Natural Product Research: Formerly Natural Product Letters

This art icle was downloaded by: [ H.R.W. Dharm arat ne] On: 05 Sept em ber 2013, At : 09: 07 Publisher: Taylor & Francis I nform a Lt d Regist ered in England and Wales Regist ered Num ber: 1072954 Regist ered office: Mort im er House, 37- 41 Mort im er St reet , London W1T 3JH, UK Natural Product Research: Formerly Natural Product Letters Publicat ion det ails, including inst ruct ions f or aut hors and subscript ion inf ormat ion: ht t p: / / www. t andf online. com/ loi/ gnpl20 Allelopathic activity studies of Mikania scandens K. G. Nelum P. Piyasena a & H. Ranj it h W. Dharmarat ne a a Nat ural Product s Programme, Inst it ut e of Fundament al St udies, Kandy, Sri Lanka Published online: 31 Jan 2012. To cite this article: K. G. Nelum P. Piyasena & H. Ranj it h W. Dharmarat ne (2013) Allelopat hic act ivit y st udies of Mikania scandens , Nat ural Product Research: Formerly Nat ural Product Let t ers, 27: 1, 76-79, DOI: 10. 1080/ 14786419. 2012. 656110 To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 14786419. 2012. 656110 PLEASE SCROLL DOWN FOR ARTI CLE Taylor & Francis m akes every effort t o ensure t he accuracy of all t he inform at ion ( t he “ Cont ent ” ) cont ained in t he publicat ions on our plat form . However, Taylor & Francis, our agent s, and our licensors m ake no represent at ions or warrant ies what soever as t o t he accuracy, com plet eness, or suit abilit y for any purpose of t he Cont ent . Any opinions and views expressed in t his publicat ion are t he opinions and views of t he aut hors, and are not t he views of or endorsed by Taylor & Francis. The accuracy of t he Cont ent should not be relied upon and should be independent ly verified wit h prim ary sources of inform at ion. Taylor and Francis shall not be liable for any losses, act ions, claim s, proceedings, dem ands, cost s, expenses, dam ages, and ot her liabilit ies what soever or howsoever caused arising direct ly or indirect ly in connect ion wit h, in relat ion t o or arising out of t he use of t he Cont ent . This art icle m ay be used for research, t eaching, and privat e st udy purposes. Any subst ant ial or syst em at ic reproduct ion, redist ribut ion, reselling, loan, sub- licensing, syst em at ic supply, or dist ribut ion in any form t o anyone is expressly forbidden. Term s & Condit ions of access and use can be found at ht t p: / / www.t andfonline.com / page/ t erm sand- condit ions Natural Product Natural ProductResearch, Research2013 Vol. 27, No.iFirst 1, 76–79, http://dx.doi.org/10.1080/14786419.2012.656110 2012, 1–4, SHORT COMMUNICATION Allelopathic activity studies of Mikania scandens K.G. Nelum P. Piyasena and H. Ranjith W. Dharmaratne*y Natural Products Programme, Institute of Fundamental Studies, Kandy, Sri Lanka Downloaded by [H.R.W. Dharmaratne] at 09:07 05 September 2013 (Received 22 August 2011; final version received 2 October 2011) Preliminary investigation of a number of plant extracts for allelopathic activity using seed germination inhibition bioassay showed a promising activity of the water extract of the aerial parts of Mikania scandens. Activity-guided fractionation of the M. scandens extract led to the isolation of the highly allelopathic active compound mikanolide, with minimum inhibitory concentration of 0.083 mM mL�1. As M. scandens is a highly abundant invasive plant in Sri Lanka and other South Asian countries, this plant could be developed as an environment friendly natural herbicide, either in crude form as shredded plant material or as pure mikanolide, which is the major constituent (�0.02%) in the plant. Keywords: allelopathic activity; seed germination inhibition; Mikania scandens; mikanolide 1. Introduction Allelopathy is one of the ways in which certain plant species reduce interspecies competition in their natural habitats, reduce pathogens and affect weed emergence. In an effort to reduce dependence on artificial herbicides, the use of plant species with strong allelopathic activities for weed control has shown promising results (Vyvyan, 2002). Allelochemicals are termed as nature’s own herbicides. Natural herbicides are attractive for a variety of important reasons, and show several benefits over synthetic compounds. A considerable number of bioactive natural products contain non-halogenated molecules, and they are at least partially water-soluble, and as a result of natural selections, they are more likely to exhibit some bioactivity at low concentrations. Natural herbicides have short half-life and they are safe in environmental toxicology standpoint. Further, due to the emergence of increasing number of herbicide-resistant weeds, artificial herbicides become less effective against the resistant weeds (Bhowmik & Inderjit, 2003). Therefore, there is an urgent need for new and effective herbicides. Some of these plants with allelopathic activity have been traditionally used as cover crops or green manure (Sturz & Christie, 2003). Appropriate manipulation of allelopathy can be used to improve crop productivity, environmental protection through environmental friendly control of weeds, pests (fungal and bacterial pathogens, nematodes and insects), crop diseases and conservation of nitrogen in cropland (Chung et al., 2002; *Corresponding author. Email: [email protected] yPresent address: National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA *Corresponding author. Email: [email protected] †Present address: National Center for Natural Products Research, School of Pharmacy, University of ISSN 1478–6419 print/ISSN 1478–6427 online Mississippi, MS 38677, USA � 2012 Taylor University, & Francis http://dx.doi.org/10.1080/14786419.2012.656110 http://www.tandfonline.com © 2013 Taylor & Francis 2 K.G.N.P. Piyasena and H.R.W. Dharmaratne Natural Product Research 77 Sturz & Christie, 2003) particularly in organic farming. Laboratory bioassays are very useful in establishing the allelopathic activity of a compound or a plant extract, but should ultimately be followed by greenhouse or field studies to see if the observations are reproducible in the natural environment. The most widely used bioassays are seed germination and seedling growth studies. Germination rate and seedling growth are monitored against control samples. Radish (Raphanus sativus L.) and lettuce (Lactuca sativa) were used as dicotyledons while Allium cepa was used as a monocotyledon in this experiment as indicator plants, due to their sensitivity to allelochemicals at low concentration (Khanh, Hong, Xuan, & Chung, 2005). Downloaded by [H.R.W. Dharmaratne] at 09:07 05 September 2013 2. Results and discussion Preliminary investigation of a number of plant extracts for allelopathic activity using seed germination inhibition bioassay showed a promising activity of the water extract of the aerial parts of Mikania scandens, which is a highly invasive twining weed (Piyasena & Dharmaratne, 2007). Aqueous leaf extracts of this plant have been used in traditional/folk medicine in treating stomach ulcers, gastric problems and a variety of other diseases. The plant is consumed as a vegetable in India and is a rich source of vitamins A, B and C. However, M. scandens has become a problematic weed (Hasan et al., 2009; Moon, Rattray, Putz, & Bowes, 1993) due to its fast growing invasive character. Mikania scandens is a rich source of mikanin, friedelin, efifriedinol and some sesquiterpene dilactones including the major constituent mikanolide (1), dihydromikanolide and deoxymikanolide. Three diterpenic acids known as kaurenic acid, butyryloxykaurenic acid, benzoyloxykaurenic acid and stigmasterol and betasitosterin have also been isolated from this plant (Hasan et al., 2009). Organic solvent extracts from the aerial parts of M. scandens were subjected to modified seed germination bioassay, and only dichloromethane extract showed 100% seed germination inhibition activity. Hence, the dichloromethane extract was subjected to column chromatography to yield 14 fractions. Seed germination inhibition bioassay was carried out for each of the above fractions, and five of them showed 100% activity. These active fractions (9.6 g) were combined and subjected to column chromatography to yield two pure compounds, and they were identified as mikanolide (1) and ergosta-5, 22-dien-3-ol (2). Of them, mikanolide showed 100% seed germination inhibition activity. Mikanolide (1) was further subjected to bioassays to find out minimum inhibitory concentrations (MICs). A concentration gradient of 0.33, 0.17, 0.08, 0.04, 0.02 and 0.01 mM mL�1 was prepared and each concentration was subjected to lettuce seed germination bioassay. Our results showed the MIC of mikanolide (1) as 0.08 mM mL�1. In this bioassay, abscisic acid (10 ppm) was used as the positive control. As 1 is the major compound (0.02%) of M. scandens, there is a great potential for using mikanolide (1) in crude form (e.g. as powdered plant material) in weed controlling. Further, pure mikanolide (1) could be developed as an environmental friendly natural herbicide, particularly in organic farming. 3. Experimental 3.1. General procedure 1 H-NMR and 13C-NMR spectra were recorded on a Varian Mercury-400BB (400 MHz for H-NMR and 100 MHz for 13C-NMR) spectrometer using TMS as internal standard in CDCl3. 1 78 K.G.N.P. Piyasena and H.R.W. Dharmaratne Natural Product Research 3 Downloaded by [H.R.W. Dharmaratne] at 09:07 05 September 2013 3.2. Plant material Mikania scandens was collected in May 2007 from Hantana in the Central Province of Sri Lanka and the plant specimens (no. MS-01-07) were deposited at the Natural Products Laboratories of the Institute of Fundamental studies, Kandy, Sri Lanka. A part of the plant material was macerated in water using a grinder and filtered. Then, the resulting solution was freeze dried using a freeze-dryer (EYELA FD-1) to get dry extract and tested for the seed germination bioassay, and found to be 100% active. The rest of the plant material was air-dried and powdered, and the powder (1.2 kg) was, respectively, extracted with n-hexane, dichloromethane, ethyl acetate and methanol using a sonicator (VWR) at room temperature. Weights of these extracts are 24.0 g (2.0%), 57.0 g (4.75%), 33.2 g (2.75%) and 15.0 g (1.23%), respectively. Of them, the dichloromethane extract was subjected to column chromatography. A silica gel (Fluka 60741) column was packed with cold n-hexane, and eluted with n-hexane using increasing amounts of dichloromethane to yield 14 fractions. After the bioassay, active fractions (9.6 g) were combined and subjected to column chromatography to yield two pure compounds 1 and 2, and they were confirmed as mikanolide (1) and ergosta-5, 22-dien-3-ol (2) using spectroscopic data and comparison with literature (Cuenca, Bardon, & Catalan, 1988; Itoh, Sica, & Djerassi, 1983). 3.3. Allelopathic activity studies In this study, seed germination bioassay was employed to determine allelopathic potential of plant extracts and pure compounds. The germination rate and seedling growth were monitored, against a control sample. As the indicator plants in this experiment, lettuce (L. sativa) was used as the model. Lettuce seeds were treated with 5% Chlorox solution for 10 min, rinsed five times with sterilised distilled water and kept for 10 min in sterile distilled water. Floating immature seeds were discarded and five seeds were kept in each Petri dish containing 5 mL of 1000 ppm of appropriate crude plant extract dissolved in distilled water, lined with Whatman No. 4 filter paper. Control dishes received 5 mL aliquots of distilled water. After the seeds were placed on moistened filter paper, Petri dishes were incubated in the dark at 25� C for 5 days. At the end of the incubation period, the number of seeds germinated in each Petri dish was counted, and the length of the roots and the shoots were measured to the nearest millimeter. Similarly, 5 mL aliquot of abscisic acid (10 ppm solution), which is a well-known compound for the seed dormancy, were used as the positive control (Hilhorst & Karssen, 1992). In the lettuce seed germination bioassay, four replicates were used for each treatment and the experiment was repeated. One way analysis of variance and the Tukey’s pair-wise comparisons were carried out to detect whether there is any significant difference in the average radicle and hypocotyls after treating with different concentrations of the plant extracts (Khanh et al., 2005; Singh, Batish, & Kohli, 2002). The statistical analysis was performed using MINITAB version 11.12. 3.3.1. Mikanolide (1) White needles (Hexane/CH2Cl2) 210 mg (0.0175%); m.p. 229–232� C [lit. m.p. 226–228� C (Herz, Subramaniam, Santhanam, Aota, & Hall, 1970)]. 1H-NMR (400 MHz, Pyridine) �; 7.87 (1H, s, H-5), 6.42 (1H, d, J ¼ 3.6 Hz, H-13), 5.94 (1H, d, J ¼ 3.2 Hz, H-13), 5.56 (1H, d, J ¼ 2.0 Hz, H-6), 5.15 (1H, m, H-8), 4.27 (1H, J ¼ 2.0 Hz,d, H-3), 3.53 (3H, m, H-1, 2,3), 2.28 (2H, m, H-9), 1.24 (3H, s, Me, H-14). 13C-NMR (100 MHz, Pyridine) �; 171.4 (C-15), 168.5 (C-12), 148.9 (C-5), 138.9 (C-11), 131.1 (C-4), 123.0 (C-13), 82.5 (C-6), 77.7 (C-8), 58.6 (C-1), 57.9 (C-10), 56.6 (C-2), 51.4 (C-7), 50.8 (C-3), 43.9 (C-9), 21.8(C-14). 4 K.G.N.P. Piyasena and H.R.W. Dharmaratne Natural Product Research 79 3.3.2. Ergosta-5,22-dien-3-ol (2) Downloaded by [H.R.W. Dharmaratne] at 09:07 05 September 2013 White needles (Hexane/CH2Cl2) (150 mg, 0.00125%) m.p.167� C. 1H-NMR (400 MHz, CDCl3) �; 5.34 (1H, m, H-6), 5.13 (1H, dd, H-22), 5.03 (1H, dd, H-23), 2.23 (2H, m, H-4), 2.04 (2H, m, H-16), 2.01 (2H, m, H-20), 1.86 (2H, m, H-2), 1.83 (2H, m, H-1), 1.82 (2H, m, H-8, 25), 1.72 (2H, m, H-7), 1.67 (3H, m, H-21), 1.54 (1H, m, H-9), 1.53 (1H, m, H-24), 1.50 (2H, m, H-15), 1.39 (2H, m, H-12), 1.15 (1H, m, H-17), 1.10 (6H, m, H-27,19), 1.06 (1H, m, H-14), 0.86 (3H, m, H-26), 0.80 (3H, m, H-28), 0.69(3H, m, H-18).13C-NMR (100 MHz, CDCl3) �; 141.0 (C-5), 138.5 (C-22), 129.5 (C-23), 121.9 (C-6), 72.0 (C-3), 57.2 (C-14), 56.2 (C-17), 51.4 (C-24), 50.4 (C-9), 42.5 (C-4), 42.4 (C-13), 40.7 (C-20), 37.5 (C-1), 36.7 (C-12), 32.1 (C-8, 25), 31.9 (C-2), 29.1 (C-7), 28.9 (C-16), 24.6 (C-15), 25.6 (C-11), 21.4 (C-21), 19.6 (C-27,19), 19.2 (C-26), 12.4 (C-28), 12.3 (C-18). References Bhowmik, P.C., & Inderjit (2003). Challenges and opportunities in implementing allelopathy for natural weed management. Crop Protection, 22, 661–671. Chung, I.M., Kim, K.H., Ahn, J.K., Chun, S.C., Kim, C.S., Kim, J.T., & Kim, S.H. (2002). Screening of allelochemicals on barnyardgrass (Echinochloa crus-galli) and identification of potentially allelopathic compounds from rice (Oryza sativa) varity. Crop Protection, 21, 913–920. Cuenca, M.D.R., Bardon, A., & Catalan, C.A.N. (1988). Sesquiterpene lactones from Mikania micrantha. Journal of Natural Products, 51, 625–626. Hasan, S.M.R., Jamila, M., Majumder, M.M., Akter, R., Hossain, M.M., Mazumder, M.E.H., . . . , Rahman, S. (2009). Analgesic and antioxidant activity of the hydromethanolic extract of Mikania scandens (L.) Willd. leaves. American Journal of Pharmacology and Toxicology, 4, 1–7. Herz, W., Subramaniam, P.S., Santhanam, P.S., Aota, K., & Hall, A.L. (1970). Structure elucidation of sesquiterpene dilactones from Micania Scandens (L.) Willd. Journal of Organic Chemistry, 35, 1453–1464. Hilhorst, H.W.M., & Karssen, C.M. (1992). Seed dormancy and germination: role of abscisic acid and gibberellins and the importance of hormone mutants. Plant Growth Regulation, 11, 225–238. Itoh, T., Sica, D., & Djerassi, C. (1983). Minor and trace sterols in marine invertebrates. Part 35. Isolation and structure elucidation of seventy-four sterols from the sponge Axinella cannabina. Journal of the Chemical Society, Perkin Transactions I, 147–153. Khanh, T.D., Hong, N.H., Xuan, T.D., & Chung, I.M. (2005). Paddy weed control by medicinal and leguminous plants from Southeast Asia. Crop Protection, 24, 421–431. Moon, M., Rattray, M.R., Putz, F.E., & Bowes, G. (1993). Acclimation to the flooding of the herbaceous vine, Micania scandens. Functional Ecology, 7, 610–615. Piyasena, K.G.N.P., & Dharmaratne, H.R.W. (2007). Allelopathic activity studies of Sri Lankan flora. Proceedings of the Sri Lanka Association of Advanced Science, 63, 201–202. Singh, H.P., Batish, D.R., & Kohli, R.K. (2002). Allelopathic effects of two volatile monoterpenes against bill-goat weed (Ageratum conyzoides L.). Crop Protection, 21, 347–350. Sturz, A.V., & Christie, B.R. (2003). Beneficial microbial allelopathies in the root zone: the management of soil quality and plant disease with rhizobacteria. Soil and Tillage Research, 72, 107–123. Vyvyan, J.R. (2002). Allelochemicals as leads for new herbicides and agrochemicals. Tetrahedron, 58, 1631–1646.