Ethnobotanical study of medicinal plants of the Ipassa-Makokou Biosphere Reserve , Gabon : Plants used for treating malaria

1 Department of Botany, Faculty of Sciences, University of Douala, Cameroon, BP 24 157 Douala, Cameroon. 2 Faculty of Sciences, University of Bangui, BP 908, Avenue des Martyrs, Bangui, Central African Republic. 3 INDEFOR, APDO 207, Bata, Equatorial Guinea. 4 Institut des Recherches en Ecologie Tropicale (IRET), Centre National de la Recherche Scientifique et Technologique (CENAREST), BP : 13 354 – Libreville, Gabon.


INTRODUCTION
Malaria is a global disease that is predominant in the tropics and caused by blood parasites, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax.The parasite is transmitted to its human hosts via various mosquito species of the genus Anopheles.The disease can cause high fever, enough to be fatal to human being.Malaria has a great morbidity than any other infectious diseases of the world (World Malarial Report, 2005;World Health Organization, 2000).Half of the world's population is at risk of malaria, with an estimated 247 million cases that caused 881,000 malaria deaths in 2006 (World Malaria Report, 2008).In tropical and subtropical regions such as sub-Saharan Africa, malaria remains a major cause of illness and death as well as a contributing factor to poverty.Each year, about 400 million people contract malaria, with 90% in Africa, and each year, the disease kills one million of them, mostly (85%) children under five years of age.Pregnant women are also among the highly vulnerable people (Saotoing et al., 2011;Tropical diseases Web Ring, 2000).Most of the current antimalarial drugs have been developed through synthesis or screening voices.These approaches have been revealed as non efficient and expensive.Due to the lack of vaccination, the chimiotherapy and chymioprophylaxy remain the best method in the struggle against malaria (Oketch-Rabah and Mwangi, 1998).P. Falciparum, the pathogenic most widespread human malaria, is becoming increasingly resistant to antimalarial drugs.This requires extra effort and continuous search for new drugs, especially with new mode of action (Saotoing et al., 2011;Oketch-Rabah and Mwangi, 1998).Ethnobotanical and ethnopharmaclogical studies are now recognized to be the most viable methods of identifying new medicinal plants (Balick, 1985(Balick, , 1990(Balick, , 1994;;Cotton 1996; King and Tempesta, 1994).
In Gabon, malaria is predominantly caused by P. falciparum and malaria transmission is recurrent and considered as hyperendemic (Elissa et al., 1999).Past reports showed that infections with P. falciparum ranged from 30 to 50% on the children consulted with fever between 1980s and 1990s throughout the country (Richard-Lenoble et al., 1986;Richard-Lenoble et al., 1987).Adults and pregnant women are also among the most affected in the country (Bouyou-Akotet et al., 2003 and2009).Although progress regarding modern treatment of malaria has improved in the country, however, many people still depend on traditional usages of medicinal plants, especially rural ones (Sassen and Wan, 2006).Despite this dependence, there is a paucity of ethnobotanical and ethnopharmaclogical studies on traditional uses of medicinal plants in the treatment of malaria on one hand and promoting local knowledge on traditional medicines on the other hand.Thus, this paper aims to document the traditional use of medicinal plants in the treatment of malaria in villages settled in the periphery of the Ipassa-Biosphere Reserve, in Makokou, the province of Ogoué-Ivindo for local knowledge promotion in the Republic of Gabon.

Study site
The Ipassa Biosphere Reserve is located in North East of Gabon, in the Ogooué Ivingo province, and at 620 km from Libreville and at about 12 km to Makokou city.Makokou itself is the regional capital of the Ogooué-Ivindo province.The Ipassa reserve was listed as a Biosphere reserve since 30 June, 1983.And since then, it is the unique Biosphere Reserve in Gabon.The Ipassa Biosphere Reserve is composed of three main areas including: a central or core area of 10,000 hectare, a 2 km 2 buffer zone, and a 3.5 km 2 transition area.The average altitude is 520 m and with an average temperature is 23.9°C, while the annual rainfall varies between 1,600 and 1,800 mm.
Makokou belongs to the Guineo-congolian phytogeographical type (White, 1983).Primary forests contain many plant species of Caesalpiniaceae, Burseraceae, and Euphorbiaceae family groups.Secondary forests contain high light demand plant species such as Pycnanthus angolensis and Scyphocephalium ochococoa.Species such as Scorodophleus zenkeri, Santiria trimera, Coula edulis, Anonidium mannii, Afrostyrax lepidophyllus, and Picralima nitida are known as Non Timber Forest Products (NTFPs) in Gabon.They are listed among the most abundant tree species in the primary forests of the Ipassa Biosphere Reserve.The reserve hosts a large variety of Wildlife species including 129 mammals and 401 bird species (Dupuy, 2008).The major ethnic groups are the Bantus and the Baka Pygmies who live side by side outside the reserve.The Bantus include the Fang, Kwélé, and Kota.Baka Pygmies live mostly scattered in small settlements, mainly in the forest at some distance from the Bantu villages and roads.Bantus and Baka pygmies undertake many livelihoods activities such as slash and burn agriculture type, hunting, fishing, gathering in the buffer and transition zones of the reserve.

Ethnobotanical survey
The method used in this study called the "method for the popular pharmacopoeia", consists of gathering data on the popular use of medicinal plants in a given area (ex.village).Data collected for this study were obtained from both semi-structured interviews and direct interviews, with the local people conducted from 5th May to 30th August, 2011 in villages settled in the periphery of the Makokou city.The survey aimed at identifying plants used in the popular pharmacopoeia among local people.The household was considered as the sample unit.In each household who accepted to answer to our questions, data were mostly recorded from adult women (mothers), because they usually knew the plants better than men and younger people.They provided useful and firsthand information on the popular use of medicinal plants.During the survey, enquiry was made to know "to what extent ailments were treated by which plant species" rather than asking "which plants were used to treat which ailments".For each health problem cited, the name of the plants and the plant parts used were carefully recorded.The vernacular names of the plants were recorded as much as possible, and the plants specimen mentioned by the informants were collected and brought to the National Herbarium of the Institut de Pharmacopé et Medecine Traditionelle (IPHAMETRA), in Libreville (Gabon) for further identification.Voucher herbal specimens were kept at the Herbarium of IPHAMETRA.The therapeutic statements were made of a specific disease, a symptom or a physiological effect.Information on the diagnosis of ailments was provided through a semi-structured interview of nurses or local health officials.

Ethnic composition of the informants
A total of 60 informants based in 7 villages prescribed plants in the treatment of malaria.Table 1 shows the informants, their age, gender, and ethnic groups.The distribution of interviews in different ethnic groups is as follows: Fang (7 informants), Kota (35), Kwélé (13), and the Baka Pygmies (5).The Kota ethnic group forms the largest portion, with more than half of the total number of informants; this is because the Kota is the dominant ethnic group in the province.

List of anti-malarial plants and recipes
A total of 61 plant species were cited, for which a total of 374 citations were made on malaria (Appendix 1).2).To investigate if the collected plants were representative of the plants used for treating malaria by people living in the periphery of the Ipassa Biosphere Reserve in Makokou, we counted the cumulative number of plants cited by additional number of informants.The informants were chosen randomly without replacement in groups of 5.The change in the number of plant species to that of informants is illustrated in Figure 1.
The curve can best be approximated equation: Y = 17.86 Ln (X) + 15.99.Y is the number of plant species; X is the number of informants.The examination of the figure shows that an increasing number of informants do not contribute to increasing the number of anti-malarial plants beyond a certain point.But this may only be true for the new informants belonging to the same ethnic groups with the sample mainly composed of the Kota ethnic group.

Characterization of recipes
Recipes are characterized by the plant part, the pharmaceutical form, the mode of administration, and the degree of association of plant species.A total of seven plant parts were cited by people living in the periphery of the Ipassa Biosphere Reserve for treating malaria including fruits, leaves, roots, seeds, stems bark, tubers.Figure 3 illustrates the relative importance of those plant parts.Leaves are the plant parts that are largely cited (62%), followed by stem barks (17%) and fruits (13%).A total of seven different pharmaceutical forms were cited (Figure 4) that include decoction, juice, maceration, oil, pounding, rapure, and trituration.Decoction (58%) and  maceration (35%) appear to be the most pharmaceutical forms used for treating malaria in the Makokou region.People living in the periphery of the Ipassa Biosphere Reserve have cited 10 different modes of administration of drugs used for treating malaria, which are bath, footwash, friction, headwash, massage, oral, rectal, scarification, suppository, and vaporation bath.Vaporation bath (41% of citations), rectal (25%) and oral voices (23%) appear to be the main modes of administration of plants used for treating malaria in the study (Figure 5).About 73% of the citations are made of combination of two, three, four, five, six, or seven plant species.Only 13 plant species (21.3%) cited are used alone.Plants species such as C. papaya, Capsicum frutescens, C. limon, Cymbopogon citratus and Manihot esculenta are largely cited as associated plant species for treating malaria.

DISCUSSION
The samples collected for ethnobotanical survey are often very poor.The number of informants may not even reach one percent (1%) of the total number of persons found in the study site (Höft et al., 1999).This is partly because informants are often reluctant to provide information.This problem is more crucial in survey of medicinal plants.To examine the validity of the sample, we examined the representativeness of the samples through a regression curve of the number of plant species by number of informants.Figures 1 and 2 show that the plants and the recipes samples recorded in this study are quite representatives of all that are used by people living in the periphery of the Ipassa Biosphere Reserve as anti-malarial.In fact, almost all the plants and recipes used for treating malaria by people living in the periphery of the Ipassa Biosphere Reserve were collected.The distribution of informants to different ethnic groups shows considerable imbalance.The Kota is the group that is largely represented (58.3% of informants), and aslo the most dominant ethnic group in the province.Baka pygmies are represented only with 08.3% of informants.The major consequence of such an imbalance in relative number of informants by ethnic group is that an intergroup comparison would not make sense.A comparison of the total sample collected with those in other African countries would be more fruitful.
Most of the citations (73%) cited for treating malaria by people living in the periphery of the Reserve are made of combination of many plant species.In traditional medicine whole plants or mixtures of plants are used rather than isolated compounds.There is evidence that crude plant extracts often have greater antiplasmodial activity than isolated constituents at an equivalent dose (Rasoanaivo et al., 2011).Leaves (62% of citations) are plant parts that are largely used.They are followed by stem barks (17%) and fruits (13%).Villagers have through experience observed that plant parts such as barks, leaves and roots are sites of bio-synthesis and sites of storage of secondary metabolites.These metabolites are responsible of biological properties of plants.The vulnerability of a plant species is linked to the rhythm of regeneration of plant parts used.The harvesting of stem barks or root barks, "always tend to kill the trees", but the harvesting of latex, leaves, fruits and oleaginous seeds do not necessarily leads to the death of adult trees and do not modify radically the specific structure of the populations of trees (Peters, 1997, Ticktin, 2004).The sporadic harvesting of few fruits will have little impact on the long term stability of the populations of the exploited trees.But an intensive harvesting of seeds for example, can lead to the progressively elimination of the tree species (Peters, 1997, Ticktin, 2004).
Decoction (58%) and maceration (35%) are the two most pharmaceutical forms used for treating malaria by people living in the periphery of the Ipassa-Makokou Biosphere Reserve.The plant species for which the recipes allow a long conservation (ex.storage) will be less vulnerable compared to those which must be used immediately (Betti, 2002a).The oil can be stored and stay more long than aqueous forms such as decoctions and macerations or plant parts freshly used (ex.pulp).The Dja herbalists as those of the Congo Brazzaville (Diafouka, 1997) do not get aseptic methods to store and conserve aqueous medicines.Their length of conservation does not exceed five to seven days.After this length, the medicine becomes unfit for consumption, not effective and sometimes toxic, due to the proliferation of pathogen agents.
Some plants largely cited by people living in the periphery of the Ipassa Biosphere Reserve are also known in other African countries for the same usages.Other plant species, which are frequently mentioned by the informants as being used for treating malaria, are well known in the literature for their active compounds for the same usages.These plant species are presented hereby, with the countries where they are cited from and roles played for treating malaria and if possible, pharmacological inquiries related to that ailment:

Alstonia boonei (Apocynaceae)
This plant specie was cited 31 times by informants belonging to the all ethnic groups for treating malaria.The plant is known for the same usage in eight African countries including Cameroon, Congo-Brazzaville, Equatorial Guinea, Nigeria, Senegal, Togo, and the Democratic Republic of Congo (Betti, 2002b).The genus Alstonia in general, was considered to be not effective against Plasmodium (Wright et al., 1993).The antimalarial tests conducted by Oliver-Bever (1986) with A. boonei were not positive.However, Zihiri Guede ( 2004) has revealed a good activity of A. bonnei (IC 50 < 4 µg/ml) against Plasmodium falciparum FcB1/Colombia. A. boonei's antimalarial activity is significant, while its toxicity is minimal (Idowu et al., 2010).This antimalarial activity can well explain the large use of that plant specie in the treatment of malaria in the Makokou region.

Carica papaya (Caricaceae)
This is the most cited plant species in the treatment of malaria by people living in the Makokou region (43 citations).The plant is largely cited for the same usage in many African countries such as Cameroon and Ghana (Saotoing et al., 2011), and Ngeria (Idowu et al., 2010).Its use against malaria is justified by the presence of high concentrations of active compounds in the seeds.For Titanji et al. (2008), there is no report on the phytochemistry of this plant with regards to its antiplasmodial activity.However, Idowu et al. (2010) reported that C. papaya has a minimal antimalarial activity and a minimal toxicity.

Citrus limon (Rutaceae)
This is cited 26 times for treating malaria.The fruits are often associated (23 citations/26) in decoction to other plant species.The fruits of C. limon contain anti-malarial substances, and the studies conducted in Cameroon showed that the plant is effective against malaria (Titanji et al., 2008).

Cymbopogon citratus (Poaceae)
This plant was cited 24 times by informants for treating malaria.C. citratus is one of the most commonly used herbs in Cameroon to treat malaria and other fevers (Titanji et al., 2008).As observed in the far north region of Cameroon, C. citratus is generally used in combination with other plant species (21 citations/24) in decoction for treating malaria/fever.The same use is mentioned in Nigeria (Idowu et al., 2010).Although, the essential oil has analgesic and pyretic properties, however, that oil is not active against malaria (Robineau, 1991).C. citratus was reported to be slightly significant and not toxic (Idowu et al., 2010).

Enantia chlorantha (Annonaceae)
This plant specie was cited three times for treating malaria by the Fang ethnic group.The plant is known for the same usage in Cameroon, Congo Brazzaville and Equatorial Guinea (Betti, 2001).E. chlorantha is active against Plasmodium yoelii nigeriensis (Agomo et al., 1992).

Vernonia amygdalina (Asteraceae)
This is cited 16 citations for treating malaria by Kota and Kwélé farmers (The plant was recorded to be frequently used against malaria by the Baka pygmies living in the periphery of the Dja Biosphere Reserve, Cameroon.The extract from leaves has shown some anti-malarial property against Plasmodium berghei (Hakizamungu and Weri, 1988) and Plasmodium falciparum (Tona et al., 1999).The "ndolé" the popular name for Vernonia leaves is widely used as relish in Cameroon.The plant is non toxic, but the excessive ingestion of leaves can produce a purgative effect (Iwu, 1993).Idowu et al. (2010) reported that V. amygdalina's toxicity is negligible.
The fact that some plant species cited by Makokou people are well recognized in literature for their antimalarial property against Plasmodium, is a credibility index which can be attributed to local knowledge of those people on pharmacopoea.This also illustrates the efficiency of the method used to identify medicinal plants used by rural people around the Ipassa Biosphere Reserve in Makokou.Thus, local knowledge on medicinal plants used for treating malaria needs to be promoted throughout the country to secure pharmacopoeia as a whole.The glaring development challenge at the background of what precedes is the pressing need to implement strategies and programmes to identify active chemical substances of other plant species which have not yet been investigated for their chemical and antimalarial activities.

Figure 1 .
Figure 1.Cumulative number of anti-malarial plants to the number of informants interviewed.

Figure 2 .Figure 3 .
Figure 2. Cumulative number of anti-malarial recipes to the number of informants interviewed.

Figure 5 .
Figure 5. Mode of administration of recipes in the treatment of malaria in the Ipassa Biosphere Reserve, Makokou region.

Table 1 .
List of informants who cited plants for treating malaria in the Makokou region.

Table 2 .
List of plants cited against malaria by people living in the periphery of the Ipassa-Makokou Biosphere Reserve.
Zihiri GuedeN (2004).Survey of anti-malarial plants of the Issia division (Côte d'Ivoire) and a descriptive study of those with a very good antimalarial activity.Revue Méd.Pharm.Afr.18:99-103.Citations of plants in the treatment of malaria by people living in the periphery of the Ipassa-Makokou Biosphere Reserve, Gabon.