Effect of NPK and Farm Yard Manure on the Accumulation of Forskolin, an Anti-Obesity Diterpenoid Molecule in Coleus forskohlii

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1 Effect of NPK and Farm Yard Manure on the Accumulation of Forskolin, an Anti-Obesity Diterpenoid Molecule in Coleus forskohlii S. Nageswara Rao, S. Gangadhar Rao, B. Bhargavi and K. Janardhan Reddy* Department of Botany, Osmania University, Hyderabad , India *For correspondence - kjreddy50@yahoo.co.in 1346 Abstract Medicinal plants growing in wild cannot ensure consistent quality due to genetic variability found in the natural populations, geographical variations and season of collection. herefore, there is a need to cultivate medicinal plants using known varieties for extraction of high yields of active principles. his can be achieved only with organic farming practices and post harvest technologies. he research conducted by institutes like CIMAP and other CSIR institutes, ICAR and Universities has led to the release of a number of high yielding medicinal plant cultivars. Coleus forskohli is a herb with fleshy, fibrous roots that grows in wild in the warm, subtropical areas like in India, Burma, and hailand. Coleus forskohlii is being grown in several parts of the country as a commercial crop. In the present investigation the effect of nitrogen, phosphorus, potassium (NPK) and farm yard manure amendments on the growth of roots and accumulation of forskolin was studied. he results revealed that 50% of NPK and 10 tonnes of farm yard manure per hectare at a harvesting time 150 days are ideal for maximum yield of forskolin. Key words: Forskolin, Farm Yard Manure, NPK, Coleus forskohlii Introduction Cultivation of medicinal plants appears to be an important strategy for research and development. More than 400 plant species used for production of medicines by the Indian Industry. Cultivation of various varieties of medicinal plants is important to isolate the active principles. he application of organic farming practices and post harvest technologies can enhance their yields so that it becomes profitable to the farmers. he potential returns to the farmer from cultivation of medicinal plants are reported to be quite high if fertilizers are used at a proper dose and time. he application of bio-fertilizers in combination with the inorganic fertilizers substitutes the required quantity of the fertilizers needed by the medicinal and aromatic crops. he bio-fertilizers due to its eco friendliness will change the phase of medicinal and aromatic crops production in the years to come (1). In Chlorophytum borivallianum, the number of tubers, fresh and dry tuber yields and steroid content increased with the application of vermicompost of 5 tonnes ha -1 (2). When Coleus forskohli was grown in FYM amended soil with Paecilomyces lilacinus, there was better root development due to control of

2 1347 nematodes (3). Earlier studies on Coleus forskohlii, with a combination of 40 kg N, 60 kg P 2 and 50 kg K 2 O ha -1 resulted in optimum fresh and dry tuber yields of tonnes ha -1 (4). Raising the level beyond this was found to reduce the tuber yield significantly. Nitrogen at 60 kg ha -1 produced higher tuber yield, high dry matter yield but maximum bulking rate along with 120 kg K 2 O ha -1 in Coleus parviflorus (5). Somanath et al. (6), in their study on influence of FYM and NPK on uptake of nutrients and growth of Coleus forskohlii found that maximum increase in fresh weight of roots was recorded in all the stages of growth (at 30 DAP kg plant -1, at 90 DAP kg plant -1 and at 150 DAP kg plant -1 ). Application of N at 100 kg ha -1 (half basal + half one month after transplanting) along with a basal application of 100 kg ha -1 P2O5 and 20 tonnes FYM ha -1 increased the fresh weight of rhizomes in Acorus calamus (7). In Aswagandha (Withania somnifera), high dry weight of roots (1.297 g plant -1 ) were obtained with 15 kg N ha - 1 (8). In Catharanthus roseus, 100 kg N, 40 kg P 2 and 50 kg K ha -1 gave maximum root yields (9). Application of N (30kg N ha -1 ) increased the root yields (2702 kg ha -1 ) in Rauwolfia serpentina (10). Highest root yield of Aswagandha was obtained with the application of 20 kg N and 40 kg P 2 ha -1 (11). But there are no reports on the influence of nutrients (inorganic and bio- fertilizers) on the content of forskolin, the main chemical constituent of Coleus forskohlii. Keeping this view in mind, the present investigation was taken up to enhance the forskolin levels under different fertilizer amendments. he aim of the present investigation is to increase the yields of roots and forskolin content under different fertilizer amendments so as to make it profitable to the farming community. Materials and methods Preparation of soil bed :he experimental area was repeatedly ploughed; finally, it was leveled and divided into plots as per the layout. he experiment was laid out in a randomized block design with 6 treatments and replicated thrice. A plot size of 3 X 2.5 m was taken for experimental work. reatments of fertilizers and farm yard to the soil for cultivation of Coleus forskohlii he details of the treatments are: 1 : Control No manures 2 : 100% NPK recommended dose 3 : 50% NPK + 10 tonnes of farm yard manure / ha 4 : 50% NPK + 5 tonnes of vermicompost/ ha 5 : 20 tonnes of farm yard manure / ha : Vermicompost 10 tonnes/ha 6 Extraction of forskolin : Coleus forskohlii roots (10 kg) were extracted with methanol (25 x 3) at room temperature. After this, methanol was distilled out to give a residue of 1.5 kg. his residue was further extracted with Petroleum- Ether (5 x 5) to give another residue of 230 g containing waxes and fats. After this, defatted residue was extracted with ethyl acetate to give a residue of 270 g, which contains 27% forskolin. Fresh and dry weights of the root : he influence of different fertilizer amendments on fresh weight and dry weights of the root [kg plant -1 ] at 30, 90 and 150 Days after Planting (DAP) of growth in Coleus forskohlii was recorded. HPLC analysis of forskolin Conditions: Mobile phase : Mehanol :Water (65:35 v/v) Column : Hypersil C-18 (250 mm* 4.6 mm ) Detection : Refractive index Flow rate : 1 µl/min Injection volume : 20 micro lit. Coleus forskohlii (roots) was cultivated under different treatment conditions, and Nageswara Rao et al

3 1348 forskolin was extracted in a soxhlett apparatus with methanol and concentrated. LC solvent system (Rf 0.45, 20% EtOAc + petroleum ether) showed the presence of forskolin. he extracts were subjected to HPLC analysis. Results Fresh weight of the root : he crop was harvested at 30, 90 and 150 days after transplantation. Light irrigation was given one day before harvesting for easy lifting up of roots. Harvesting was carried out manually by digging and uprooting the individual plants. he roots were separated and washed to remove the soil. Forskolin content was determined in roots 150 days after transplantation. he influence of different fertilizer amendments on fresh weight of the root [kg plant -1 ] at 30, 90 and 150 DAP of growth in Coleus forskohlii is shown in table 1. Maximum increase in fresh weight of roots was recorded in treatment number 3 in all the stages (at 30 DAP kg plant -1, at 90 DAP kg plant -1 at 150 DAP kg plant -1 ) of growth. Dry weight of the root and content of forskolin : Influence of various fertilizer amendments on dry weight of the root [kg plant -1 ] at 30, 90 and 150 DAP of growth in Coleus forskohlii is shown in able 2. In treatment number 3, maximum fresh weight was observed i.e., at 30 DAP 9.1 g plant -1, at 90 DAP 18.9 g plant -1 at 150 DAP 60.0 g plant -1. he fresh and dry weights of roots were measured under different nitrogen regimes and are shown in able 3. he treatment 50% NPK + 10 tonnes of farm yard manure gave the best dry root weight among the different treatments (able 3). Samples were collected under different treatments for quantification of forskolin. Forskolin was extracted in a soxhlett apparatus, concentrated and used for HPLC analysis (Fig. 1). HPLC analysis revealed that application of 50% NPK + 10 tonnes of farm yard manure ha -1 gave maximum yields of forskolin after 150 days of harvest. Discussion Somanath et al. (6), in their study on influence of FYM and NPK on uptake of nutrients and growth of Coleus forskohlii observed maximum increase in fresh weight of roots in treatment number 3 (at 30 DAP kg plant -1, at 90 DAP kg plant -1 at 150 DAP kg plant -1 ). Application of N at 100 kg ha -1 (half basal + half one month after transplanting) along with a basal application of able 1. Effect of various fertilizer amendments on fresh weight of root (g per plant) at different stages of growth in Coleus forskohlii reatments Days after planting : Control No manures : 100% NPK recommended dose (N- 40 kg ha -1 ) : 50% NPK + 10 tonnes of farm yard manure/ha : 50% NPK + 5 tonnes of vermicompost/ha : 20 tonnes of farm yard manure/ha : Vermicompost 10 tonnes/ha

4 1349 Current rends in Biotechnology and Pharmacy able 2. Effect of various fertilizer amendments on dry weight of root (g per plant) at different stages of growth in Coleus forskohlii reatments Control No manures % NPK recommended dose (N- 40 kg ha ) 50% NPK + 10 tonnes of farm yard manure/ha 50% NPK + 5 tonnes of vermicompost/ha 20 tonnes of farm yard manure/ha Vermicompost 10 tonnes/ha Nageswara Rao et al Days after planting

5 1350 able 3. Fresh and weights of roots grown under different nitrogen regimes. N = Replicates, M = Mean, SE = Standard error 30 days 90 days 150 days reatment N M-SE M+SE M-SE M+SE M-SE M+SE 1 Fresh weight of root Dry weight of root Fresh weight of root Dry weight of root Fresh weight of root Dry weight of root Fresh weight of root Dry weight of root Fresh weight of root Dry weight of root Fresh weight of root Dry weight of root = control; 2 = 100% NPK; 3 = 50% NPK + 10 tonnes of farm yard manure; 4 = 50% NPK + 5 tonnes of vermicompost; 5 = 20 tonnes of farm yard manure; 6 = 10 tonnes of vermicompost. 100 kg ha -1 P2O5 and 20 tonnes FYM ha -1 increased the fresh weight of rhizomes in Acorus calamus (7). Sheu et al. (12) also observed higher yield (23 tonnes ha -1 ) in Dioscorea doryophora with organic and inorganic fertilizer amendments. In treatment number 3, maximum fresh weights were recorded (able 3 and Fig. 4.). In Aswagandha (Withania somnifera), dry weight of roots (1.297 g plant -1 ) were obtained with 15 kg N ha -1 (Maryada et al., 2001). In Catharanthus roseus, 100 kg N, 40 kg P 2 and 50 kg K 2 O) ha -1 gave maximum root yields (Hegde, 1988). Highest root yield of aswagandha was obtained with the application of 20 kg N and 40 kg P 2 ha -1 (11). In aswagandha, application of 40 kg N ha -1 and 60 kg P 2 ha -1 and 20 kg K 2 O ha -1 gave the highest dry root yields of kg ha -1 (13). Root characters like number of roots per plant and length of roots increased in Chlorophytum borivallianum, along with the number of tubers, fresh and dry weights of tuber yield and steroidal content with the application of vermicompost of 5 tonnes ha -1. he steroidal content was the highest with vermicompost application (2). Maheshwari et al. (1988) also observed increase in the root yield in Rauwolfia serpentaina with the application of N (30 kg N ha -1 ). Application of NPK at 30:30:0 kg ha -1 recorded the highest dry root yields of Aswagandha but further increasing the levels of

6 1351 fertilizers had no effect on the yields of dry roots (14). Highest root yield of aswagandha was obtained with the application of 20 kg N and 40 kg P 2 ha -1 (11). HPLC analysis revealed that application of 50% NPK + 10 tonnes of farm yard manure ha -1 gave maximum yield of forskolin. he statistical analysis revealed that fresh and dry weights of roots, number of roots plant -1 and root length were significant compared to controls and positively correlated with each other. It is suggested that appropriate dose of application of fertilizers at appropriate time will result in higher tuber yield and forskolin content. Our results also suggest that time of harvest for optimum yields of tuber is also important. References 1. Srinivasappa, K.N., Anuradha, M.N., Farooqi, A.A., Kathiresan, C., Suresh, H.C., and Shivananda,.N. (2007). Use of biofertilizers in cultivation of medicinal and aromatic plants - a review. BIOMED, 2: Paturde, J.K., Wankhade, S.G., Khode P.P, Chatol, P.U, Deo, D.D. and Bhuyar, S.A. (2002). Effect of organic manures and plant population on yield of safed musli (Chlorophytum borivallianum). Agricultural Science Digest, 22: Sivakumar, C.V., and Vidhyasekaran, P. (1990). Control of Meloidogyne incognita on Coleus Forskohlii with Paecilomyces lilacinus in farm yard manure amended and non-amended soil. Journal of Biological Control, 4: Veeraraghavathatham, D, Venkatachalam, R., and Sundararajan., S. (1988). Effect of various levels of N, P and K on the tuber Yield of Coleus forskohlii. South Indian Horticulture, 36: Geetha, K., and Madhavan Nair., K.P. (1993). Effect of N and K in Coleus. Journal of ropical Agricultural, 31: Somanath, I., Bhaskar, S., and Sreenivasmurthy C.A. (2007). Influence of FYM and NPK on uptake of nutrients and growth of Coleus forskohlii. BIOMED, 2: iwari, J.P., iwari, A.B., iwari, G., and Singh A.K. (2000). Effect of nitrogen application on growth and yield of Acorus calamus. Proceedings of the National Seminar on the Research and Development in Aromatic Plants. Current trends in biology, uses, production and marketing of essential oils, Lucknow, India, July Journal of Medicinal and Aromatic Plant Sciences, 22: Maryada, M, Kaushal, S.K and Kaistha, B.P. (2001). Response of aswagandha (Withania somnifera) to nitrogen fertilization under mid hill conditions of Himachal Pradesh. Himachal Jourmal of Agriculture Research, 27: Hedge, D.M. (1988). Response of periwinkle (Catharanthus roseus L.) to nitrogen, phosphorus and potassium fertilization. Agricultural Research Journal of Kerala, 26: Maheshwari, S.K., Yadav, S, Gangrade, S.K and rivedi K.C. (1988). Effect of fertilizers on growth, root and alkaloid yield of rauwolfia (Rauwolfia serpentina). Indian Journal of Agricultural Sciences, 58: Maheshwari, S.K. and Yadav, S. (1981). Response of Withania somnifera to variable levels of N and P rainfed. Paper presented at the IV All India Workshop on Medicinal Nageswara Rao et al

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