Ethnobotanical survey in Tampolo forest (Fenoarivo Atsinanana, Madagascar)

Background Madagascar shelters over 14,000 plant species out of which 90% are endemic to the region. Some of the plants are very important for the socio-cultural and economic potential. Tampolo forest is one of the remnant littoral forests hinged on by the adjacent local communities for their daily livelihood. However, it has considerably shrunk due to anthropogenic activities forming forest patches. Thus, documenting the useful plants in and around the forest is important for understanding the ethnobotany in this area. In this study, we 1) collected and identied useful plants utilized by local communities. Voucher specimens were collected following the information given by interviewees. 2) recorded the collection activities and the consumption methods through semi-structured interviews of the local inhabitants. 3) did a phytochemical screening to identify the active compounds and the potential healing metabolites of the medicinal plants. antidiarrheal contains polyphenols, also Tetracera madagascariensis Willd. ex Schltdl., used to treat oral candidiasis, contains polyphenols and tannins which are antiseptics. indepth into ecacy.


Abstract Background
Madagascar shelters over 14,000 plant species out of which 90% are endemic to the region. Some of the plants are very important for the socio-cultural and economic potential. Tampolo forest is one of the remnant littoral forests hinged on by the adjacent local communities for their daily livelihood. However, it has considerably shrunk due to anthropogenic activities forming forest patches.
Thus, documenting the useful plants in and around the forest is important for understanding the ethnobotany in this area.

Methods
In this study, we 1) collected and identi ed useful plants utilized by local communities. Voucher specimens were collected following the information given by interviewees. 2) recorded the collection activities and the consumption methods through semistructured interviews of the local inhabitants. 3) did a phytochemical screening to identify the active compounds and the potential healing metabolites of the medicinal plants.

Results
A total of 65 people between 25 to 75 years old were interviewed. Surveys recorded 123 species used as timber, food or medicine. Among them, 92 were forest species and 31 were ruderal species. Medicinal plants were mostly used to cure diarrhea, stomachache, and fever with leaves being the most used plant part. Phytochemical analyses of 20 endemic medicinal species showed the presence of compounds that could be responsible for the therapeutic effects of the plants.

Conclusions
Tampolo forest proves to be an important littoral forest highly utilized by the adjacent local communities due to the presence of high number of useful plants which are mostly endemic to the region. Hence, our investigation assessed the importance of these species in the locality and this can be used for further study on ecology, conservation and valorization of these species.

Background
Tropical forests harbor a rich diversity of species which have a high productive and protective natural values [1,2]. They are also a driver of a signi cant social and economic development as a result of the exploitation of the existing natural resources [2,3].
Additionally, humans also depend on the forests for food, shelter and medicines [4]. However, there is a rapid loss of tropical forests through deforestation driven by the increased land use change, natural resource overexploitation and climate change.
Madagascar has a remarkable wealth in terms of vegetation and endemic species. The Island is composed of a variety of natural environments, which harbor a unique and globally important assemblage of plant species. It is home to over 14,000 plants species, out of which 90% are endemic to the region [3,5]. Among the 490 tree genera on the island, 161 are endemic [6]. However, the increasing intense population growth has led to rapid deforestation as land is cleared for agricultural elds and for fuel. The rainforest cover in Madagascar has recorded a gradual decrease from 5,254,306 hectares in the year 1990 to 4,489,248 hectares in the year 2005 [7], and a further loss of 4,345,000 hectares in the year 2013 [8], which translates to one million hectares loss in 15 years.
Tampolo forest is part of the eastern littoral forest remnants of Madagascar which have considerably shrunk due to anthropogenic activities hence forming forest patches [5]. The adjacent local communities majorly depend mostly on farming and shing which generally do not generate enough income hence the improper exploitation of forest's natural resources to supplement the daily income. Due to this direct addition into the wellness of the adjacent community, there is a greater risk of extinction of many endemic animal and plant species such as Daubentonia madagascariensis Gmelin "Aye-Aye)" (Daubentoniidae) and Dalbergia baronii Baker (Fabaceae) which inhabit the forest. This biodiversity loss is greatly propelled by the forestry sector which have since the colonial period, focused on the wood production potential of the sites, rather than focus on the region's plants and their practical uses through the traditional knowledge of the local culture and people's perspective [9][10][11][12][13][14].
As rural communities, local people of Tampolo depend on natural resources for their daily livelihood [13,15,16], especially for their healthcare [17]. In many parts of the world, traditional knowledge has always been transferred orally from generation to generation [18]. However, there is a risk of loss of information over the years, hence the importance of gathering them through ethnobotanical studies [4,[19][20][21][22][23][24]. In terms of traditional cures, despite the lack of written documents, forest medicinal plant species were used to treat various types of diseases. Unlike other parts of the island where works were completed [17,21,25,26], no related works were available for this present area of study. Therefore, this paper is aimed at lling the gap of the previous literature available and to document the oristic list of useful forest plants with emphasis on medicinal endemic species.
Additionally, to evaluate the signi cance of most salient plant families, genera and species and their uses among the participants for the conservation of the biological resources and their sustainable utilization.

Study area
Tampolo forest is located on the eastern coast of Madagascar covering 360 ha (Fig. 1). It is about 110 km from Toamasina, the capital district and 10 km from Fenoarivo Atsinanana, in Analanjirofo region [27]. It is bordered by Lake Tampolo and the village of Rantolava to the North, the village of Ampasimazava to the South, the Indian Ocean to the East and the National Road number 5 and the village of Tanambao Tampolo to the West. It is classi ed as low altitude dense evergreen humid forest belonging to the series of Anthostema and Myristicaceae by Humbert and Cours-Darne [28] and as coastal forest by DuPuy and Moat [29], recording over 360 plant species [30]. Three types of soils can be found in the forest station: South of the station, the ground sandy, In the North and West, the soil is generally clayey-loamy, on the hills, the soil is ferralitic in Nature.Tampolo region has an average annual temperature of 23 O C with the coldest month being July with 19 0 C and December is the hottest month with 26.5 O C. The region receives 3406mm of rain per year with average rainy days of 241 registered per year. In terms of human population, nearly 6,000 people are distributed within the following "fokontany": Andapa II, Tanambao Tampolo, Rantolava and Takobola, which belong to the rural "commune" of Ampasina Maningory in 2014.

Ethnobotanical data collection
Five eldtrips for ethnobotanical and biological surveys were carried out from February to November, 2012. Ethnobotanical surveys were done by using the methods of Martin [31] to achieve open or semi-open interviews which means that questionnaires were asked in manner that could not in uence the answers of the participants and by following the International Society of Ethnobiology (ISE) code of ethics [32]. Researchers started their interaction with each prospective respondent by rst explaining the aims and objectives of the project in order to solicit their consent and co-operation before any ethnobotanical data were gathered. Interviews were conducted with the selected informants to determine and explore the ethnobotanical knowledge regarding the utilization of plant species, their usefulness, their utilized parts, mode of preparation, or method of processing the plants. The ethnobotanical data for this research were gathered from altogether 53 farmers, 3 traditional healers, 3 shermen, 2 chiefs of Fokontany, 1 forest guard, 1 Tangalamena (traditional chief of the village), 1 retired nurse and 1 blacksmith. The participants were selected based on their consent to be interviewed and their a rmation on the use of or having knowledge on at least one of the uses of the forest plant species. At rst before any interview, we introduced ourselves to those responsible for the village; after their agreement, participants were asked if they can be interviewed in the context of our study. They were free to participate or not i.e., voluntary. Preliminary engagement has shown that younger people are ignorant of traditional knowledge; in contrast, the elderly interviewees depend mostly or entirely on natural resources.
Investigations were conducted in the three villages surrounding the forest: Tanambao Tampolo, Andapa II and Rantolava where informants of the age 25 and above were interviewed and with their prior consent. The Nagoya protocol on access and bene tsharing [33] has been followed.
The importance value of the use of each species by local population were assessed by calculating its Use Index by using the formula of Lance et al. [34]: where I (%) is the percentage index of use, n is the number of people citing the species, and N is the total number of people surveyed. The given species is heavily used if the value of I (%) is between 60 and 100% and moderately used if I (%) is between 30 and 60%; and if I (%) is less than 30%, it is rarely used.

Specimen collection and taxonimic identi cation
Voucher specimens were collected with the help of the eld guides and following the information given by interviewees relying on the plant species vernacular names. Some of the plant species were identi ed in the eld and the remaining ones identi ed at the Herbarium of Tsimbazaza Park (TAN), acronym according to the Index Herbariorum list [35]. Species names were checked using Tropicos, International Plant Name Index (IPNI), the Plants Of the World Online (POWO) and Plant List databases [36][37][38][39]. An aqueous extract was prepared by mixing 1 g of leaf powder with 20 ml of distilled water, then the solution was boiled and cooled [40][41][42]. Four drops of salted gelatin 1% was mixed with 0.25 ml of the aqueous solution, the formation of a precipitate indicates the presence of tannins. On the other hand, phenolic compounds were detected when the color of the mixture switches to dark blue or blue-green by mixing four drops of ferric chloride in methanolic solution with 0.5 ml of the extract [42]. Then, Anthraquinones were detected by using the Bornträger reaction [43,44], a 0.5 ml of the aqueous solution was mixed with 1 ml of benzene. After decantation, 0.5 ml of ammoniac 25% was added, the turn to red of the solution indicates the presence of Anthraquinones. The presence of desoxyoses were also detected by using 0.5 ml of the aqueous solution with consecutively 0.5 ml of cold acetic acid, 0.5 ml of ferric chloride 10% and 0.5 ml of sulfuric acid (H 2 SO 4 ) 36 N where "N" is the number of particles in the substance (reaction of Keller-Kiliani [45]). The formation of a purplish ring at the interface of the tube con rms the presence of desoxyoses [46]. Iridoids were detected by adding some drops of hydrochloric acid (HCl) 12 N to 0.5 ml of the aqueous solution. The mixture was boiled in a water bath for 30 minutes, then a dark green or dark blue precipitate or color appears if these compounds are presents. For saponins, after dissolving in water, there should be a formation of a foamy solution after strongly shaking for 30 seconds. [40], if the convoluted foam persisted within 30 minutes, it contained Saponins.
Chloroform extract was used to detect the presence of steroids and terpens. One gram of leaf powder was mixed with chloroform, stored in cold place for one night, then ltered. The Libermann-Burchard test [47] was used by mixing 1 ml of the extract with 1 ml of acetic anhydride. After shaking, 1 ml of H 2 SO 4 was then added. The formation of a purplish red ring indicates the presence of terpens while the presence of steroids was indicated by the formation of a green color at the upper level of the solution. Also, the presence of sterols was detected by using the reaction of Salkowski [48,49], The phase at the bottom of the test tube turns in red if they were presents when 0.5 ml of H 2 SO 4 34 N and three drops of anhydrous acetic were added to 0.5 ml of chloroformic extract.
After that, 1 g of leaf powder was mixed with 10 ml of hydroethanol (75%) then stored in a cold place for one night. To detect the presence of avonoids, the Wilstater procedures [50,51] was used by adding 4 drops of HCl 12 N and 2 Magnesium turnings to 2 ml of the extract. The color change to red indicates the presence of avonoid compounds. Then, the detection of anthocyanins followed the procedures of Bate-Smith [52]. A mixture of 2 ml of the plant extract and 0.5 ml of HCl 12 N was boiled for 30 minutes, and when cooling, a red color appeared.
Finally, 1 g of leaf powder was mixed with 10 ml of HCl 2 N and marinated for one night. Then, 1 ml of the acid extract were then mixed with four drops of reagent of Mayer [53], Wagner [54] or Dragendorff [55] and produced a white precipitate or a occulation if Alkaloids were presents in the solution.

Demographic Variables
During the study, 65 local inhabitants were surveyed, 41 (63.08%) were male and 24 (36.92%) were female. The age of the informants ranges from 25 and 75 years old ( Table 1). The survey was done either by individual interviews (one-on-one consultations) or through focus groups. The interviewees consisted of 53 farmers, 3 traditional healers, 3 shermen, 2 chiefs of Fokontany, 1 forest guard, 1 Tangalamena (traditional chief of the village), 1 retired nurse and 1 blacksmith. The participants were selected based on their consent to be interviewed and their a rmation on the use of or having knowledge on at least one of the uses of the forest plant species. At rst before any interview, we introduced ourselves to those responsible for the village; after their agreement, participants were asked if they can be interviewed in the context of our study. They were free to participate or not i.e., voluntary. It was observed in the survey that their knowledge of useful plants mostly medicinal plants was passed down from their ancestors through oral traditions. Preliminary engagement has shown that younger people are ignorant of traditional knowledge; in contrast, the elderly interviewees depend mostly or entirely on natural resources. The use of traditional medicine is an important part of healthcare of the Tampolo community.

Plant utilizations
The following ethnobotanical information are reported for each species: the scienti c name, the family name, the growth form, the plant part used, and uses. During these interviews, 123 plant species distributed within 62 families and 112 genera, including ruderal species, were cited as useful in the locality of Tampolo of which 59 were medicinal (48%), 54 for timber and rewood (44%) and 10 were edible (8%) (Fig. 2

Growth form of the plants
Trees (56 Species) and shrubs (25 species) were cited by the participants to be the most exploited (Fig. 3) while climbers (7 species), Epiphytes (3 species) and Herbs (1 specie) were least cited. Four species such as Uapaca thouarsii Baill., Intsia bijuga Kuntze, Dalbergia baronii Baker and Mimusops coriacea Miq. had their use index greater than 60% because they were highly valued by the local people as timber. Moreover, U. thouarsii was used as medicinal plant. Few forest species were edible and fruits were the major part that were eaten by the local people. Furthermore, these species were also eaten by lemurs and birds.

Medicinal plants
Among the useful forest plants, 43 species were medicinal having therapeutical values, However, among the 43 species, 3 species were also used as timber as well as food while 37 of them were endemic to Madagascar ( Table 2). The most frequent diseases that are treated with plants were diarrhea, stomachache, oral, dental, genital infections and non-malaria fever (Table 3). In the case of malaria, all of the interviewees a rmed that they consult a doctor. However, leaves were the most frequently used parts (68%) that were used in the cure of most of the diseases as shown in the Fig. 4. The other parts or components of the plant such as stem, root, bark and latex or mixture of two or more of them were also used in low proportions.  Bremek. - Griseb.

Discussion
All the informants who participated in this study were over 25 years old, mostly dwelling in Tampolo which is a rural area. Rural communities have been known to utilize the natural resources to satisfy their daily needs [56]. This is due to the low income, lack of alternative sources of income and lack of modern healthcare facilities within the regions [57,58].
Despite its small size of about 1/6500 (0.015%) of the total cover the Malagasy Rainforests, the ora of Tompolo forest is highly diverse having 360 plant species [30] which represents 2.6% of the ora of Madagascar [3]. The assumption that local communities use forest plant species as timber, rewood and especially for medicinal purpose was demonstrated in this work. We found out that most of the illness encountered with these regions have been treated with plant materials. Their dependence on natural resources for their livelihood and their basic healthcare were due to their economic, lack of health facilities in the remote regions of the country and their socio-cultural situation [59][60][61]. These species are utilized by the local adjacent communities to ful ll their daily livelihood needs [9,12,15,62,63].
Our ndings were in sync with other botanical surveys that showed the importance of forest species to the local communities [13].
Non-severe health problem such as fever and digestive disorder were the most commonly treated with medicinal plants. Similarity in the mode of use and the recorded healing properties of several species were observed in different areas across Madagascar [64]. For example, leaves of Phyllarthron bojeranum DC (Bignoniaceae) were also used as treatment of fatigue in Analangazaha Farafangana [26,65], in Ambalabe Vatomandry [21], in Antananarivo [25,66] and in Tampolo. This is also the case of Anthocleista madagascariensis Baker which were an antidiarrheal [25]. Phytochemical analysis revealed the presence of active secondary metabolites which have been linked to treatment of various diseases [67] in the 20 selected forest species. However, more studies need to be undertaken to test their e cacy. The healing properties of the medicinal plants are in part due to the presence of the secondary metabolites such as alkaloids, saponins, avonoids, tannins, glycosides, anthraquinones, steroids and terpenoids [68]. Phytochemical screening was used to detect the presence of them following the procedure of Cordell [41], Hemingway and Karchesky [42] and Bruneton [40]. Some secondary metabolites such as phenols, tannins, avonoids and quinones show antidiarrheal effects [69][70][71][72]. Compounds such as alkaloids, phenols, tannins, iridoids, avonoids, steroids and terpenes have been shown to have anti-in ammatory, antioxidant, antiseptic properties [69][70][71][72][73][74]. Based on these previous literatures, their presence can justify the speci ed therapeutic properties of the plant. In this study, Saldinia axillaris (Lam. ex Poir.) Bremek., indicated as antidiarrheal contains polyphenols, also Tetracera madagascariensis Willd. ex Schltdl., used to treat oral candidiasis, contains polyphenols and tannins which are antiseptics. However, further analysis should be done to prove an indepth understanding into their e cacy.
While the medicinal plant gathering by local people are non-destructive because the quantity of the collected leaves is relatively small and it is only for daily dose and for family use. The same holds true for the need of rewood because only the dead woods can be collected and that is under the control of the protected area managers. Moreover, although most of the population did not

Conclusion And Recommendations
This survey showed that one third of the whole forest species which is about 360 species were useful plants, while 84.78% of them were endemic. This documentation of ethnobotanical knowledge provides a catalog of useful plants of the Tampolo, and will serve as a physical record of their culture for the education of the future generation. It will also strengthen their culture by recognizing their traditional knowledge on medicinal plants and providing scienti c basis for it. However, the overexploitation may disturb the ecological balance of the area which subsequently can lead to the disappearance of these species. Hence, further efforts on environmental education still should be provided because Tampolo is one of the last remnant littoral forests of the East of Madagascar thus this could help conserve this area/forest. Necessary measures should also be taken to protect these most exploited species to avoid their future extinction. The current nding can be used as a reference point for various studies within the forest to help reconcile the local livelihood needs with forest conservation. Based on the ndings, we recommend further studies regarding ecology, conservation and chemistry of the remaining species which constitute the ora of the littoral forest of Tampolo. Declarations