PARIS POLYPHYLLA SMITH: AN IMPORTANT HIGH VALUE HIMALAYAN MEDICINAL HERB

Mohd Tariq* 1 , Shilpi Paul 1 , I.D. Bhatt 1 , K.Chandrasekar 1 , Veena Pande 2 and S.K.Nandi 1 . 1. G.B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora263 643, Uttarakhand, India. 2. Department of Biotechnology, Kumaun University, Bhimtal Campus, Bhimtal263 136, Uttarakhand, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

This study describes various aspects related to in vitro multiplication, pharmacology and recent biotechnological methods adopted for conservation of this species. Since emphasis on propagation and conservation strategies needs to be framed using various biotechnological and ecological methods (ENM), hence information on furthering research in IHR would be considered essential to suggest conservation strategies for this threatened species. Due to high demand for medicinal purposes, the population of this species has decreased substantially. A study from Eastern Himalaya shows that low density of population ranged between 0.4 to 1.48 individuals m -2 (Paul et al. 2015). Further population status of species from Western Himalaya is not known. Hence, future studies need to focus on assessment of populations in the wild.
Steroidal saponins of P. polyphylla i.e., Polyphyllin D could serve as an agent for breast cancer treatment (Lee et al., 2005). It was found that treatment of MCF-7 and MDA-MB-231 cells with Polyphyllin D resulted in the inhibition of viability and induction of apoptosis in a dose dependent manner. Mechanistically, Polyphyllin D dissipates the mitochondrial membrane potential, induces a down regulation of anti-apoptotic Bcl-2 expression (apoptosis regulating protein) and an up regulation of pro-apoptotic Bax-expression and activate caspase 9; this further suggest that saponins of this species may be potentially used as an anticancer agent against human breast cancer.
The bioactive natural products from the rhizome of P. polyphylla, i.e., polyphyllin D, polyphyllin C and Saponin -I showed mild to moderate anti-leishmanial activities (Devkota et al., 2005). Moreover, roots also exhibited antibacterial action against Bacillus dysenteriae, Bacillus paratyphi, Escherichia coli, Staphylococcus aureas, Haemolytic streptococci, Meningococci and some other bacteria (Anonymous, 2002). Further research is needed to screen out the phytochemical compounds responsible for the pharmacological activities of this herb. The phytochemical bioactive compounds of P. polyphylla and their derivatives can therefore be used as a potential source for the discovery of new drugs.

Diosgenin: synthesis:-
Diosgenin is a naturally occurring steroid also present in P. polyphylla and is known to suppress fatty acid synthase expression in over expressing breast cancer. It has the potential to advance as chemo-preventive and chemotherapeutic agents for cancers. Structurally, it is similar to cholesterol and other steroids ( Figure 1a). Since its discovery, diosgenin is the single main precursor in the manufacture of synthetic steroids in the pharmaceutical industry (Djerassi et al., 1952). Structurally, diosgenin [(25R)-spirost-5-en-3b-ol] is a spirostanol saponin consisting of a hydrophilic sugar moiety linked to a hydrophobic steroid aglycone ( Figure 1b). Although, mevalonic acid (MVA) and isopentenyl diphosphate (IPP) pathway have been well studied however, the synthesis of Diosgenin starting from squalene synthase is still not very clear ( Figure 2). Limited studies have been reported in the area of pathway identification of diosgenin synthesis. It would be important to find out the key genes and enzymes that regulate the biogenesis of diosgenin, thus helping in pathway engineering, and subsequently enhancing the in vitro 852 production of pharmaceutically and potentially bioactive compounds of this important herb. Therefore, there is a need to take up further studies on diosgenin biosynthesis pathway.

Loss of diversity:-
According to World Health Organization (WHO), approximately 80% of world's population in developing countries depend on traditional medicines for primary healthcare (WHO, 2002) and in modern medicine too, nearly 25% are based on plant derived drugs (Tripathi, 2002). Habitat fragmentation and illegal trade from wild is considered one of the major factors for loss of plant number and diversity, and P. polyphylla is no exception with reckless and frequent removal; this directly affects plant dispersal, thus, reducing gene flow, increasing endogamy, and consequently disturbing the competitive capacity and adaptation of a species (Heywood et al., 2003, Oostermeijer et al., 2003. Therefore, due to high medicinal value, there has been large scale and unrestricted harvesting of the plant from the wild to meet its ever increasing demand for drug formulation by pharmaceuticals industries. The plant based rapid development of medicinal formulations in China resulted in increased illegal transportation from India has further led to reduction in natural populations of this species (Anonymous, 2002). Moreover, no such sustainable cultivation and attempts to replenish it in the wild have been reported.
Preserving the genetic diversity of species is one of the primary goals of conservation planning because long-term survival and evolution of species depend on the maintenance of sufficient genetic variability among populations to accommodate new selection pressures brought about by environmental changes (Barrett and Kohn, 1991). Population genetic diversity is affected by a number of factors including breeding system, seed dispersal, genetic drift, geographic range as well as the genetic isolation of populations and natural selection. P. polyphylla is an out crossing species that has nectarines and is entomophilous (Li, 1998;Sage et al. 2001), and is considered to possess high population genetic diversity. Under natural conditions the plants grow as short-lived perennials, which defoliate in the winter and grow from rhizome in the following year. Besides above mentioned different anthropogenic and changing climatic conditions, the vulnerability of the plant is further aggravated by its characteristics nature such as long seed dormancy period of more than 2 years, poor germination, slow growth and annual fluctuations in the production of seeds (Li, 1984;1986;Zhou et al. 2003). Of these various factors, breeding system is the primary factor that affects genetic diversity both among and within populations (Hamrick et al. 1990).

Propagation, cultivation and market scenario:-
Under natural conditions the plant propagates via seeds and rhizomes; these methods are well recognized method for multiplication and conservation of any endangered species. In addition, in vitro method can also be adopted for multiplication of this species as this technique has proven to be an alternate and effective means of rapid propagation that can facilitate the recovery of Rare Endangered Threatened (RET) Himalayan herbs (Chandra et al. 2006;Nadeem et al. 2000;Nandi et al. 2002;Pandey et al. 2004;Purohit et al. 2015). Recently, in vitro plant regeneration through direct somatic embryogenesis for this species as well as protocol using thin cell layer culture were developed (Raomai et al. 2014(Raomai et al. , 2015. Propagation by conventional as well as in vitro methods can be employed for mass scale multiplication of quality propagules for systematic cultivation in locations closer to their natural habitats (in alpine and sub-alpine areas) near village clusters. Keeping in view the applicability of the above mentioned methods it is desirable to apply simple methods like enhancing seed germination or propagation via rhizome segments, their hardening, acclimatization and subsequent field transfer. It must be emphasized that greater awareness needs to be created and the local inhabitants should be motivated to cultivate medicinal plants as an alternative source. In view of higher economic returns and commercial demand, cultivation packages need to be developed for the farmers. In order to implement such programmes effectively nurseries need to be established for supplying healthy and elite planting material. Non-government organizations, State Forest Departments, Village communities and voluntary bodies can be encouraged and suitably funded to undertake conservation (through cultivation) by planting clonally propagated as well as plants of seed origin in their natural habitat. Commercial cultivation of this species in India is yet to commence, although it is being practiced in China. Therefore, combined and sustained efforts with full participation of the local communities are required to reduce the pressure on natural populations. The plant is generally harvested in the senescence stage (October) because the yield of bioactive constituents is high at this time. It was found to be traded in bulk amount in the past to the cities in Nepal, namely, Pokhara and Kathmandu and presently in the different villages of Kumaun Region, Uttarakhand (India). It has been informed (following discussion with local inhabitants of Khati Village, Bageshwar, Uttarakhand) that species is being exported to neighbouring countries. Due to high price value of the rhizome along with increasing trade, villagers are now attracted towards collection of Paris polyphylla, a relatively easy option for livelihood.

Modern tools for conservation:-
The physiological characters of the seeds along with habitat fragmentation are some of the reasons for low genetic diversity of the natural population of P. polyphylla. However, in the long run, it would be pertinent to conserve this species and reintroduce them in its natural habitat by the application of suitable methods. One such method is Ecological Niche Modelling, also popularly known as Species distribution modelling, is a recent tool which uses computer algorithms to generate predictive maps of species distributions in a geographic space by correlating the point distributional data with a set of environmental raster data. A study relating to the prediction of the ecological niche of P. polyphylla species across Western Himalayas (in Uttarakhand) needs to be undertaken using Maxent modelling approach as reported for other species (MaxEnt version 3.3.3e; Phillips et al., 2006). The different bioclimatic variables can be used to predict the potential distribution of P. polyphylla in the study area. Jackknife test will be used to evaluate the importance of the environmental variables for predictive modelling. Predictive distribution models aid in forecasting the spatial occurrence of species, especially habitat suitability or realized niche based on the data from field work in relation with climate and topographic factors (such as slope, elevation, and precipitation) (Pearson, 2007). The habitat suitability or niche prediction is done through various algorithms or principle which usually integrates the species occurrence information and environmental data to find out the possible favourable places. To obtain a continuous supply of plant material in vitro techniques can be used for multiplication and genetic improvement. It must be mentioned that at present a number of biotechnological approaches are being used for the conservation and improvement of plant species for desired traits.
The following areas of research on P. polyphylla have not been addressed so far and needs to be undertaken: 1. Quantitative assessment of geographical distribution and status of population using Ecological Niche Modelling. 2. Species-specific conservation plan and identify the bottlenecks that leads to the dispersal of plant from nature. 3. The morphological, biochemical and genetic diversity. 4. Standardization of plant propagation techniques (biotechnological and conventional) and scaling up production. 5. Development of suitable methods for reintroduction of the species in their natural habitat.

Conclusion:-
Biotechnological and ecological approaches are extremely important and essential for setting up of conservation and management step or plan for plant species that are mainly rare and endangered. Paris polyphylla is an economically and medicinally important species and is categorized as endangered (CAMP), and thus needs such approaches for its long term survival and sustainable utilization. Considering the ecological features and population status of this species, proper designing and implementation of effective conservation program need to be undertaken using Ecological Niche Modelling. The use of tissue culture (in vitro) biotechnological and conventional methods of multiplication for their reintroduction in natural habitats and niche areas of the species will be a significant initiative for its conservation. Since, the genus is important and has potential commercial application, hence immediate attention should be focused on its management, conservation and sustainable utilization. A thorough look of the summary of work depicted in Fig.3 clearly reveals various studies already carried out and other areas that remain unattended. Furthermore, keeping in view the demand and status of this species, it is necessary to create awareness programmes for conservation, develop agro-technologies and conduct further research in order to overcome the problem being faced by this species.