This protocol is for a widely used technique to determine whether dried or fresh leaf material contains cyanogenic glycosides. We use the technique when we wish to screen large numbers of samples. If one wishes to quantify the concentration of cyanide following a positive test then there are many methods, often based on picric acid or acid hydrolysis but also using ion-selective electrodes. Useful references include Hosel and Moller (1981), Adsersen et al (1988), Haskins et al (1988) and Haque and Bradbury (2002).
Cyanogenesis is a classic plant defence especially in places where nitrogen is not limiting. According to Haque and Bradbury (2002), at least 2650 species of plants produce cyanoglycosides and usually also a corresponding hydrolytic enzyme (beta-glycosidase). The two interact to protect the plant when mechanical damage, often due to a herbivore, disrupts the plant’s cell structure. The cyanoglycoside decomposes to a sugar and a cyanohydrin, which quickly decomposes further to hydrogen cyanide and an aldehyde or a ketone (Hosel 1981). Most plant species either produce cyanide compounds or they do not. There are, however, some species, most notably white clover that are polymorphic. In the case of white clover, some produce cyanoglucoside and enzyme while others may show any combination of the two characters including no cyanoglucoside and no enzyme (Hughes 1991). This polymorphism is important from an analytical point of view because tests may fail to detect plants that produce cyanoglucosides but no enzyme.
Two of the methods for screening large numbers of plant samples for cyanide include the picrate method (Adsersen et al 1988)and the Feigl-Anger spot test (van Wyck 1989). The latter method relies on endogenous enzyme catalysing the hydrolysis of cyanoglucoside to cyanohydrin. If the plant does not produce the enzyme then the test will produce a low value or even a negative result. The same will occur if tannins bind the enzyme and reduce its activity (Goldstein and Spencer 1985) (a distinct possibility in eucalypt leaves). One can circumvent the possible lack of enzyme by adding b-glucosidase to the test. It may be possible to inactivate the tannins and not interfere with the test by adding polyethylene glycol too.
- dried, ground leaf material
- sodium phosphate buffer (0.1 M, pH 7.1)
- Vacutainer ™- evacuated glass tubes with a rubber stopper for collecting blood.
- Feigl-Anger paper
- beta glucosidase from almond (Prunus amygdalis; B-D-glucoside glucohydrolase)
- copper ethyl acetoacetate
- Whatman No. 3 MM chromatography papers
- cupric sulphate
- ethyl acetoacetate
- sodium acetate
- Add 150 L of sodium phosphate buffer (0.1 M, pH 7.1) to 0.20g (+/-0.01g) of dried, ground leaf in a vacutainer. Alternatively, crush fresh leaves. Crushed apple seeds are a useful indicator that the test is working.
- Suspend strips of Feigl-Anger paper in the tops of the vacutainers, and seal the containers.
- Allow samples to stand for 24 hours. The Feigl-Anger paper turns blue in the presence of cyanogenic glycosides. If no activity occurs within 24 hours, stop the trial to avoid false positives (because of the possibility of CN from cyanogenic bacteria (Brinker and Seigler 1989).
- Create new samples for all negative species, adding a small quantity (ca 1 mg) of beta glucosidase from almond “(Prunus amygdalis; B-D-glucoside glucohydrolase) to each to test for cyanogenic potential, separate from the activity of endogenous enzymes.
- Grade colour according to Methuen Handbook of Colour.
Preparation of papers
- Prepare the following solutions in chloroform:1% (w/v) of 4,4-tetramethyldiaminodiphenylmethane
1% (w/v) of copper ethyl acetoacetate
- Mix equal volumes.
- Soak strips of Whatman No. 3 MM chromatography papers for about 2 min and then dry. Store for months in a plastic bag at room temperature.
Preparation of copper ethyl acetoacetate
- Gently heat a solution of cupric sulphate (1.30 g, 30mL H2O).
- Add a solution of ethyl acetoacetate (2.60g, 10mL ethanol).
- To the mixture add a warm solution of sodium acetate (1.8g, 20mL H2O).
- Heat the mixture for 10 min, cool on ice and collect the crystals.
- Recrystallise from ethanol.
- Dry overnight in an oven (50∘C).
Notes and troubleshooting tips
- If no activity occurs within 24 hours, stop the trial to avoid false positives (because of the possibility of CN from cyanogenic bacteria (Brinker and Seigler 1989).
Links to resources and suppliers
Adsersen, A, Adsersen, H and Brimer, L (1988). Cyanogenic constituents in plants from the Galapagos islands. Biochemical Systematics and Ecology 16:65-77.
Brinker, AM and Seigler DS (1989). Methods for the detection and quantitative determination of cyanide in plant materials. Phytochemical Bulletin 21:24 -31
Goldstein, WS and Spencer, KC (1985). Inhibition of cyanogenesis by tannins. Journal of Chemical Ecology 11:847-857.
Haque, M. R. and Bradbury, J. H. (2002). Total cyanide determination of plants and foods using the picrate and acid hydrolysis methods. Food Chemistry 77:107-114.
Hosel, H. (1981). The enzymatic hydrolysis of cyanogenic glucosides. In: Vennesland, B, Conn, EE, Knowles, CJ, Westley, J and Wissing, F (eds), Cyanide in biology. London:Academic Press pp217-232.
Hughes, MA (1991). The cyanogenic polymorphism in Trifolium repens L. (white clover). Heredity 66: 105-115.
Van Wyck, BE (1989). The taxonomic significance of cyanogenesis in Lotononis and related genera. Biochemical Systematics and Ecology 17: 297-303.
Health, safety & hazardous waste disposal considerations
Read appropriate material safety data sheets (MSDS) and be aware of local laws regulating disposal. Particular care should be taken when handling chloroform. Work in a fumehood and use a breathing apparatus.