Nitrification Inhibitors from the Roots of Leucaena leucocephala Andrew J. Erickson, † Russel S. Ramsewak, † Alvin J. Smucker, ‡ and Muraleedharan G. Nair* ,† Department of Horticulture and National Food Safety and Toxicology Center and Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824 The nitrification inhibition (NI) bioassay guided fractionation of the methanol extract of lyophilized and milled roots of Leuceana leucocephala resulted in the isolation of four compounds, 1)4, as confirmed from their 1 H and 13 C NMR spectral data. Compound 1, gallocatechin, was the most active NI inhibitor at 12 μg/mL. Epigallocatechin, 2, and epicatechin, 4, isolated as mixtures, were not assayed individually for their NI inhibitory activities against the nitrification bacterium Nitrosomonas europaea. Keywords: Leuceana leucocephala; Nitrosomonas europaea; gallocatechin; epigallocatechin; cat- echin; epicatechin; nitrification inhibitors INTRODUCTION Nitrification is carried out by bacteria whereby am- monia is converted to nitrate. It occurs in almost all soils and also in marine and freshwater sediments. The process is three step and is caused by two sets of bacteria. In the first set, consisting of Nitrosomonas europaea, Nitrosolobus multiformis, and several other species, ammonia is oxidized to hydroxylamine and then to nitrite (eqs I and II). The second set consists of Nitrobacter agilis, which converts nitrite to nitrate (eq III). Nitrification is important because much fertilizer is applied as ammonia, and the nitrification process reduces fertilizer efficiency. There is considerable evidence to show that plants produce secondary metabolites that inhibit nitrification. Early work demonstrated that grasslands displayed low nitrification rates (Theron, 1951; Stiven, 1952; Munro, 1966). Further research showed that specific plant compounds inhibited nitrification, and tannins and gallotannins were reported to inhibit nitrification (Ba- sabara, 1964; Rice, 1965, 1969). A large body of research provided evidence that phenolic acids and some fla- vonoids inhibit nitrification, including chlorogenic acid, gallic acid, caffeic acid, quercetin, and karanjin (Rice, 1964, 1965; Rice and Pancholy, 1974; Sahrwat and Mukerjee, 1977). However, there are published reports questioning the nitrification inhibition activity of naturally occurring compounds. Gallic and caffeic acids were tested for nitrification inhibition in both soil and pure Nm. euro- paea, Nl. multiformis, and Nitrospira cultures but were found to be inactive (McCarty and Bremner, 1986; McCarty et al., 1991). It was also shown that phenolic acids inhibited nitrification only at levels much higher than those found in the soil (Turtura et al., 1989). Although questions remain as to whether plants produce secondary compounds that influence the nitri- fication process, there is much evidence indicating that plant secondary compounds play a variety of roles in protecting plants from a variety of stresses. The present research describes the isolation and characterization of nitrification inhibiting compounds from the roots of Leucaena leucocephala, using an in vitro nitrification inhibition (NI) assay. MATERIALS AND METHODS General Experimental Procedures. NMR spectra ( 1 H and 13 C) were recorded on a Varian INOVA 300 spectrometer (300 MHz for 1 H and 75 MHz for 13 C) or a Varian VXR 500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C). Chemical shifts were recorded in DMSO-d6 or CD3OD and the values are in δ (ppm) based on δ residuals of DMSO-d6 2.49 and 39.5 for 1 H and 13 C NMR or δ residuals of CD3OD 3.30 and 49.0 for 1 H and 13 C NMR, respectively. Coupling constants, J, are in hertz. All positive controls and chemicals used in the antioxidant assay were purchased from Sigma Chemical Co. unless otherwise stated. All organic solvents were of ACS reagent grade (Aldrich Chemical Co., Inc., Milwaukee, WI). Plant Material. L. leucocephala seeds, obtained from Nigeria, Africa, were germinated in the Plant and Soil Sciences Building greenhouses in 12-in. plastic pots. Plants were later transferred to the Bioactive Natural Products Laboratory (BNPL) greenhouses, subjected to a 12-h photoperiod, watered once daily, and maintained at 75 °F. For extractions, whole plants were harvested, and root, stem, and leaves were collected separately and stored at -20 °C. A total of 391 g of fresh root material was collected for extraction. The roots were lyophilized using a tray lyophilizer (model TD-3B, FTS Sys- tems, Inc., Stone Ridge, NY) at 5 °C for 48 h to yield 185 g of dry weight. Dried roots were ground to a fine powder in a Wiley mill (mesh size ) 2 mm, Thomas-Wiley, laboratory mill, model 4) prior to extraction. Extraction and Isolation of NI Compounds. Lyophi- lized, powdered L. leucocephala roots (165 g) were extracted sequentially at room temperature by soaking with hexane (2 × 500 mL; 24 h), ethyl acetate (2 × 500 mL; 24 h), and * Author to whom correspondence should be addressed [telephone (517) 353-2915; fax (517) 432-2310; e-mail nairm@ pilot.msu.edu]. † Department of Horticulture and National Food Safety and Toxicology Center. ‡ Department of Crop and Soil Sciences. NH 3 + O 2 + 2e - + 2H + f NH 2 OH + H 2 O (I) NH 2 OH + H 2 O f NO 2 - + 5H + + 4e - (II) NO 2 - + H 2 O f NO 3 - + 2H + + 2e - (III) 6174 J. Agric. Food Chem. 2000, 48, 6174-6177 10.1021/jf991382z CCC: $19.00 © 2000 American Chemical Society Published on Web 10/21/2000