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Ecophysiological Study of Centella Asiatica (Linn.) Urban of Nepal

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Ecophysiological Study of Centella Asiatica (Linn.) Urban of Nepal Ecophysiological study of Centella asiatica (Linn.) Urban of Nepal By Anjana Devkota A Dissertation Submitted to Central Department of Botany Institute of Science and Technology, Tribhuvan University, Nepal For the award of Doctor of Philosophy (Ph.D) in Botany 2011 This work has been dedicated to my parent Babu Ram Devkota and Netra Kumari Devkota and my husband Chandra Kanta Bhandari iii ACKNOWLEDGEMENTS I am greatly indebted to my research supervisors Dr. Pramod Kumar Jha (Professor, Central Department of Botany, Tribhuvan University, Kathmandu, Nepal) and Dr. Gabriella Innocenti (Professor, Department of Pharmaceutical Sciences, Padova University, Padova, Italy) for their continuous guidance, encouragement and support throughout my work. University Grants Commission, Nepal provided partial scholarship for the support of this thesis work and Tribhuvan University granted study leave for one year during preparation of this dissertation. Exchange programme between Tribhuvan University, Nepal and Padova University, Italy provided me an opportunity to perform phytochemical analysis at the Department of Pharmaceutical Sciences, Padova University, Italy. I am very grateful to my colleague Dr. Bharat Babu Shrestha (CDB- TU) for his continuous helps and valuable suggestions during my entire research period. I am thankful to Dr. Krishna Kumar Shrestha, Professor and Head, Central Department of Botany, TU, for his encouragement and administrative supports. I am thankful to Dr. Ram Kailash Prasad Yadav (Central Department of Botany, TU) for critical comments and suggestion on the first draft of this dissertation. I am thankful to Dr. Suresh Kumar Ghimire (CDB- TU) for his valuable suggestions and in some statistical analyses. I am grateful to my colleague, Susanna Phoboo for her continuous encouragement and suggestion. I am indebted to Dr. Stefano Dall’Acqua and Dr. Stefano Comai of the Department of Pharmaceutical Sciences, Padova University, for their hospitality, helps and cooperation in phytochemical analysis. I am thankful to Prof Dr. Margherita Lucchin , Dr. Sabrina Canova and Dr. Marzia Salmaso, Department of Agronomy, Agripolis, Padova University, Italy for their kind support and cooperation in molecular study. I am also grateful to Prof Dr. Ram Prasad Chaudhary for his encouragement. Thanks are also due to Dr. Sangeeta Rajbhandary and Dr. Chitra Baniya for their kind suggestion. I am thankful to Dr. Deepak Raj Pant (Amrit Science Campus, TU) for his suggestion on molecular study. Thanks are due to Jyoti Gajurel, for his kind help in study of herbarium specimens. I am very grateful to NARC (Nepal Agricultural Research Council), Khumaltar, Lalitpur for providing me working place for molecular analysis. Thanks are also due to Dr. Hari P Bimb and Dr. Jwala Bajracharya of NARC for their help during molecular study. Dr. Raj Kumar Niroula, Scientist, NARC helped in molecular data analysis. Sambu Bista , Dhan Raj Kandel, Bishnu Prasad Baral, Saraswati Aryal, Kusum Sharma, Sanjeev Mishra, Salik Kalathoki, Prem Bhandari, Lila N Sharma, Basant Bhandari and Padam Regmi accompanied me in field samplings. Thanks are due to my former students Ravi Kumar Sharma, Bishnu Prasad Baral, Devendra Kunwar, Ravi iv Mohan Tiwari and Mitra Pathak who assisted me in my work. Thanks to Bal Krishna Ghimire, who sent me several references from Korea. I am thankful to administrative staffs and gardeners of Central Department of Botany, TU, for their cooperation and various helps. I am grateful to Department of Plant Resources (DPR), Thapathali, for allowing me to use Clevenger’s apparatus. I am thankful to National Herbarium Godavari (KATH) for allowing me to study herbarium specimens. Following local people at various locations helped variously during field samplings: Bal Bahadur Gharti, Raj Kumar Chaudhary, Tulsi Shrestha, Kalu Lama, Seeta Ram Gharti and Bishnu Gharti. I am deeply indebted to my sister- Bimala Paudel and brother- in – law Prem Prasad Paudel accompanied me during field samplings and for encouragement. I would like to thank to my husband- Chandra Kanta Bahandari for his moral support. I am deeply indebted to my lovely son -Spandan Bhandari for his patience during my work. Finally I am always indebted to my parent, brother, sisters, other family members and relatives for their continuous encouragements and supports. Anjana Devkota v ACRONYMS %: Percentage °C: degree Celsius a.u: Arbitrary units Asl: Above Sea level C: Central CDB: Central Department of Botany cm: centimeter CTAB: Hexadecyl-trimethylammonium bromide d.w: Dry weight DAD: Diode Array Detector DNA: Deoxy ribonucleic acid DPR: Department of Plant Resources E: Eastern ELSD: Evaporative Light Scattering Detector FYM: Farmyard Manure g: gram GA3: Gibberellic acid GC-FID: Gas Chromatography- Flame Ionisation Detector GC-MS: Gas Chromatography -Mass Spectrometry h: Hours HPLC: High Performance liquid Chromatography m: meter MAPs: Medicinal and Aromatic Plants MOFSC: Ministry of Forests and Soil Conservation MPs: Medicinal Plants N: Nitrogen OC: Organic Carbon OM: Organic matter PCR: Polymerase Chain reaction ppm: Parts per million RAPD: Random amplified polymorphic DNA RT: Room temperature SLA: Specific Leaf Area (cm2/g) TCM: Traditional Chinese Medicine TTC: 2,3,5-triphenyltetrazolium chloride TU: Tribhuvan University UPGMA: Unweighted pair group method with arithmetic averages UV light: Ultraviolet light W: Western WHO: World Health Organization vi ABSTRACT Centella asiatica (L.) Urban, commonly known as Indian Pennywort, is an important ethnomedicinal plant of tropical to subtropical region. It is a clonal perennial herb with a wide range of enthomedicinal uses such as blood purifier, memory enhancer, anticancer, antidepressive etc. Distribution pattern, abundance, life history traits, leaf nitrogen content, genetic diversity of population, and quantification of eight secondary metabolites of 21 populations of Centella asiatica from different habitats and regions of Nepal were studied. Effects of different environmental factors (moisture, soil texture, light and shading) and integrated manuring on growth traits and yield of C. asiatica were determined in pot-grown plants treated under randomized block design. Centella asiatica grows in a wide range of habitats from shady grassland, open grassland, to open agricultural land in tropical to temperate area in Nepal. In terms of density and plant biomass, partially shaded grassland was the most suitable natural habitat for C. asiatica. Density and biomass yields varied significantly with habitat types, with mean value of 72.53 pl/m2 and 37.95 g/m2, respectively. The flowering peak was recorded in April-June and it little varies in different habitats. The freshly collected seeds had the highest viability which declined progressively as the duration of storage increased. Pretreatment like soaking seeds with GA3 prior to sowing reduced the time required for initiation of germination. Seed germination of C. asiatica was affected adversely by salinity. Aqueous extract of some invasive plants viz. Chromolaena odorata, Parthenium hysterophorus, Ageratum conyzoides and Xanthium strumarium had inhibitory effects on germination, which threaten the population density of C. asiatica in nature. The pot experiment with different moisture levels (125% FWC (surplus water), 100% FWC, 70% FWC and 30% FWC) showed that the dry matter production and yield in Centella asiatica was highest in 100% FWC, followed by 70% due to higher growth characteristics such as number of primary branches, leaves, and leaf chlorophyll contents. Growth under four levels of shade (0, 30, 50 and 70%) showed that dry matter production and yield was significantly higher in 30% shade, followed by 50 %. Asiatic acid was significantly higher in 70% shading. There was no significant effect of shading on other measured secondary metabolites. Growth traits and yield of C. vii asiatica was significantly higher in sandy loam soil than clay-loam and pure sand. All measured secondary metabolites were significantly higher in sand than in clay-loam and sandy loam. The pot experiment with different integrated manuring conditions (Urea: FYM, 100:0; 75:25; 50:50; 25:75; 0:100; and control - no manure) showed higher yield and better growth traits of Centella asiatica in integrated manuring . Concentration of all secondary metabolites measured in present study was significantly higher in open agricultural land than in shady and open grasslands. Mean concentration of asiaticoside, the most important bioactive component of C. asiatica, was 1.8% (dw). Concentration of secondary metabolites was higher in samples from central Nepal than western and eastern Nepal. Asiaticoside content was inversely related with the altitude of samples collected. Asiaticoside content was higher in samples collected from 150- 600 m asl. Thus, a negative correlation was observed between altitude and asiaticoside content while the opposite was for quercetin-3-O- glucuronide content. Concentration of secondary metabolites was higher in wild than in transplanted samples. Essential oil yield of Centella asiatica from different habitats ranged from 0.10 (open grassland) to 0.12% (shady grassland). Total yield of essential oil was higher in samples from partially shaded habitat, but concentration of major components was higher in open agricultural land. Genetic diversity study of 21 different populations of Centella asiatica was carried out by morphological and molecular marker (RAPD). Morphological characters were significantly
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