol.

The antibacterial and antifungal activity of the product already shown in literature was confirmed by this study with a minimum inhibitory concentration (MIC) of nearly 250 micrograms per ml. The microbes most sensitive to this product are Staphylococcus aureus, Pseudomonas aeruginosa, Microsporum canis, Trichophytom mentagrophytes and Candida albicans.

No other activity, antiviral or pharmacological (cardio-vascular), was noted in this plant during our study. However, bibliographical research has shown that pyrogallol has a hepatoprotective activity which is observable when the same amount of doses are used as those showing antibacterial effect. This triphenol shares that action with other phenols of similar structure, catechins and tannins.

To conclude, we established that the chemotherapeutic activity of Rubus rigidus used by Rwandese traditional practitioners is mainly due to the presence of pyrogallol. From the medico - pharmaceutical point of view, pyrogallol already has several uses especially in external usage. References which we consulted mention also antibacterial ointments with doses of 2 to 10%. However, it is known that the plant has some toxic effect when used internally. We therefore advised traditional practitioners to put more emphasis on its external uses.

2. Lantana trifolia L.

It is a verbenaceous plant widely found in Rwanda where it is known as "umuhengeri". This plant had previously shown an antibacterial activity especially in its leaves. Traditional practitioners use it to treat many syndromes.

It is the antibacterial activity that gives promising results while other biological activities investigated do not give any results that can justify further investigation in other areas.

The chemotherapeutic antibacterial study on the active fraction made it possible to isolate and identify a series of products which have promising activities. These products are: two saturated chains aliphatic hydrocarbons (C₃₃H₆₈ and C₃₅H₇₂), saccharose, two pentacyclic triterpenes of the ursane group (alpha-amyrine, urs-12-ene-3-one), a new polymethoxy flavonoid (5-hydroxy-6,7,3',4',5'-pentamethoxynavone) that we named "umuhengerine" following the Kinyarwanda, name and finally diospyrin which is a binaphtoquinone related to juglone. "Umuhengerine" was isolated from these plants for the first time whereas diospyrin had only been identified in the different genera of Diosypros (Ebenaceae).

Among the products isolated, only the last two showed any antibacterial activity worth investigating. Umungerine has a small antibacterial and antifungal spectrum at concentrations of 300 micrograms. Diospyrin has a wide spectrum on gram positive and gram negative bacteria and some fungi, with a predilection against Mycobacteria (for example the causal agents of leprosy and tuberculosis) whose representatives show sensitivity to an MIC of nearly 2.5 micrograms per ml.

As far as this plant is concerned, even if the comparison of MIC is not the only parameter taken into consideration, diospyrin is active in a similar concentration (perhaps even better) compared with many antibacterial products used in therapy; such is the case for its action against M. fortritum (MIC = 2.5 mg ml) compared to the control, Neomycin, which is only active with a MIC of 32 micrograms/ml. Umuhengeri has a weaker antibacterial spectrum but according to its chemical structure, it could be more active at the level of lipophile balance, a factor presently recognised as determining the activity of chemical molecules against gram negative and gram positive bacteria. In addition, its identification clearly contributes to the chemical knowledge of this species.

According to the literature on Lantana camara, another Verbenaceous plants which resembles very much the preceding plant, Lantana camara has toxic products especially against the liver and the skin, such as those which show some photosensitization. An example of these structures is lantadene A.

We did not isolate these products in the active fraction of L. trifolia. However, we cannot conclude that they are absent in all parts of the plant. It is probable that these toxic products can be demonstrated by other chemical methods which do not take the biological activity as a major indicator. Bibliographic research on the genus Lantana advises some caution in the use of this plant.

3. Vernonia amygdalina Del.

It is an Asteraceous plant belonging to the subfamily of Vernonieae, which is very common in tropical and subtropical Africa. It is called "Umubilizi" in Rwanda and in some neighbouring countries like Uganda and Burundi. Its use in traditional medicine ranges from treating hepatitis, cardiac ailments, poisoning, malaria, stomach pains, snakebites and eczema. The authors of the "Communautes Africaines" journal have confirmed recently the use of V. amygdalina as food for humans in Cameroon. We had earlier on stated this use of the plant in East and Southern Africa.

Concerning the biological activity, especially of the antitumour and cytotoxic nature already identified in this plant, this study identified other structures that had never been stated before and demonstrated other therapeutic activities such as antiviral, and the pharmacological effect at the level of platelet aggregation and cardiac ailments.

The study was done on extracts of dried flowers of the plant prepared and fractioned according to procedures. Given its importance in chemotherapy, the antiviral activity guided the separation and purification of active molecules. Some chemical structures were isolated and identified, for example:

· 11 saturated aliphatic C₂₂ to C₃₂ fatty acids;
· 5 esters of fatty acids derived from glycerol;
· a sesquiterpene lactone known as vernolide;
· a series of flavonoids, that is:

quercetine (3,5,7,3',4'-pentahydoxyflovone), 3-methoxyquercetine (3-MQ), 3,3'-dimethoxyquercetine (3,3'-DMQ), rutine, quercetine -3-0-1-beta-D-glucose-6-1-alpha-L-rhamnose) and kaempherol (3,5,7,7,4'-tetrahydroxyflavone (K).

3-MQ was isolated with a yield close to 1% compared with the powder of dried flowers at ambient temperature.

A detailed study of therapeutic activity of isolated products showed that flavonoids and vernolides are the active principles, while the fatty acids and esters are aliphatic products which are associated with these active principles. Four groups of biological activities were studied. These are the cardiovascular activity, antiparasitic activity, antiviral activity as well as the verification of some lexicological aspects of products that can be clinically used.

1. Cardiovascular activity

1. Effect on platelet aggregation. All technical details of the procedure for demonstrating this activity have been described elsewhere especially in "Revue Medicale Rwandaise" in 1986.

The technique used demonstrated that quercetine flavonoids, 3-methylaquercetine and rutine, to a small degree inhibit platelet aggregation, lipoxygenase activity, and cyclooxygenase, at a concentration of 100 micrograms (110 M) per ml. This concentration is 1,000 times higher than that which shows an important antiviral effect.

Vernolide also shows a completely reversible inhibition of platelet aggregation induced by arachidonic acid, but this activity is very small.

2. Other cardiovascular activities.

At doses of 10 micrograms per millilitre, 3-methoxyquertine shows a positive chronotropic effect on the right auricle and an antiarythinic activity on the left auricle of the isolated guinea-pig's heart.

3. Antiparasitic activity

This was indirectly demonstrated, especially by vernolide. In fact during our research, another group working independently isolated the same product from Vernonia colorata and showed that the product has an antiparasitic action especially against Entamoeba histolitica at nearly the same level as antiparasitics used clinically, such as metronidazole. By demonstrating this product in V. amygdalina we were justifying, at the same time, the use of this plant against intestinal parasites.

4. Antiviral activity

3MQ and 3,3'-DMQ have an important antiviral activity which was shown even at concentrations as low as 10 nanograms. These products have a selective effect since they prevent the formation of ARN and viral proteins without interfering with the metabolism of the host cell. They are especially active against the virus of poliomyelitis, the coxcachie virus, the vesicular stomatitis virus (VSV), the Rhino virus, and against other virus of African origin like Bangin and Bunyamwera.

The importance of this plant in antiviral chemotherapy is thus obvious, especially since even in more developed European medicine, there is no medicine in this area. Fortunately, the family of products isolated from this plant allows us to foresee further research with some hope of success in treating other groups of virus, such as retrovirus. Proof exists some of which is very recent. For example in 1979 Mr. Apple and his colleagues demonstrated inhibition of reverse transcriptase of encornavirus by some flavonols of vegetable origin.

In May of the same year the Japanese group ONO Katsuhiko with French researchers reexamined the action of some flavones related to quercetine as inhibitors of reverse trascriptase, enzymes that were associated with human immuno-deficiency syndrome.

Even if further research was to demonstrate the absence of any important activity in this area, the importance of Vernonia amygdalina in semi-purified extracts as well as pure products is obvious given the low level of toxicity in the plant. One can foresee the therapeutic use of this species in future. Before this stage of the study, we explored some toxicological aspects of the main active principle.

5. Preliminary toxicological study of 3-MQ in internal usage

As described above, we tried to establish the importance of toxic manifestations that can occur when using the plant as an ointment in treating dermatological diseases such as eczema. The study shows that the application of an ointment with 5% of a semi- purified extract of V. amygdalina does not produce any detectable irritation among rabbits in the laboratory treated with it. The same applies to rabbits that only get the expient.

General conclusion and discussion

In reporting the results of this research we have underscored, once again, that the value of African medicinal plants in general, and Rwandese plants in particular, in the treatment of all kinds of diseases does not need to be demonstrated any more. The use in traditional therapy of plants that are the focus of this paper is justified by the demonstrated biological activity of their products. There are plants which have activities already known but of which we did not know the presence in the plant under study, for example pyrogallol. There are products which were very well-known in chemistry as being inactive or nearly so. However the research showed us that they had a very useful activity which was sometimes unknown elsewhere in the medico-pharmaceutical sector. An example of this group is 3-methoxyquercetine isolated from V. amygdalina. Finally we found toxic or inactive products in relation to the activity under investigation. The demonstration of these products contributes very much in toxicological or phytochemical knowledge of plants under study.

As expected, the plants under investigation do not have the same importance in developing further the socio-sanitary sector. The activity of R. rigidus is very low. Its main importance is mostly in justifying its use in traditional therapy. Lantana trifolia, however, has activities similar to those of the most active antibiotics but because the plant is very toxic, the products could be purified and then used in new medicines in bacterial chemotherapy. Vernonia amygdalina is hardly toxic and grows spontaneously in many of our regions. Its varied activity, very obvious in antiviral chemotherapy and as an anthelmintic, suggests that we should develop quickly research on its use, even without isolating active molecules. One could use its semi-purified extract.