Published: June 27, 2011 r2011 American Chemical Society 8065 dx.doi.org/10.1021/jf200754f | J. Agric. Food Chem. 2011, 59, 8065–8069 ARTICLE pubs.acs.org/JAFC A Simple Analytical Method for Dhurrin Content Evaluation in Cyanogenic Plants for Their Utilization in Fodder and Biofumigation Gina Rosalinda De Nicola, Onofrio Leoni, Lorena Malaguti, Roberta Bernardi, and Luca Lazzeri* Agricultural Research Council À Industrial Crop Research Centre (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy ABSTRACT: Cyanogenic plants have some potential as biocidal green manure crops in limiting several soilborne pests and pathogens. Sorghum (Sorghum bicolor (L.) Moench) and Sudangrass (Sorghum bicolor subsp. sudanense (P.) Stapf), in fact, contain the cyanogenic glucoside p-hydroxy-(S)-mandelonitrile-β-D-glucoside (dhurrin) as a substrate of its secondary defensive system able to release hydrogen cyanide following tissue lesions due to biotic or abiotic factors. Given that dhurrin content is correlated with the biofumigant efficacy of the plants, a high dhurrin content could be a positive character for utilization of sorghum and Sudangrass as biocidal green manure plants. For chemical characterization of the available germplasm, a simple, safe, and accurate method is necessary. In this paper, a new method for dhurrin analysis, based on methanol extraction and high-performance liquid chromatography, is reported and discussed. The feasibility of this analytical procedure was tested by evaluating dhurrin level in roots and stems during cultivation of four different sorghum and Sudangrass varieties in agronomic trials performed in 2008 in the Po valley (Italy). The dhurrin content ranged from 0.16 ( 0.04 to 7.14 ( 0.32 mg g À1 on dried matter (DM) in stems and from 1.38 ( 0.02 to 6.57 ( 0.09 mg g À1 on DM in roots, showing statistical differences among the tested germplasms that could be linked to the efficacy of their utilization as biofumigant plants. The method also opens new perspectives for the characterization of sorgum plants as fodder, for which the presence of dhurrin is considered to be negative for its well-known toxicity. KEYWORDS: biofumigation, dhurrin, sorghum, Sudangrass, hydrogen cyanide, cyanogenic glycosides, p-hydroxybenzaldehyde ’ INTRODUCTION Sorghum (Sorghum bicolor (L.) Moench) and Sudangrass (Sorghum bicolor subsp. sudanense (P.) Stapf) are heat- and drought-tolerant cover crops with interesting potential for im- proving soil quality, suppressing weeds, and controlling diseases and nematode damage. 1,2 The cells of Sudangrass and sorghum contain the cyanogenic glucoside p-hydroxy-(S)-mandelonitrile- β-D-glucoside (dhurrin) which can degrade into hydrogen cya- nide (HCN) in a process known as cyanogenesis. Figure 1 shows the two-step enzymatic hydrolysis process that gives rise to the release of hydrogen cyanide. Dhurrin is first hydrolyzed by the endogenous β-d-glucoside glucohydrolase (dhurrinase) (EC 3.2.1.21) to produce glucose and p-hydroxy-(S)-mandelonitrile. This latter compound is unstable and quickly converted to free HCN and p-hydroxybenzaldehyde (p-HB) by the endogenous enzyme R- hydroxynitrile lyase or at basic pH values. 3 HCN is a powerful nematicidal compound 4À8 that plays a role in the low sensitivity of these plants to several pests and pathogens. As observed for the glucosinolateÀmyrosinase system in Brassicaceae plants, 9 in intact plant tissues enzymes and substrate are kept separated in the cells: dhurrin is located in the vacuole of the epidermal cells, whereas the catabolitic enzymes are in the mesophyll cells. 3 Only when plant tissues are lesioned or destroyed, as a consequence of biotic or abiotic factors, do enzymes and substrates come into contact, releasing the bioactive compound that is involved in limiting plant infection. Due to the negative role of cyanogenic glucosides in human nutrition and fodder, various methods, including direct high- performance liquid chromatography (HPLC) 10,11 and indirect evaluation of released HCN after hydrolysis, 12 have been used for evaluating these compounds in plants. For example, HCN lib- erated from plant material can be assessed by colorimetric methods such as the succinimide/barbituric method 13 and the alkaline picrate method. 14 However, all of the reported indirect methods are described as time-consuming and are not practical for screening a large number of samples without the use of a hazar- dous cyanide salt for calibration curve determination, whereas in most cases the direct HPLC methods require the use of an expensive cyanogenic glucoside standard that is, in addition, not always easily available on the market. With the aim of characterizing a large number of genotypes and tissues of Sudangrass and sorghumÀSudangrass hybrid plants (viz., from agronomic trials or breeding trials) a simple and accurate direct analytical method has been defined. The proposed method is also cheap and safe and allows a quantitative and fast determination of dhurrin in plants. It can be easily performed for routine analysis by means of the use of an HPLC instrument equipped with an automatic injector. This new method was applied in the evaluation of dhurrin content on different varieties of sorghum and Sudangrass during cultivation in agronomic trials performed in Italy in 2008, confirming its feasibility and practicality. ’ MATERIAL AND METHODS Reagents. Dhurrin standard (CAS Registry No. 499-20-7) was obtained from Extrasynth ese (Lyon, France) and p-hydroxybenzalde- hyde (CAS Registry No. 123-08-0) from Sigma-Aldrich Chemie (Steinheim, Germany). Activated carbon used in this study was charcoal, Received: February 25, 2011 Revised: June 23, 2011 Accepted: June 27, 2011