Application of Ethnobotanical Indices to Assess the Diversity of Medico-magic Knowledge on Four Herbaceous Species in Benin.

Background Ethnobotanical knowledge on four herbaceous species, Phyllanthus amarus, Spilanthes uliginosa, Momordica charantia and Acalypha ciliata in Benin was investigated. Methods Thirty-six herbal medicine traders in six different markets were interviewed using a semi-structured questionnaire. The Relative Frequency Citation, Fidelity Level, Use Value and Rahman Similarity Index were calculated to assess the diversity of medico-magic knowledge. All informants traded P. amarus, M. charantia and A. ciliata and the majority traded S. uliginosa. Purchase in traders’ own markets was the predominant source of P. amarus, M. charantia and A. ciliata while S. uliginosa was mostly purchased in other more distant markets. A noticeable proportion of traders also collect P. amarus and M. charantia from wild populations. Phyllanthus amarus was the species most demanded by customers followed by M. charantia. Traders conrmed the scarcity of all species in recent years and climate change as well as destruction of natural habitats for logging were the most cited causes. The entire plant of P. amarus was used mainly to treat malaria, diabetes and constipation and decoction with oral administration the most frequent preparation for malaria treatment. To treat diabetes, informants mixed P. amarus with M. charantia used as a decoction with oral administration. Momordica charantia was also used to treat measles and chicken pox. Spilanthes uliginosa and A. ciliata were mostly used for their spiritual use for luck, predominantly by chewing fresh leaves or owers, and by bathing with the ground plant mixed with soap, respectively. Overall, M. charantia had the greatest use value followed by P. amarus. The majority of traders do not plant the species.


Introduction
The green medicine is obviously of major importance to mankind [1][2][3]. Gathering of high value products including medicinal plants from the wild continues in developing countries [3,4]. Moreover, medicinal plants harvested from the wild serve as raw materials for commercial pharmaceutical factories [3,5]. Thus, medicinal plants are important in both developed and developing countries and there is an increasing need to gather knowledge related to their exploitation and to de ne conservation strategies for long-term exploitation. Amongst medicinal plants, herbs have a great importance [6,7] as they are accessible to many people.
Ethnobotanical indices have been applied to evaluate ethnobotanical knowledge on plant species in other parts of the world [8][9][10] but not in Benin. Indeed, quantitative ethnobotany is concerned with measuring the importance of plants and vegetation for human well-being, as well as it relies on cultural signi cance indices as quantitative tools for qualitative data [11][12][13]. Because this approach is likely to generate data that lend themselves to hypothesis testing, statistical validation and comparative analysis [10], applying speci c ethnobotanical indices may either serves in analysing the range of information related to plants used for medico-magic purposes. The present research focused on four herbaceous species known as medicinal plants in Benin, Phyllanthus amarus Schumach. & Thonn., Spilanthes uliginosa Sw., Momordica charantia L. and Acalypha ciliata Farssk.. These species have been reported as used in green medicine in many parts of the world [14][15][16][17]. There is a lack of ethnobotanical information on them in Benin although Boko-Haya et al. [18] reported the ethnic differences in use of P. amarus in Northern Benin. Many years ago, this species was also tested for its antiplasmodial activity in Benin [19]. The aim of the present research was to document the medico-magic knowledge held by herbal medicine traders on these herbaceous species in Southern Benin through the application of four ethnobotanical indices i.e. Relative Frequency Citation [20,21,, Use Value [20,21,25], and the Rahman Similarity Index [26].

1-Study species
Phyllanthus amarus Schumach. & Thonn., (Hinlinwé in Fon), belongs to Euphorbiaceae, is an annual herb, 60-75 cm tall and quite glabrous. Its roots are stout and woody; the stems are branched at the base and angular with numerous leaves. The plant naturally grows in tropical and subtropical climates on well drained sandy-loam soils [27]. It protects the liver and limits the effects of viral hepatitis A, B, and C, reduces type 2 diabetes, helps ghting cancer and detoxi e the body [28].
Spilanthes uliginosa Sw. (Awélékpékpé in Fon), as an Asteraceae, is an annual herb with simple leaves and pungent owers [29], liking humid places and super cially drained Sudanian swamps, and sometimes ruderal. The species is used for dental problems and for its antiseptic, homeostatic, analgesic, insecticidal and anti-in ammatory activity [30].
Momordica charantia L. (Yinsinkin in Fon), from Cucurbitaceae, commonly called bitter-melon or ampalaya, grows 12 to 20 cm long. Its fruits ripen from green to yellow and ripe fruits are ornamentally attractive but malodorous [31]. It is a pantropical vegetable originated in tropical Africa [32]. Fruit also is carminative, tonic, stomachic, aphrodisiac, anthelmintic, astringent to bowels and useful in treatment of syphilis, rheumatism, spleen troubles, and effectiveness in treating diabetes [33].
Acalypha ciliata Farssk. (Viviman téton in Fon), is an Euphorbiaceae occurring widely in Africa. It is uncommon crop weed in heavily watered areas [34,35], a monoecious annual herb tilled up to 1 m with simple leaves at 2 mm long stipules linear at blade ovate. Fruits are 3-lobed capsule, smooth, almost glabrous [36]. Foliar powder or infusion is used for maggot-infested wounds [37].

Ethnobotanical data collection
Two markets in each of three districts of Benin were surveyed, Pahou and Zobê in Ouidah District, Cococodji and Godomey in Abomey-Calavi District, Vêdoko and Dantokpa in Littoral District. Six herbal medicine traders were randomly selected in each market and ethnobotanical data were gathered from them on each of the four study species. A total of 36 traders were thus surveyed. A semi-structured questionnaire was used in collecting data on the trade, sources and importance of each species. Traders were asked to rank the four species according to consumer demand and the availability versus scarcity of each species in recent years. The various causes of scarcity were also recorded. In addition, herbal medicine traders were asked to list all diseases, disorders and magic issues treated using each species as well as all types of preparation used for these purposes. In order to assess traders' contribution to the research species conservation, the plantation or in-situ conservation of each species by traders and their motives were investigated. Each time an informant con rmed the trade of a species, they were asked to show a sample for positive identi cation. All interviews were conducted in the local language, Fon, well spoken and understood by all informants.

Ethnobotanical indices calculation for data analysis
(a) The Relative Frequency Citation (RFC) [20,21], was calculated using the following formula: where n i is the number of informants who mentioned the concerned species, thing or aspect and N is the total number of surveyed traders.
(b) The Fidelity Level (FL) [22 -24] was determined for the diseases as follows; where I p is the number of informants who mentioned the use of a species for a speci c disease, disorder or magic issue and I u is the total number of informants who mentioned the species for any use. The FL was also calculated in a similar way for the types of preparation recorded for each disease, disorder or magic issue. The FL served to assess the informants' preference for a species to treat a speci c disease, disorder or magic issue. It also served to assess the preference for a speci c type of preparation. FL ranges from zero to a hundred per cents and a value close to 100% means a high preference.
where n d is the total number of use reports (diseases, disorders or magic utilization) mentioned for a species and I u is the total number of informants who mentioned the species for any use. The use value served to compare the relative importance of the study species in terms of uses.
(d) The ethnobiological Rahman Similarity Index (RSI) [26], to assess the similarity between species in terms of uses, was calculated as follow: This formula is similar to the Jaccard Similarity Index. Rahman et al. [26] considered an ailment recorded in two communities and treated with a number of medicinal plant species, species commonly used in both communities. In the present research, this approach was adapted to similarity of uses of pairs of species with, respectively, n a and n b as number of use reports in species a and b and, the number of common uses recorded for the two species as n c . RSI can range from 0 to 100% and an RSI lower than 50% means low similarity in terms of uses between the two species while an RSI higher than 50% indicates a high similarity of uses between the two species.

Results
1. Sources of the species traded.
All informants traded P. amarus, M. charantia and A. ciliata while 95% of informants traded trade S. uliginosa. Samples of each species were shown by each trader as proof. With regards to the sources of each species traded (Table 1), six different sources were recorded and the majority of informants purchase P. amarus, S. uliginosa and M. charantia in both their own markets and other distant markets. In addition to these sources, a relatively large proportion of traders collect plants from wild populations of P. amarus and M. charantia. Traders mostly got A. ciliata by purchasing it in their own markets and collecting the species from the wild. The single most important source of each species reported by herbal medicine traders was their own market for P. amarus (44% of traders), M. charantia (50% of traders) and A. ciliata (39% of traders), while for S. uliginosa it was distant markets (39% of traders). Table 1 Sources of the study species according to traders.

Purchase in their markets
Purchase in nearby markets 2. Ranking of the species in terms of demand and availability.
With regards to their demand, P. amarus was ranked as rst by 72% of traders, S. uliginosa by 22%, M. charantia by 6%, and A. ciliata by 0% ( Table 2). The majority of surveyed traders con rmed the scarcity rather than easy availability of all four species in recent years. The percentage of traders reporting scarcity was 94% for M. charantia, 89% for P. amarus, 83% for A. calypha and 67% for S. uliginosa. Eight different factors causing scarcity were reported by the herbal medicine traders (Table 3). Climate change and destruction for logging were the top two causes for all four species with overexploitation also mentioned as an equal second cause for S uliginosa.  3. Uses recorded for each species.
Ten uses were recorded for P. amarus parts or the entire plant ( Table 4). The most frequently (FL in %) reported use was to treat malaria using the entire plant as medicine, followed by treatment of diabetes, while the same proportion of traders reported the use of this species to treat constipation and stomach aches. Most informants (14 out 16; 88%) who mentioned use of P. amarus to treat diabetes insisted on its combination with M. charantia. Three spiritual and one medicinal uses were recorded for S. uliginosa (Table 4). All herbal medicine traders reported the spiritual utilization of this species for luck, but it was also used to treat mouth sores. Fifteen uses were recorded for M. charantia (Table 4). All traders mentioned the use of this species to treat measles and chicken pox. All informants who mentioned the use of this species to treat diabetes (39%) insisted on its mixture with P. amarus. Acalypha ciliata was recorded as used to treat eight diseases, disorders and magic issues ( Table 4). The most reported use of this species (FL = 86%) was its spiritual use for luck.  4. Relative importance of species and their similarity in terms of uses.
All species exhabited a Use Value lower than 0.5 (Table 5). However, M. charantia was relatively the most important in terms of uses. The Rahman Similarity Index for uses of pairs of species (Table 5) con rmed an absence of similarity of use between P. amarus and both A. ciliata and S. uliginosa, and also between M. charantia and S. uliginosa. However, there was a moderate similarity of use between P. amarus and M. charantia with both used to treat stomach complaints, malaria, diabetes and constipation. There were very low similarities of use between M. charantia and A. ciliata, and between S. uliginosa and A. ciliata with these pairs of species used in common to treat malaria and for good luck, respectively.

Phyllanthus amarus
Based on the Fidelity Level, the three major conditions treated with this species were malaria, diabetes and constipation, the last chosen in this table instead of stomach ache although having equal FL (Table 6). Four types of preparation were mentioned by informants for malaria treatment and three for the other two conditions. For each condition, the majority of traders suggested the use of the entire plant as a decoction and the route of administration was oral. An infusion of the entire plant in a traditional alcoholic drink or in hot water was also mentioned by a minority of the traders to treat all three conditions. A mixture of the dried, powdered plant in an alcoholic drink was reported as a treatment only for malaria.

Spilanthes uliginosa
Only the spiritual use of this species for luck was considered. Seven preparations were mentioned by medicinal plant traders (Table 6). Most commonly reported was the chewing of the leaves followed by the chewing of owers early in the morning to request for good luck in any kind of business

Momordica charantia
The three major conditions treated using this species were measles, chicken pox and diabetes (Table 6). Informants insisted that the rst two diseases shared the same preparations. Seven modes of preparation were recorded for each of these diseases and the most common was grinding the plant (leaves and stems) in the traditional alcoholic drink, 'sodabi'. Regarding diabetes, similarly to the ndings for P. amarus, with which this species was usually combined, three types of preparation were noted, the most common being the decoction of the plant (leaves and stems) with oral administration.

Acalypha ciliata
Only the magic use of this species for luck was considered and six preparations were reported (Table 6). Based on the Fidelity Level, the most common preparation, mentioned by a majority of the traders, involved grinding the plant (leaves and stems) and mixing it with soap for bathing. So the route of administration was herbal bathing. This was said by the traders to bring good luck to business activities.

Traders' contribution to species conservation
The majority of the traders did not plant the four research species (Table 7). However, a low proportion of traders con rmed that they planted S. uliginosa and P. amarus for trade and personal use. In-situ conservation of the naturally grown populations of all species at home except S. uliginosa was reported by a small proportion of traders. All informants con rmed the trade of P. amarus, M. charantia and A. ciliata while 94% traded S. uliginosa. These ndings con rmed the medico-magic importance of these four herbaceous species to indigenous people in Benin. In addition, the trade of these species may generate a considerable income to traders. There is an increasing demand for plants for herbal drugs, natural health products and secondary metabolites throughout the world [38]. The majority of traders purchased the four species in their own markets, meaning a close relationship and collaboration between medicinal plant harvesters and local traders. Elsewhere, the relationship between wholesalers and traders of medicinal plants in South Asia seemed to be exploitative [39]. There is a need to better understand this relationship in Africa, especially in Benin. In all cases, there were fewer traders reporting purchasing the four medicinal plants in distant markets. The collection from wild populations of three of the four species, P. amarus, M. charantia and A. ciliata, con rmed the connection of local traders with their environment. Moreover, since there is no control of herbaceous medicinal plant collection from the wild, this harvesting may threaten the conservation of the concerned species. Consequently, further investigation is needed on the harvesting and trade of herbaceous medicinal plants to ensure their long-term sustainable conservation.

Ranking of the four species in terms of demand and availability
The ranking of P. amarus as most demanded by the greatest number of traders showed the importance of this species in health care of the local population. Similarly, M. charantia also has a high importance. The high demand for these two species suggests that there is a likely to higher pressure on the conservation of these species than on the other two. The use of species belonging to the genus Phyllanthus as green medicine is becoming more popular [15]. Momordica charantia is widely used and in demand all over the world for its medicinal potency [40] resulting in over exploitation of its wild populations. Species belonging to the genus Spilanthes are widely used in traditional medicine in various cultures [41]. Knowledge related to the impact of climate change on plant species availability [42] was con rmed by local traders who perceived climate change to be a contributing factor to species scarcity. Additionally, the destruction of species habitats for logging reported by traders con rmed threats to forest biodiversity due to both habitat loss and degradation of forest ecosystems ( [43,44]. Not all medicinal plant species are affected in the same way by harvesting pressure so research is needed to assess the factors affecting the scarcity of each species studied. Herbal traders who reported the planting of S. uliginosa in other gardens con rmed the ex-situ conservation of this species. As a result, further investigations should be conducted in gardens to assess the ecological patterns and horticultural practices of these plantings. This will serve to promote cultivation of the species planting throughout the country in gardens.

Diversity of diseases, disorders and magic utilization of species
The large number of uses recorded for P. amarus and M. charantia revealed that herbal traders hold a diversity of knowledge on these species. The medicinal potency of M. charantia has been widely reported [40,45,46] including its use to treat diabetes as reported in the present study. The use of the species to treat measles was also reported in Nigeria [16], as well as its action against in ammatory diseases [17]. The combination of P. amarus with M. charantia for the treatment of diabetes corroborates ndings of Sarin et al. [15] who mentioned diabetes among ailments treated with Phyllanthus species. As recorded in the present research, indigenous people in Nigeria used fresh leaves of M. charantia in various preparations to treat measles [16].
The medicinal use of this species seems common along the West African coast. Since diabetes is mentioned among the ve most lethal diseases in the world [47], further research is needed on the pharmacognosy of P. amarus, M. charantia and their combination in treating diabetes. Similarly to the present results, the use of Phyllanthus species in the treatment of malaria, liver disorders and constipation was also staded by Sarin et al. [15]. Malaria is one of the most lethal diseases in tropical countries so the long-term conservation of medicinal plants, especially P. amarus, active in treating malaria in Benin, provides a guarantee for the treatment of this dreadful disease.
Few species of the genus Spilanthes have received research attention for their uses [41]. The magic utilization recorded for the species in this research was a proof of traditional belief that African populations assign to plants. Acalypha ciliata, similarly to S. uliginosa, was known as magically e cient by herbal traders. This con rmed the connection of Africans with their nature and the conservation of traditional beliefs across generations. In fact, similar to our ndings, Ma misebi and Oguntade [48] reported magico-religious practices using plant species in Nigeria. Recently, Dossou-Yovo et al. [3] reported the magico-religious practices and rituals using mangrove plant species in Benin. Since people believe that those species bring good luck, this is a strong argument to be used to promote their cultivation and conservation throughout the country.
4. Relative importance of species and their similarity in terms of uses.
Despite being the most frequently demanded, P. amarus did not have the highest use value. In contrast, M. charantia ranked second for demand had a higher use value than P. amarus. The relative importance and high demand con rmed the need for sustainable conservation strutegies for P. amarus and M. charantia as medicinal plants. Spilanthes uliginosa and A. ciliata were the least important in terms of uses. Although there was a low similarity in terms of use between P. amarus and M. charantia, the RSI value indicated that 19% ailments and disorders could be treated using either P. amarus or M. charantia. Spilanthes uliginosa and A. ciliata with an RSI value of 9% also had a low similarlity of use. However, the possible substitution of one medicinal plant for another justi es further study as it might contribute to conservation by relieving pressure of exploitation on wild populations of scarce and most threatened medicinal plant species in tropical countries.

Preparation and routes of administration
Similar to the present ndings, aqueous extracts of P. amarus were reported as more e cient than ethanol extracts for the treatment of malaria by Ajala et al. [14]. The preference of traders for decoctions with an oral route of administration against malaria matches western medical knowledge and con rmed that indigenous knowledge on green medicine has a great role to play in ghting dangerous diseases worldwide. Phyllanthus amarus was also recorded as the most commonly used in diabetes treatment, predominantly as a decoction with oral route of administration by Thai communities [47]. Constipation is a stomach disorder very often related to dysfunctioning of the liver. Given that excessive alcohol consumption is damaging to the liver [49], the authors question the wisdom of recommending extracts of medicinal plants in alcohol for the treatment of this condition. Spilanthes uliginosa and A. ciliata were recorded mainly for their spiritual use for luck and the chewing of leaves or owers, and the mixture of the ground plant with soap for bathing were the predominant methods of use. Elsewhere, species of the genus Spilanthes were reported as used for more than 60 disorders including parasitic diseases [41]. Herbal bathing as route of administration recorded in this research was also reported by Sabran et al. [50]. The harvesting of fresh owers of S. uliginosa for chewing compromises the reproductive capacity of the species since owers are reproductive organs.

Traders' contribution to species conservation.
There is evidence of the important role that in-situ and ex-situ conservation plays in the conservation of plant genetic resources [43]. However, the majority of traders do not plant the medicinal plant species that they sell. Therefore, there is an urgent need to promote the species plantation or the insitu conservation of naturally grown populations of the study species. Traders should be trained and sensitized on tools for the planting of P. amarus whenever possible in order to reduce pressure on its wild populations. Momordica charantia, exhibiting a relative high importance in this study, should also be conserved by traders wherever it grows. Although few uses were recorded for S. uliginosa and A. ciliata, it is recommended that traders also contribute to the in-situ and ex-situ conservation of these species. Those traders who con rmed the planting and conservation of naturally grown populations of the research species should be encouraged to contribute to training and dissemination of conservation tools for these species.
Herbaceous species, especially those used as medicine in Benin, are often overlooked by conservation programs. The authors strongly suggest that greater attention should be paid to herbaceous medicinal plants in Benin in order to contribute to their conservation.

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