Received , revised

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1 Indian Journal of Traditional Knowledge Vol. 13 (3), July 2014, pp Ethnopharmacological survey, Phytochemical screening and Antibacterial activity measurements of high altitude medicinal plants of Nepal: A bioprospecting approach Janardan Lamichhane* 1, 2, Surya B Chhetri 1, 3, Mukund Bhandari 1, Sameer Pokhrel 1, Anaya Pokharel 2,3 & Jae Kyung Sohng 2 1 Department of Biotechnology, Kathmandu University, Dhulikhel, Kavre, Nepal; 2 Department of Pharmaceutical Engineering, SunMoon University, Asan-si, Chungnam, South Korea; 3 Research Institute for Bioscience and Biotechnology (RIBB), Kathmandu, Nepal ljanardan@ku.edu.np Received , revised The ethnopharmacological survey was undertaken to collect information about the use of seven most used medicinal plants, viz. Gentiana depressa D. Don, Rhododendron setosum D. Don, Rhodiola spp., Elsholtzia strobilifera (Benth.) Benth., Hedychium spicatum Sm., Eriophyton wallichii Benth., Rheum L. spp, in the region which is defined as a most potent area of high altitude medicinal plants by the Government of Nepal. The medical indication of each plant was reported on the basis of their uses by traditional healers and ethnic groups. Following the survey result, extraction of the samples was carried out by solvent extraction process in petroleum ether and cold methanol in view to compare their activity preliminarily. Phytochemical tests, cytotoxic effect (LC 50 ) and zone of inhibition (ZOI) against 13 different bacteria and 5 different fungi indicated the medical value of these plants that supports their traditional uses. The results of this study reinforce bioprospecting of the plants as a potential and promising drug candidate for the future pharmacological industries. Keywords: Gentiana depressa, Rhododendron setosum, Rhodiola spp, Elsholtzia strobilifera, Hedychium spicatum, Eriophyton wallichii, Rheum spp, LC 50, Antimicrobial IPC Int. Cl. 8 : A61K 36/00, CO1, CO7, C12Q Plants with medicinal value offer us new sources of drugs which have been used for centuries in traditional medicine. There are many compounds used in medicines today which are derived from plants. Undoubtedly, plant kingdom has many plants containing substances of medicinal value which are yet to be discovered (WHO Monographs on Selected Medicinal Plants Vol. 1, 1999). Topographical characteristics of the Himalayas have resulted in a variety of ecological niches that host diverse medicinal plants 1. The earliest written record of plants used as medicine originating from the Himalayas are found in the 6, yrs old texts of the Rigveda 2, followed by Atharvaveda (2000- BC) and Ayurveda ( BC) 3-4. Medicinal herbs have been collected in the Nepali Himalayas since 4 BC; written Ayurvedic knowledge was traveling back and forth between the Nepali Himalayas and South Asia as early as 879 AD 5. Ayurveda, the ancient *Corresponding author South Asia medical tradition associated with Hinduism, has often relied on medicines obtained from the biodiversity of the Nepali Himalayas. Owing to the history of Ayurveda and the complex topography with rich floral diversity, Nepal possesses several drug compounds that have been sought for modern day clinical research. Nepal extends along the Himalayan range between the altitudes 26 22' 30 27' and longitudes 80 04' 88 12'. Altitudes vary from less than 60 m in the lowland of Terai in the South to the crest of the Himalaya reaching 8848 m in the North. Moreover, the country ranks between 25 th and 30 th on the global scale and 11 th on the continental scale for richness in floral diversity. Nepal's flora comprises 2.5% of the total global flora. Of the flowering species, 5% are endemic to Nepal and 30% are endemic to the Himalayan Mountains. The complex topography and geology together with the varied climatic conditions have enabled a wide spectrum of vegetation types. These include small areas of subtropical forest

2 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL 497 (below m) Oaks, chirpiness, maple, fir, blue pine, hemlock spruce and (notably the incredibly versatile lichens) 6. Nepal Himalayas include about 2,000 species with medicinal and aromatic values, and more than 1,400 of these are known to be used locally particularly as medicines 10. The dialectical relationship between indigenous knowledge and practices shape, the ecosystem and affects the constituent plant populations 12. By incorporating indigenous knowledge and use in the process of scientific research, new hypotheses for the sustainable conservation of the resources can be developed 13. Indigenous knowledge and use have to be analyzed to develop appropriate management measures that build on both scientific and local knowledge 14. Due to changing perception of the local people, and the ever increasing influence of global commercialization and socio-economic transformation, indigenous knowledge on plant resource use is constantly diminishing Lying in the Himalayan range, Langtang National Park (LNP, Nepal) is a unique habitat for a number of highly valuable medicinal and aromatic plants (MAPs) 17. It belongs to Rasuwa district, Bagmati Zone of Central Nepal, enclosing the catchments of two major river systems of Nepal- the Trisuli River and the Sun Koshi River. It is the Himalayas that are often the wellspring for this medicinal and other ethnobotanical knowledge and the products produced from this knowledge are a rather large cog in the local economies. Various species of Rhododendron make up the main forest species. Above these alpine scrub and grass (up to 0 m) give way to rocks and snow. Culturally the area is mixed, the home of several ethnic groups. The majority of people are Tamang followed by Sherpa, an ancient Nepalese race groups. They consider the medicinal herbs as the main ingredients of their local medicine, and the traditional healthcare system as the main lifeline. Higher uses of plants as ethnomedicine at higher altitude was attributed to the absence of modern medical facilities 18 that intensify the uses of plants as remedies in mountain areas. High altitude medicinal plants provide quality products 19-20, and this is the reason why they are often the first choice of local users as immediate therapy and by pharmaceutical companies as precious ingredients. Materials and methods An Ethnopharmacological survey starting from Syabrubesi was carried out in Langtang National park (situated at Latitude: 28 13' 0 N, Longitude: 85 34' 60 E) in the month of September and October Subsequently, several samples were collected after the identification of their importance on the basis of the interaction with the healers and the old age people. The botanical names of each plant were authenticated from the Royal Botanical Gardens, Godavari, Kathmandu Valley, Nepal and deposited in the Department of Biotechnology, Kathmandu University as stated. The samples were then extracted, following different biological assays in view to compare whether there is any preliminary matching of their value as traditional medicines or not. Survey and sample collection In total 34 healers and old ages households were interviewed in this survey; 15 healers in Dhunche village of Syabrubesi area, 3 Chandan bari village, 7 in ThuloSyabru village, 4 in Lama Hotel village, 3 in Langtang village and 2 in Bamboo village, regarding the medicinal and other related uses of the plants available around them. The healers were asked about their process of using, parts of the plant used and the doses for concerned diseases. Though the conversations were focused to build the trust for the common goal of utilizing and increasing the knowledge of medicinal plants of the country to improve the health situation of the country and best utilization of available resources of the country, in most of the cases healers were reluctant to explain the exact doses of plants usage for specific disease. This might be due to the secrecy of their endangered job. In respect of tradition, small gifts bought from Kathmandu and money was bestowed upon the traditional healers. The interviews were performed in Nepali language along with their Native Tamang and Sherpa language for which interpreters were used. Based on the interviews and suggestions, different plant samples were collected from different altitudes of the surrounding areas. In some cases, a long travel and hard rock climbing was performed with the help of the local people there, just to find the specified plant. The seven different high altitude medicinal plants, viz. Gentiana depressa D.Don, Rhododendron setosum D. Don, Rhodiola spp., Elsholtzia strobilifera

3 498 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 3, JULY 2014 (Benth.) Benth, Hedychium spicatum Sm., Eriophyton wallichii Benth, Rheum spp, were chosen as best plant samples of this survey though many other plants were also come to know as best medicinal value but due to the seasonal difference many of the plants were already away from the scene and could not be collected. They were already lost or covered by the snow as the snow fall was already started to some parts of the collection site chosen during the time. The plant samples collected belongs to the region from Chandann bari (3,584 m), Lauri Binayak (3,900 m) Gosaikunda (4,360 m) to Suryakunda (4,800 m) and rocky hills around the area. Specifically, Eriophyton wallichii Benth, Rhodiola spp. and Rhododendron setosum D. Don were collected from Suryakunda, Gentiana depressa D.Don was collected from Gosaikunda, Elsholtzia strobilifera (Benth.) Benth and Rheum spp were collected from Lauribinayak and Hedychium spicatum Sm.was collected from Chandanbari (Fig. 1). The scientific name of the plants was identified with the help of senior taxonomist and the National Herbarium centre, Godaavari, Nepal. All the plants samples were kept as herbarium in the Department of Biotechnology, Kathmandu University. The herbarium of each plants were deposited to Biotechnology department of Kathmandu University and specimen number was provided as Gentiana depressa D.Don ( KU), Rhododendron setosum D. Don ( KU), Rhodiola spp. ( KU), Elsholtzia strobilifera (Benth.) Benth ( KU), Hedychium spicatum Sm. ( KU), Eriophyton wallichii Benth ( KU) and Rheum spp ( KU). The importance of the plants for research purpose was identified based on the literatures and supportive documents from bibliographic record of Medicinal Plants of Nepal by HMG/N (1976) including other pertinent taxonomic literature. The plant samples collected belongs to the region from Gosaikunda (4,360 m) to Suryakunda (4,800 m) and Godatabala (3,100 m) to Kyanjin Gumba (3,800 m) region of Langtang. All these points belong to the Langtang National park buffer zone region of Rasuwa district, Nepal. Extraction The dried whole plant sample was taken and crushed in grinder to convert it in powder form. Weighed gram of crushed sample was taken and measured volume of petroleum ether was poured on it and was shaken regularly. After 24 hrs, filtration was done and fresh petroleum ether was again added followed by storing of filtrate. Upon the completion of petroleum ether extraction, the sample was dried and further extraction was carried out in methanol following the similar process. The numbers of extractions were determined by visualizing the color of the filtrate. Finally, the collected filtrate was vaporized using rotavapor and water bath at 40 0 C. The final extracted amount was weighed to calculate the extract yield. Phytochemical screening Different tests were performed to check the presence of active chemical constituents such as alkaloids (Mayer s test), glycosides (NH 4 OH test), terpenoids and steroids (Leibermann- Burchard s Test), reducing sugar (Fehling s test), tannin and polyphenols (Ferric chloride test), flavonoids, flavones and flavonones (Methanol solution with Mg and HCl) and saponin (vigorously shaking the extract and checking its thick froth) (Table 1). Brine shrimp assay Brine shrimp assay was carried out by preparing test sample of each plant species in different concentrations (1,000, 100, 10 ) and test their activity against brine shrimp nauplii. Hatching of brine shrimp was carried out by preparing artificial sea water in test tubes and lethal concentration 50 (LC 50 ) value of each sample was calculated against the number of death of nauplii (Table 2). Antimicrobial assay Antimicrobial assay was performed by diluting the plant extracts in different concentrations (1,000 & ) and tested against different pathogenic and non-pathogenic bacteria and fungi using different sterilized growth medium at suitable temperature (usually 37 C for bacteria and C for fungus) for hrs zones of inhibitions were measured with the help of inhibition zone reader by measuring zone of inhibition from the circumference of the disc. Results Hedychium spicatum Sm. (Zingiberaceae) Hedychium spicatum with the local name Kapoor kacharo or Panee saro is a rhizomatous fragrant leafy herb with robust stem, and is up to 1.5 m tall. Rhizomes have strong aromatic odour and bitter

4 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL 499 Fig. 1 Collected plants in this part of the research and their habitat altitudes

5 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 3, JULY 2014 Table 1 Phytochemical screening of plant samples Plant Anthraquinone Saponin Cardioglycoside Tannin R. sugar Alkaloids Flavonoids Rheum spp Gallic tannin + - Flavones E.wallichii Benth E. strobilifera (Benth.) Benth G. depressa D.Don Catecholic tannin (Green black) + + orange color flavones Hedychium Spicatum Sm Rhododendron (Greenish ) + + Faint orange setosum D. Don Catecholic tannin Rhodiola Catecholic tannin species(leaf) (Green black) Rhodiola species(root) - - Pinkish ring persistent Catecholic tannin red Extract Rheum spp. Eriophyton wallichii Benth E.strobilifera (Benth.)Benth G. depressa D.Don Hedychium spicatum Sm. R. setosum D. Don Rhodiola spp. (leaf) Rhodiola spp. (root) Extract conc () (A) Table 2 Brine Shrimp Bioassay for cold methanol extract (24 hrs) Log (A) Initial nauplii Nauplii survival n=1 Nauplii survival n=2 Nauplii survival n=3 Mean survival Death % death camphoraceous taste. Leaves are up to 30 cm long, lanceolate, with green, obtuse bracts. The traditionally Hedychium spicatum Sm is found to be used as a home remedy to treat several diseases. Based on the survey report the use of it for indigestion (50%) and skin infection (20.5%) are the most typical reported ailments followed by snake bites (14.7%), and several others were used for blood purification purpose as well (Fig. 2). The three most used mode of preparation were decoction(51.7%), powder (34.4%) and juice was prefered by (13.8%) (Fig. 3) and sometimes as a drink as well. Moreover, the frequent part used for this plant were rhizome (55.1%) and roots (34.4%), while the fruits accounted for decently low (10.3%) (Fig. 4) for remedial purpose. The phytochemical screening and biological activities test in cold methanol extract (2.158% yield) and petroleum ether extract (0.683% yield) was found

6 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL 501 to contain cardioglycosides and alkaloids as major chemical constituents. The LC 50 value for 24 hrs was 100 whereas an outstanding zone of inhibition was observed against pathogenic bacteria, Enterobacter aerogenes (8 mm), Citrobacter freundii (5 mm), Salmonella typhi (4 mm), Bacillus thuriengiensis, S. aureus (4 mm), E. coli (4 mm) and Pseudomonas aeruginosa (4 mm) at 1,000 (Table 3). A significant inhibition was observed against fungus, Candida albicans (2 mm) and Rhizopus oryzae (2 mm) at 1,000 (Table 4). Elsholtzia strobilifera (Benth.) Benth. (Lamiaceae) Elsholtzia Strobilifera with the local name Ban bawari or Thangnenoba is erect, annual herb with ovate, crenate or serrate, stalked leaves and dense, cylindrical spikes of small, pink-purple flowers concealed by overlapping involucres of broad, obcuneate, truncate bracts. Stem is 5-15 cm tall; branches and stem is brown, crisped white pilose. Traditionally the healers were found to use Elsholtzia strobilifera (Benth.) Benth as a remedial purpose against headache that accounted for (58.82% reported ailments) while wounds and boils (14.70%) followed by skin infection (5.88%) (Fig. 2). In the curing process, the mode of preparation was primarily in powdered form seed (48.14%), plant paste (40.74%) and the rest of others aggregated to 11.11% (Fig. 3). Furthermore, part of the plant that most frequently accounted was entire plant (62.96%), seed (22.22%) and stem (14.81%) (Fig. 4). The phytochemical screening and biological activities test in cold methanol extract (yield 6.833%) of and petroleum ether (4.634% yield) showed positive for alkaloids and saponin. The LC 50 value for 24 hrs was whereas the zone of inhibition against pathogenic bacteria such as Citrobacter freundii (5 mm) in, Bacillus thurengensis (4 mm) in and Klebsiella pneumoniae (3 mm) at were excellent (Table 3). A significant zone of inhibition was observed against fungus, Rhizopus oryzae (2 mm) and Alternaria alternata (2 mm) at 1,000 (Table 4). Rhodiola spp. (Crassulaceae) Among the distinguishing characters of the genus Rhodiola, are two series of stamens totaling twice the number of petals free or nearly free petals; a stout rhizome from whose axils the flowering stems rise; and a basal rosette of leaves. Traditionally Rhodiola spp. Is found to be used for various stimulator Fig. 2 Main medical indications for each plant Fig. 3 The main methods of preparation of each plant to use as medicine Fig. 4 The parts of the plant used for the medical purpose purposes including the remediation against many diseases as well. It is reported to be used particularly the roots for fertility and sexual function (47%) while depression accounted for (32.35%) and surprisingly tuberculosis (8.82%) (Fig. 2), and few of them reported for cancer as well though the kind of cancer was not proved as the healers themselves don t know the term clearly. Rhodiola leaf was found to be used especially for fever that accounted for 26.47%, and scurvy (20.58%) followed by the bolstering process of

7 502 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 3, JULY 2014 immune system (8.82%). During treatment, the mode of preparation most preferred was decoction and powder (53.33%), followed by plant paste (33.33%) and others (13.33%) (Fig. 3). Plant parts mostly opted was root (50%), leaf (33.33%) and the flowering stem followed (16.66%) (Fig. 4). Cold methanol extract (8.935% yield) of Rhodiola spp. (root) and petroleum ether extract (3.105%) showed positive for cardioglycosides, reducing sugar, flavonoids, steroids and catholic tannin as major chemical constituents. The LC 50 value for 24 hrs was 2398 whereas the zones of inhibition against pathogenic bacteria such as Citrobacter freundii (5 mm) at, Salmonella typhi (4 mm) at and Styphylococcus aureus (3 mm) at were very good. A comparatively high zone of inhibition was shown against fungus, Candida albicans (3 mm) and Penicillium (2 mm) at. Similarly, Cold methanol yield (4.675%) Rhodiola spp. (leaf) and petroleum ether yield (1.071%) was found positive for steroids and catholic tannin. The LC 50 value for 24 hrs was 6456 and zone of inhibition against Klebsiella pneumoniae (5 mm), Citrobacter freundii (4 mm) at 1,000 and Pseudomonas aeruginosa (4 mm) at (Table 3) whereas a significant zone of inhibition was observed against fungus, Penicillium (2 mm) and Alternaria alternata (2 mm) (Table 4). Eriophyton wallichi Benth. (Lamiaceae) Eriophyton wallichii Benth locally named as Kapase Jhar, Myiashae (Tibetan name) is tuft flowering plant with stem erect, purple- reddish color perennial plant growing cm. It flowers from July to September (late summer), and the seeds ripen from September to October. Traditionally Eriophyton wallichi Benth is used as a home curative in defense against several medical problems. Primarily, as anti inflamatory (44.11%) and antiseptic (20.58%); while 5.88% importantly reported for antitumor (Fig. 2). The method of preparation preferred was mostly in the powder form (55.17%) while others accounted for 44.83% (Fig. 3). In the treatment process, the entire plant was predominantly used consequently accounting for (100%) rather than any specific parts of the plant (Fig. 4). Cold methanol extract (2.4% yield) and petroleum ether (2.3% yield) of Eriophyton wallichi Benth was found positive for steroids and alkaloids compound. The LC 50 value for 24 hrs was where as the zone of inhibition against pathogenic bacteria such as Shigella dysenteriae (7 mm) for, Salmonella typhi (5 mm) and 4 mm for and 1,000 respectively, while Citrobacter Freundii (4 mm) at (Table 3). A little zone of inhibition was observed against fungus, Rhizopus oryzae (2 mm) at and Penicillium sp.(2 mm) for both at and 1,000 (Table 4). Gentiana depressa D.Don (Gentianaceae) Locally named as Glagengaga, is a perennial herb which is 3-6 cm tall. Stolons to 15 cm, much branched, stem less or very short stems, erect, simple. Stem leaves are crowded and surrounded by flowers; petiole 5-12 mm. Traditionally Gentiana depressa is used as an appetizer and digestive (41.17%) whereas cough cold reported for 17.64% followed by fever (14.70%) (Fig. 2). The most common mode of preparation for the purpose of home remedy was accounted by juice (48%), decoction (32%) followed by powder (20%) (Fig. 3). Moreover, among most of the plant parts utilized by the traditional healers, the two most prevalent remedial methods was employed using the flower (60%) while the other one employed was typically entire plant (40%) (Fig. 4). Cold methanol extract (6.115% yield) and petroleum ether (2.315% yield) of Gentiana depressa showed positive test for reducing sugar, alkaloids, saponin, catholic, tannin, flavonoids and steriods. The LC 50 value for 24 hr was whereas the zone of inhibition against Salmonella typhi (5 mm) at and, Citrobacter freundii (4 mm) at and, Salmonella paratyphii (4 mm) at (Table 3). A significant zone of inhibition was observed against fungus, Rhizopus oryzae (2 mm), Penicillium sp. (2 mm) and Alternaria alternata (2 mm) at (Table 4). Rheum L. spp. (Polygonaceae) Locally named as Padamchal, is herb which is about 1.5m high with woody rootstock. Leaves stalked, orbiculate or broadly ovate, cordate, entire, glabrous above, sparsely hairy on the veins beneath. Flower is dark purple. Fruit is oblong, ovoid, purple, winged, notched at the apex. Traditionally Rheum spp. is used as a natural remedy for various medication purposes. Main medicinal indication reported by the study was antiseptic (47.05% of the reported ailments), constipation (20.58%) and importantly as an antitumor (11.76%) (Fig. 2). Decoction (44.44%) and a preparation of powder (29.63%) are the most commonly used preparations

8 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL 503 R.setosum D. Don Rheum spp. Table 3 Antibacterial activities against plant samples H. spicatum Sm E.strobilifera (Benth.)Benth R. spp. (leaf) G. depressa D.Don E.wallichii Benth R. spp. (root) Salmonella typhi Bacillus thuriengiensis Cytobacter fruendii Pseudomonas aeroginosa Proteus mirabilis S. aureus Eshcherichia coli Bacillus cereus Kleibsella pneumonae Shigella dysenteriae S.paratyphii E. aerogenes B. subtilis Sample Table 4 Antifungal activities against plant samples Candida albicans Aspergillus flavus Penicillium Alternaria Rhizopus R. setosum D. Don 0 0 1mm 0mm 2mm 1mm 2mm 2mm 1mm 1mm Rheum spp H.spicatum Sm E. strobilifera (Benth.) Benth Rhodiola spp. (leaf) Rhodiola spp. (Root) G. depressa D.Don E. wallichii Benth Nistatin (Standard Antifungal) 5 mm 10 mm 5 mm 10 mm 5 mm DMSO (control) 0 mm 0 mm 0 mm 0 mm 0 mm while the paste method accounted for 25.92% (Fig. 3). In as much as, the most frequent part of the plant used in the process were dried leaves (59.25%), and roots (22.22%), and others (18.51%) (Fig. 4). The phytochemical screening and Biological activities measurment of cold methanol extract (4.69% yield) and petroleum ether (0.505% yield) of Rheum spp. was positive for anthraquinone, reducing sugar, flavonoids, saponin, steroids and gallic tannin. The LC 50 value for 24 hrs was whereas the zone of inhibition against pathogenic bacteria such as E. coli (5 mm), Citrobacter freundii (4 mm) and Pseudomonas aeruginosa (4 mm) at 1,000 were very high (Table 3). Significant zone of inhibition was observed against fungus, Penicillium sp. (2 mm) at 1,000 (Table 4).

9 504 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 3, JULY 2014 Rhododendron setosum D. Don (Ericaceae) Rhododendron setosum with the local name Jhuse sunpati or silu or siru is erect shrub which is m tall, usually intricately branched and young shoots densely scaly with flaky scales. Petiole is 1 3 mm, scaly, whereas leaf blade is ovate, elliptic, and oblong to obovate, cm long and base is rounded. Traditionally Rhododendron setosum is found to be used by traditional healers as a natural home medication in our survey. Ergo, the most frequently reported purpose was as an herbal tea (44.11%) for improving the efficacy of immune system while against diarrhea (17.64% of the reported ailments for Rhododendron setosum D. Don) followed by fever and antiseptic (14.70%) (Fig. 2). The two most commonly opted modes of preparation were powder (42.30%) and preparation of a decoction and powder (34.61%) and rest is of others forms (23.07%) (Fig. 3). Besides, the frequently preferred parts of plant subsume dried petals (46.15%) and stem entails (38.46%) and other parts of the plant accounted for 15.38% (Fig. 4). High yield in cold methanol extract (4.56%) of Rhododendron setosum D. Don as compared to petroleum ether (1.96%) showed to contain reducing sugar, alkaloids, saponin and tannin in Phytochemical screening with the LC 50 value of 1,862 at 24 hrs in brine shrimp assay whereas the zone of inhibition against some pathogenic bacteria such as E. coli (8 mm), Salmonella paratyphii (6 mm) and Pseudomonas aeruginosa (5 mm) was outstanding at the extract concentration of 1,000 (Table 3) and a significant zone of inhibition against fungus, Penicillium sp. (2 mm) and Alternaria alternata (2 mm) (Table 4). Discussion Hedychium spicatum Sm. was found to contain varieties of sesquiterpenes with their potoent anticancer properties 21 which is menifested by the presence of alkaloids and cardiogycoside as phytochemicals in this study with their high activity against pathogenic bacteria such as Salmonella typhi, Staphylococcus aureus, Kleibsella pneumonae, Shigella dysenteriae and Bacillus subtilis including some other microbials (Table 3). The cytotoxicity (LC50) against brine shrimp bioassay (100 ) indicated it as a potent source of drug. The monoterpenoids, oxygenated monoterpenoids as a major constituents as secondary metabolites present in the essential oil of Hedychium spicatum 22 approves its importance in traditonal therapy. The essential oil of Elsholtzia strobilifera contains oxygenated monoterpenes with neral and geranial as major sesquiterpenoids 23. Similar finding has been reported as major constituents in the essential oils from Elsholtzia eriostachya var. pusilla 24, Elsholtzia ciliata 25 and Elsholtzia cristata 26. Presence of saponin and alkaloids as phytochemical and a significant zone of inhibition against Bacillus thurengensis and Kleibsella pneumonae bacterial strains along with Rhizopus oryzae and Alternaria alternate fungus observed in this reserch is in allign of it traditional uses. Rhodila spp. (Rhodila rosea) is a popular in traditional medicine system with a reputation for stimulating the nervous system, decreasing depression, enhancing work performance, eliminating fatigue and preventing high altitude sickness 27. Rhodila is popularly used for anxiety and other mood related disorders Rhodiola has been traditionally used to mitigate depression and its root have been found to inhibit monoamine inhibitors 30. Ethonopharmacological survey, Phytochemicals (presence of cardioglycoside, tannin Reducing sugar and flavonoids in the root and tannin in the leaf) along with antibacterial activities against Cytobacter fruendii, Kleibsella pneumonae and S. paratyphii establishes the activities of Rhodiola spp. Eriophyton wallichi is found to contain Iridoid glycosides and glycosidic constituents four of which showed moderate DPPH free radical scavenging activity 31. The high antibacterial activity of Eriophyton wallichii Benth against Salmonella typhi and Shigella dysenteriae along with high LC50 value (10 ) against Brine shrimp showed its potential activity as stated in the survey. The aerial parts of Gentiana depressa D.Don contains the flavones, isoscoparin, isoorientin and iridoid glucosides, iridoid depressoside and depressine The observed saponin, tannin, Reducing sugar, alkaloids and flavonoids as phytochemicals with high activity against Salmonella typhi and Cytobacter fruendii antibiotics and funguses is in agreement to the research study. Rheum spp. with anthraquinone, tannin, reducing sugar and flavonoids content as phytochemical screening and moderate activities against different bacterial species except E.coli and fungus species is in align with the traditional uses. The low content of oxalate and malate in this species can have its traditional value 35. Very less research finding was observed in Rhododendron setosum D. Don that is found to contain saponin, tannin, Reducing sugar, alkaloids and flavones. The high antibacterial activity against Pseudomonas aeroginosa, E. coli,

10 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL 505 S. paratyphii and Shigella dysenteriae, a moderate cytotoxicity effect in Brine shrimp bioassay indicates its importance as antiseptic anti-diarrheal uses as observed in ethnopharmacological survey. The high altitude medicinal plants contain secondary metabolic compounds as an adaptive strategy to reduce the damaging effects The beneficial medicinal effects of plant materials typically result from the secondary products present in the plant although, it is usually not attributed to a single compound but a combination of the metabolites. The medicinal actions of plants are unique to a particular plant species or groups are consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct 36. Plant extracts are potential sources of novel antimicrobial compounds especially against bacterial pathogens. In vitro studies in this work showed that the plant extracts inhibited bacterial growth but their effectiveness varied. Intensive use of antibiotics often resulted in the development of resistant strains 37, these create a problem in treatment of infectious diseases, furthermore antibiotics sometimes associated with side effects 38, whereas there are some advantages of using antimicrobial compounds of medicinal plants such as often fewer side effects, better patient tolerance, relatively less expensive, acceptance due to long history of use and being renewable in nature 39. Because of this, the search for new antibiotics continues unabated. These findings support the traditional and indigenous knowledge of local users though it is a preliminary scientific validation for the use of these plants for antibacterial and antifungal activity to promote proper conservation, and sustainable use of such plant resources. The indigenous knowledge and practice of usage of medicinal plants in rural areas of Nepal is passed down through oral tradition and personal experiences 40. Due to changing lifestyles, perception as well as social transformation, the plant resource and indigenous knowledge of utilization are being severely degraded 41. This impact is inevitable to the Nepal Himalayas and plant resources are in great peril. Indigenous knowledge systems are not only of value for the cultures from which they evolve, but also for scientists and planners striving to improve the living conditions in rural societies 42. Indigenous plant based traditional knowledge and use has become a recognized tool in search for new sources of drugs and pharmaceuticals 43. In countries like Kenya 44 and Nepal 45, where the indigenous knowledge is predominantly used for utilization of plant resource for various purposes and high priority needs to be given to the documentation of indigenous knowledge and use of plant resources to help their conservation. Conclusion The literature and the ethnopharmacological survey are in agreement with the traditional uses and importance of these plants in local community of the Hilly people. However, the impacts of the plants as drug need to be approved by the further study that could verify the specific use of each plant components for the diseases. This study enhances the interest of researchers to disclose various traditional techniques and plants materials used in remote villages of this country. A promising phytochemical and bioprospecting results were obtained for all the eight extracts of seven different medicinal herbs collected from the high altitude of Nepal. Along with the different test and TLC result (not shown) of methanol extract also confirmed the presence of various constituents as revealed by the multiple spots on the plates which were subjected to brine shrimp, antibacterial and antifungal assay for the evaluation of its biological activities. The results obtained from this survey and preliminary tests related to their validation, of course stimulate further research on these plants and their safety concerns in human health. The great medicinal importance of these medicinal plants provokes the serious concerns for its preservation and conservation from the concerned authority. Equally, it is important to aware the local community for their protective production and work together with the healers and herb practicing peoples in hand to hand for the maximum use of such indigenous and unique plants of Himalaya. The output of this research can have a milestone for the researchers in future that will ultimately reflect their importance to the society for their social and economic growth. Acknowledgement Authors are grateful to those traditional healers and old age groups of the community who gave us an unforgettable opportunity and to the local young men who not only helped to find the location of the plant but also helped as interpreters. This part of the work has been done is support of University Grant Commission (UGC), Nepal under the name of Faculty Research Grant-2011.

11 506 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 3, JULY 2014 References 1 Singh J S & Singh S P, Forests of Himalaya: Structure, Functioning and Impact of Man, Gyanodya Prakashan, Nainital, India, Malla S B & Shakya P R, Medicinal plants of Nepal, Nepal Natures'Paradise, edited by Majupuria T C, (White Lotus Ltd, Bangkok), 1984, Nambier V P K, Improved harvesting, processing and storage of medicinal plants: their role in conservation and quality of plant based drugs, Proceeding of Sharing Local and National Experience in Conservation of Medicinal and Aromatic Plants in South Asia, (2001) January 21 23, edited by Bhattarai N & Karki M, (Government of Nepal, IDRC and MAPPA), 2002, Kunwar R M, Nepal B K, Kshetri H B, Rai S K & Bussmann R W, Ethnomedicine in Himalaya: A case study from Dolpa, Humla, Jumla and Mustang districts of Nepal, J Ethnobiol Ethnomed, 2 (2006) IUCN Nepal: National Register of Medicinal Plants, Kathmandu, IUCN, Dalip K U, Pradeep K D & Sanjeeva N, Commercial and Ethnic use of Lichens in India, Econ Bot, 59 (2005) Shrestha G L & Shrestha B, An overview of wild relatives of cultivated plants in Nepal, In: Wild Relatives of Cultivated Plants in Nepal. Proceedings of National Conference on Wild Relatives of Cultivated Plants in Nepal, June 2 4, 1999, edited by: Shrestha R, Shrestha B, (The Green Energy Mission/Nepal), 1999, Tiwari N N, Wild Relatives of Cultivated Medicinal and Aromatic Plants (MAPs) in Nepal, In: Proceedings of National Conference on Wild Relatives of Cultivated Plants in Nepal, June 2 4, 1999, (The Green Energy Mission/Nepal), 1999, Shrestha K K, Tiwari N N & Ghimire S K, Medicinal and aromatic plants database of Nepal, Proceeding of Nepal- Japan Joint Symposium on Conservation and Utilization of Himalayan Medicinal Plant Resources, Nov 6 11, (2000), Kathmandu, Nepal, 2000, Manandhar N P, Plants and People of Nepal, Timber Press, Oregon, USA, Baral S R & Kurmi P P, Compendium of Medicinal Plants in Nepal, (Rachana Sharma publishers, Chabahil, Kathmandu, Nepal), Ghimire S K, McKey D & Aumeeruddy-Thomas Y, Heterogeneity in ethnoecological knowledge and management of medicinal plants in the Himalayas of Nepal: implications for conservation, Ecol Soc,9 (3) (2004) Henfrey T B, Ethnoecology, Resource Use, Conservation and Development in a Wapishana Community in the South Rupununi Guyana. [PhD dissertation] (University of Kentucky, UK), Ticktin T & Johns T, Chinanteco management of Aechmea magdalenae: implications for the use of traditional ecological knowledge and traditional resource management in management plans, Econ Bot, 56 (2002) Gadgil M, Birkes F & Folkes C, Indigenous knowledge of biodiversity conservation, Ambio, 22 (1993) Kunwar R M & Adhikari N, Ethnomedicine of Dolpa district, Nepal: the plants, their vernacular names and uses, Lyonia, 8 (2005) Shrestha N & Shrestha K K, Vulnerability assessment of high-valued medicinal plants in Langtang National Park, Central Nepal, Biodiversity, 13 (2012) Uniyal S K, Kumar A, Lal B & Singh R D, Quantitative assessment and traditional uses of high value medicinal plants in Chhota Bhangal area of Himanchal Pradesh, Western Himalaya, Curr Sci, 91 (2006) Mikage M & Mouri C, Altitudinal variations of Berberine content in the bank of Berberis plants from West Nepal, Newsletter of Himalayan Botany, 26 (2000) Iwashina T, Omori Y, Kitajima J, Akiyama S, Suzuki T & Ohba H, Flavonoids in translucent bracts of the HImalayan Rheum nobile (Polygonaceae) as ultraviolet shields, J Plant Res, 117 (2004) Suresh G, Poornima B, Suresh B K, Ashok Y P, Suri A R M, Siva B, Rajendra P K, Lakshma N V & Ramakrishna S, Cytotoxic sesquiterpenes from Hedychium spicatum: Isolation, structure elucidation and structure-activity relationship studies, Fitoterapia, 86 (2013) Verma R S & Padalia R C, Comparative essential oil composition of different vegetative parts of Hedychium spicatum Smith from Uttarakhand, India, Int J Green Pharm, 4 (2010) Melkani A B, Dev V, Beauchamp P S, Negi A, Mehta S P S & Melkani K B, Constituents of essential oil of a new chemotype Elsholtzia strobilifera Benth, Biochem Syst Ecol, 33 (2005) Pant A K, Dev V, Parihar R, Mathela C S, Rauscher J & Vostrowsky O, The essential oil from Elsholtzia eriostachya var. pusilla, J Essential oil Res, 4 (1992) Dung N X, Hae L V, Hai L H & Leclercq P A, Compositioin of the essential oil from the aerial parts of Elsholtzia ciliata (Thunb.) Hyland, J Essential oil Res, 8(1) (1996) Thapa R K, Agrawal S G, Dhar K L & Hai L H, Some useful aromatic plants of Vietnam, Indian Perfum, 40 (1) (1996) Gregory S K, Rhodiola rosea:a Possible Plant Adaptogen, Alternative Med Rev, 6(3) (2001) Panossian A, Wikman G & Sarris J, Rosenroot (Rhodiola rosea): Traditional use, chemical composition, pharmacology and clinical efficacy, Phytomedicine 17 (2010) Kiefer D, Rhodiola rosea for general anxiety disorder, Alternative Medicine Alert, 11 (2008) van Diermen D, Marston A & Bravo J, Monoamine oxidase inhibition by Rhodiola rosea L roots, J Ethnopharmacol, 122 (2009) Fan Q-L, Tan C H, Liu J, Zhao M M, Han F-S & Zhu D-Y, Iridoid glycosides and glycosidic constituents from Eriophyton wallichi benth, Phytochemistry, 72 (2011) Chulia A J, Vercauteren J & Mariotte M, Iridoids and flavones from Gentiana depressa, Phytochemistry, 42 (1996) Chulia A J & Kaouadji M, Depressoside nouvel iridoide isole de Gentiana depressa, J Nat Prod, 48 (1985) Chulia A J, Vercauteren J & Kaouadji M, Depresteroside, a mixed iridoid-secoiridoid structure from Gentiana depressa, Phytochemistry, 36 (2) (1994) Rumpunem K & Henriksen K, Phytochemical and morphological characterization of seventy-one cultivars and selections of culinary rhubarb (Rheum spp.), J Hort Sci Biotechnol, 74 (1) (1999)

12 LAMICHHANE et al.: SCREENING OF HIGH ALTITUDE MEDICINAL PLANTS IN NEPAL Wink D A, Vodovotz Y, Grisham M B, DeGraff W, Cook J C, Pacelli R, Krishna M & Mitchell J B, Antioxidant effects of nitric oxide, Methods Enzymol, 301 (1999) Sydney S, Lacy R W, Bakhtiar M, In:The Betalactam antibiotics Penicillin and Cephalosporin in perspective, (Hodder and Stongton, London), 1980, Cunha B A, Antibiotics side effects, Medicinal Clinics of North America, 85 (2001) Vermani K & Garg S, Herbal medicine for sexually transmitted diseases and AIDS, J Ethnopharmacol, 80 (2002) Bhattarai N K, Traditional medicines: role of medicinal plants in present and future health cure, In: Prospects of Medicinal Plants, edited by Gautam P L, Raina R, Srivastava V, Raychaudhuri S P & Singh B B, (Indian Society of Plant Genetic Resource, New Delhi, India), 1998, Silori C S & Rana A R, Indigenous knowledge on medicinal plants and their use in Narayan Sarovar Sanctuary, Kachchh, Ethnobotany, 12 (2000) Warren D M, Indigenous knowledge and development. Background paper for seminar session on sociology, Natural Resource Management and Agriculture Development, December 3, (1990), The World Bank, Sharma P P & Mujundar A M, Traditional knowledge on plants from Toranmal Plateau of Maharastra, India, Indian J Tradit Knowle, 2 (2003) Heine B, Heine I & Koenig C, Plant use and concepts an ethnobotanical survey of the semi-arid and arid lands of east Africa, Part V, Plants of the Samburu (Kenya), Koelner Beitraege zur Entwicklungsforschung, 10 (1988) Kala C P, Dhyani P P & Sajwan B K, Developing the medicinal plant sector in northern India: challenges and opportunities, J Ethnobiol Ethnomed, 2 (2006)