Indigenous use and bio-efficacy of medicinal plants in the Rasuwa District, Central Nepal

Background By revealing historical and present plant use, ethnobotany contributes to drug discovery and socioeconomic development. Nepal is a natural storehouse of medicinal plants. Although several ethnobotanical studies were conducted in the country, many areas remain unexplored. Furthermore, few studies have compared indigenous plant use with reported phytochemical and pharmacological properties. Methods Ethnopharmacological data was collected in the Rasuwa district of Central Nepal by conducting interviews and focus group discussions with local people. The informant consensus factor (FIC) was calculated in order to estimate use variability of medicinal plants. Bio-efficacy was assessed by comparing indigenous plant use with phytochemical and pharmacological properties determined from a review of the available literature. Criteria were used to identify high priority medicinal plant species. Results A total of 60 medicinal formulations from 56 plant species were documented. Medicinal plants were used to treat various diseases and disorders, with the highest number of species being used for gastro-intestinal problems, followed by fever and headache. Herbs were the primary source of medicinal plants (57% of the species), followed by trees (23%). The average FIC value for all ailment categories was 0.82, indicating a high level of informant agreement compared to similar studies conducted elsewhere. High FIC values were obtained for ophthalmological problems, tooth ache, kidney problems, and menstrual disorders, indicating that the species traditionally used to treat these ailments are worth searching for bioactive compounds: Astilbe rivularis, Berberis asiatica, Hippophae salicifolia, Juniperus recurva, and Swertia multicaulis. A 90% correspondence was found between local plant use and reported plant chemical composition and pharmacological properties for the 30 species for which information was available. Sixteen medicinal plants were ranked as priority species, 13 of which having also been prioritized in a country-wide governmental classification. Conclusions The Tamang people possess rich ethnopharmacological knowledge. This study allowed to identify many high value and high priority medicinal plant species, indicating high potential for economic development through sustainable collection and trade.


Background
Ethnobotany reveals historical and present plant use to fulfil a wide variety of human needs [1,2]. Documenting indigenous knowledge through the ethnobiological approach is important for species conservation and sustainable resource use [3]. Furthermore, such studies are often significant in revealing locally important plant species, sometimes leading to the discovery of crude drugs [4,5], or contributing to economic development.
Globally, millions of people in the developing world rely on medicinal plants for primary health care, income generation and livelihood improvement [6]. Indigenous people living on their traditional territory largely rely on medicinal plants for healthcare and they are therefore rich in ethnopharmacological knowledge. The interest in phytomedicine has been renewed over the last decade and several medicinal plant species are now being screened for pharmacological potential. According to Laird and Pierce [7], the world market for herbal remedies was worth 19.4 billion US$ in 1999. The global demand for medicinal plants is increasing and, in India alone, the market is expanding at an annual rate of 20% [8,9]. Scientific research is needed to determine the active principles of traditional medicinal recipes and to evaluate their effectiveness, so that benefits could be equally shared among local peoples in the spirit of the Convention on Biological Diversity [10].
Medicinal plants play vital roles in the Nepalese livelihood [11] and the use of medicinal plants is frequent in several Nepalese regions [12][13][14]. It is estimated that only 15-20% of the population of Nepal -living in and around urban areas -have access to modern medicinal facilities, whereas the rest depend on traditional medicines [11]. Nepal is a natural storehouse of medicinal plants [12,15,16]. Each year thousands of tons of raw material are exported, mostly to India, but also to Asia, Europe and America [17]. The government of Nepal aims to promote medicinal plant use and conservation programmes for livelihood improvement and poverty alleviation through various policies [11]. However, the contribution of this sector to the national economy is still nominal.
Several ethnopharmacological studies have been conducted in Nepal [13,15,16,18,19], but many parts of the country remain unexplored. Few studies have attempted to estimate use variability of Nepalese medicinal plants or to evaluate their bio-efficacy [20,21]. Therefore, this study was conducted in order to achieve the following objectives: 1

Study area
A field study was carried out in the Chilime VDC of the Rasuwa district of Central Nepal. The district lies between 27°2' and 27°10' N and 84°45' and 85°88' E, with altitude ranging from 792 to 7245 m a.s.l. The Rasuwa district presents some of the best examples of graded climatic conditions in Central Himalaya. Pronounced altitudinal gradients, coupled with complex topography and geology, have resulted in a rich biodiversity and unique vegetation patchwork [22]. Therefore, the district harbours a rich diversity of medicinal plants. The Chilime VDC lies in the northern part of the district, bordering the Tibetan part of China, and comprises temperate to alpine climates within 2000-4700 m altitude (Fig. 1). The local inhabitants are part of the Tamang indigenous people, which comprises 98% of the total Chilime VDC population [23]. People from the Tamang ethnic group have a rich culture and possess sound traditional knowledge. However, they are economically and socially marginalized and far from having their basic needs fulfilled.

Methods
Ethnopharmacological data was collected by conducting interviews and focus group discussions with local people (50% > 40 years old), from July to September 2007. A total of 50 household heads (56% male and 44% female) from the Chilime VDC of the Rasuwa district participated in the study. Participants were purposively selected to include key informants [24] like plant collectors, medicinal plant cultivators, traditional healers, and traders. Respondents were all from the Tamang ethnic group, predominant (65%) in the Rasuwa district. The majority (62%) of the respondents had no formal education, 18% had primary school education, 12% had secondary education, and 8% had university level education. Prior informed consent was obtained with the help of community workers [25] that also facilitated interviews and discussions with the local people. Consent was granted by the local people for the dissemination of their traditional knowledge.
Guidelines for the interviews and group discussions were developed to facilitate the collection of information. Interviews and group discussions were conducted to gather information on plant uses, parts used, and modes of utilization. A checklist was developed and used to determine what species were used to treat what kinds of diseases/disorders. Herbarium specimens were collected for those species for which field identification was not certain and brought back to the lab to facilitate identification using reference collections [26][27][28][29] and expert knowledge. The specimens are deposited at the Tribhuvan University Central Herbarium (TUCH).
The informant consensus factor (F IC ) was calculated to estimate use variability of medicinal plants [30,31]. F IC values range from 0.00 to 1.00. High F IC values are obtained when only one or a few plant species are reported to be used by a high proportion of informants to treat a particular ailment, whereas low F IC values indicate that informants disagree over which plant to use [30]. High F IC values can thus be used to pinpoint particularly interesting species for the search of bioactive compounds [31]. F IC is calculated using the following formula [30,31]: where N ur is the number of individual plant use reports for a particular illness category, and N t is the total number of species used by all informants for this illness category.
Medicinal plant species were ranked according to prioritization criteria developed by the Herbs and Non- Criteria accounting for availability, local knowledge and use, and market demand were given more weight. Indigenous uses determined from interviews and discussion groups, and phytochemical and pharmacological properties determined from a review of the available literature were compared for all species for which information was available.

Results
The ethnobotanical survey identified a total of 56 medicinal plant species used to prepare a wide variety of remedies (Additional file 1). Angiosperms were predominant, with 44 species belonging to 29 families (Fig. 2 followed by Pteridophytes (6 species from 4 families), Gymnosperms (3 species from 3 families), Lichens (2 species from 1 family) and Fungi (1 species). The prevailing life form was herbs, followed by trees, shrubs, lichens, climbers and fungi (Fig. 3). Almost all plant parts were used to prepare different medicinal formulations: roots, rhizomes, tubers, bark, leaves, flowers, fruits, pollen, young shoots, and whole plants (Additional file 1). The most frequently used plant parts were roots, followed by leaves, whole plants, fruits, and rhizomes ( Fig. 4). Use of multiple plant parts was also recorded in a few cases (Additional file 1).

Ailments treated and remedies formulation
Cuts and wounds, respiratory problems, gastro-intestinal disorders, cough and cold, musculo-skeletal problems, fever and headache, weakness and dizziness, menstrual disorders, dermatological infections, ophthalmological problems, and tooth ache were the main ailments treated with medicinal plants. Gastro-intestinal disorders, fever and headache, cuts and wounds, cough and cold, and musculo-skeletal problems were treated with the highest diversity of medicinal plant species (Additional file 1, Table 1). Although most species were only used to treat one ailment (34/56), some were found having up to four different medicinal uses (Table 1). Most people who participated in interviews and group discussions were familiar with the species used to deal with common ailments like cough and cold, digestive problems, fever, headache, skin infection, and in such cases plant based medicinal remedies were used on a regular basis. For complex problems like chest pain, menstrual disorders, rheumatism, or eye and kidney problems, people took advice from local traditional healers. Traditional healers believe in a form of sanctity of the curative power of medicinal plants. They thus keep secrecy over remedy formulation, believing that the medicines would lose their potency if revealed to other people.  A total of 60 medicinal formulations were prepared from the 56 medicinal plants identified in this study. Two formulations were prepared using five different species, while all other formulations were prepared using a single species. Preparation methods included paste, juice, decoction, infusion and chewing the raw plant (Table 2, Fig. 5). The majority of formulations were prepared as paste or juice. Crushing, pounding, and grinding are executed using a pestle in a mortar made of hard stone.

Informant consensus factor
The level of informant agreement was high for most ailment categories (mean F IC = 0.82) and total consensus (F IC = 1.00) was even obtained for ophthalmological problems, tooth ache and kidney problems ( Table 3). Gastro-intestinal disorders, as well as fever and headache showed relatively low levels of consensus (F IC = 0.53 and 0.61, respectively).

Prioritization of medicinal plant species
Respondents were asked to prioritize medicinal plant species based on the criteria developed at the First National Trade Show and Seminar on Herbs, Herbal Products and Spices (see Methods and [32]). After consultations with the local people, district forest staff, NGO representatives, and researchers, a final list of 16 high-priority species was obtained (Table 4).     traditional use with known phytochemical and pharmacological properties showed complete or partial correspondence for 27 of the 30 plants ( Table 5). Twelve of the 30 plants for which phytochemical/pharmacological information was found in the literature were high-priority species in the Rasuwa district (Tables 4 and 5).

Traditional use of medicinal plants in Chilime
Altogether, 56 species of medicinal plants were identified as being used in traditional medical systems in the Rasuwa district of central Nepal. As indicated for the Dolkha district, having more or less the same economic, social and ecological characteristics, reliance on medicinal plants for health care was associated with poverty, lack of accessibility to modern healthcare facilities, and traditional belief about plant effectiveness [20]. Herbs are the primary source of medicinal plant species, followed by trees, most likely because herbs are more abundant. It is believed that the more abundant a plant is, the more medicinal virtues it may possess [20,33]. The ease with which plants can be collected, stored, and transported and the ease with which bioactive compounds can be extracted are also factors that contribute to the preference for herbs [20]. Moreover, most species used in the traditional health care system of the Chilime VDC are harvested from the wild. This is common practice all over the world, as was observed in Cameroon [34], Uganda [35] and Peru [36], for example. The preference for root to prepare traditional remedies follows the scientific reasoning that roots generally contain high concentrations of bioactive compounds [37].

Informant agreement
The average F IC value for all ailment categories was 0.82, indicating a high level of informant agreement compared to similar studies conducted in Mexico [30,31,38], Belize [39], and India [40], for example. Particularly high F IC values were obtained for ophthalmological problems, tooth ache, kidney problems, and menstrual disorders ( Table 3), indicating that the species that are traditionally used to treat these ailments are worth searching for bioactive compounds: Berberis asiatica, Astilbe rivularis, Juniperus recurva, Swertia multicaulis, and Hippophae salicifolia. The latter three species, as well as Valeriana jatamansi, are also of interest as they are traditionally used to treat three or four different ailment types (see Table 1). Total 81* 307 -*Some taxa were reported in more than one ailment category (see Table 2).

Aconitum ferox
Root paste is taken for joint pain. Alkaloid extract may possess anti-inflammatory properties [48].

Yes
Acorus calamus* Rhizome is used for cough/cold, and throat pain.

Amaranthus spinosus
Root paste is applied on cuts and wounds.

Yes
Artemisia indica Leaf paste is applied on cuts and wounds.

Asparagus racemosus*
Tuber paste is used for fever, stomach ache, and diarrhoea.
Ethanol and aqueous extracts from the tubers exhibit significant antidiarrheic activity [51].

Yes
Berberis asiatica Cambium paste is used for rheumatism and pith paste is used for eye problems.
Widespread use as an extract in eye drops for conjunctivitis [21]. Effective as an antipyretic, anaesthetic, and antihypertensive [52].

Yes
Bergenia ciliata* Whole plant juice is taken to treat indigestion, fever, diarrhoea, and dysentery.

Yes
Cannabis sativa Plant paste is taken for stomach problems.

Cordyceps sinensis
Whole plant juice is taken as a tonic. Largely recognised as inducing sexual power and vitality [16,54,55].

Eupatorium adenophorum
Leaf juice is applied on cuts and wounds.

Fraxinus floribunda
Bark infusion is used for body pain. Anti-inflammatory, anti-oxidative and skin regenerating activities [56].

Fritillaria cirrhosa*
Plant juice is taken for stomach disorders.

Hippophae salicifolia
Fruit juice is taken for cough, diarrhoea, and menstrual disorder.
Contains high levels of flavonoids (with antimicrobial properties and effectiveness against menopausal symptoms), carotenoids and vitamin C [58].

Hippophae tibetana
Fruit juice is taken for stomach disorders.

Juglans regia
Fruit juice is taken as a tonic. Seeds are diuretic and a nervous system depressant [59].

No
Lindera neesiana Fruit juice taken for diarrhoea. Essential oil extracted from fruits possess significant antimicrobial activity [60].

Lycopodium clavatum
Pollen paste is used on cuts and wounds.

Nardostachys grandiflora*
Whole plant juice is taken to treat headache and high altitude sickness.
Ethanol extract from roots showed anticonvulsant activity and are a nervous system stimulant [62].

Onychium japonicum
Used for skin problems.

Yes
Potentilla fulgens Root paste is used against gastritis. Possess antibacterial and anti-inflammatory properties [67].

Yes
Rheum australe* Root juice is taken for fever, indigestion, diarrhoea, and stomach ache.

Rubia manjith
Root paste is applied over scabies and other skin diseases.
Comparison of the information on traditional medicinal plant use in the Rasuwa district with ethnobotanical studies conducted in other areas of Nepal [15,18,20,41] shows similar results for many species. This is of significance because identical plant use by different people from different areas may be a reliable indication of curative properties. Like in other rural communities of Nepal [15,20,42,43], knowledge about traditional uses of medicinal plants is transferred from the household seniors and other elders. In many cases, this knowledge is transmitted orally, from generation to generation, and remains confined to a limited group of people [44]. Documentation efforts undertaken by Nepalese researchers in order to document traditional use of medicinal plants [13][14][15]20,45] should continue, especially as the results presented here place traditional and scientific knowledge on equal footing.

Sustainable management and use of medicinal plants
The criteria used to identify priority medicinal plant species [32] are very practical and useful in the regional context and are highly reliable as they were synthesized by experts based on national and international data. The 16 priority species identified here are highly valued on national and international markets [46]. Importantly, 13 of the 16 species prioritized in the present study are also priority species identified by the central Government of Nepal, which recognized 30 medicinal plant species for promotion of commercial use and trade [32]. Therefore, it is important to consider these species to implement policy and to guide management authorities of the Rasuwa district for proper management and use of medicinal plants to benefit local people in their traditional healthcare delivery systems and income generation activities [6,47].
Unsustainable harvesting, over-exploitation and habitat degradation have been identified as major threats to the sustainability of medicinal plants in the district. The medicinal plants sector has the potential to achieve sustainability, given the availability of resources, people's willingness to participate in conservation programmes, and the priority given to the sector by the government and other organizations. Sustainable harvesting, effective domestication methods, community participatory management, and the provision of information, education and awareness programmes to the community are key strategies that can help optimize the benefits of the medicinal plants sector in Nepal [45].

Conclusions
The Tamang people of the Rasuwa district of central Nepal possess rich ethnopharmacological knowledge and therefore use several medicinal plant species in their traditional healthcare delivery system. The striking coincidence between traditional plant use and scientifically-proven phytochemical and pharmacological properties shows that the traditional remedies are an important and effective part of indigenous healthcare systems in the district. However, published information on phytochemical and pharmacological properties are still limited for many plant species used in the district. Detailed phytochemical and pharmacological studies of traditionally-used medicinal plants is thus an important line of research to pursue, especially for species showing high informant consensus, like Astilbe rivularis, Berberis asiatica, Hippophae salicifolia, Juniperus recurva, and Swertia multicaulis. Medicinal plants provide huge opportunities for community development and livelihood improvement. However, local people are often deprived of the benefits from these resources [45]. Proper management of high-value and high-priority medicinal plants could serve as a sustainable income source for the communities. This would in turn help generate incentives for biodiversity conservation, thus ensuring the long-term availability of medicinal plants for indigenous and commercial uses.