Ian Wallis, Jens Maintz
This protocol provides a guide to the chemical analysis of leaf samples, covering the extraction of phenolics, preparation of an internal tannin and acid butanol assay for proanthocyanidins. We use this assay sparingly because the results do not neccessarily relate to animals. In other words, a high concentration of proanthocyanidins (PA) does not always correlate with binding of protein (i.e. tannin activity) and with deterrence of herbivores. Part of the explanation is that PA may also bind alkaloids, another secondary plant chemical that plants produce to defend themselves. Thus, for a functional measure of tannins we use the available nitrogen assay (DeGabriel et al., 2008) and we may use this technique to further examine plant chemistry. Although this assay tends to be slow it gives excellent calibration equations with near infrared spectroscopy (NIRS), which means that those interested can analyse many samples quickly and cheaply.
SAFETY: Before starting, read all of the related Material Safety Data Sheets (MSDS) and make sure you understand any dangers in the assay. If there is anything that you do not understand then ask.
Units, terms, definitions
There are many complicated terms and definitions associated with the chemistry of plant phenolic compounds that writers use interchangeably.
Extraction of phenolics
Background – Extraction of phenolics
The following describes the extraction of phenolics from leaf samples according to the procedure of Cork and Krockenberger (1991) and in the “Tannin Handbook” by Hagerman (2002), with appropriate adjustment to the amounts of sample and chemicals. Note that the sample mass depends on the purpose. If you want to prepare the internal tannin, then you will need to start with 500 mg of sample and later you may need to dilute samples or use less of the extract for reading on the spectrophotometer.
Materials/Equipment – Extraction of phenolics
50% aqueous acetone (MilliQ water:acetone, 50:50) containing AR acetone (Ajax Finechem, Australia) and MilliQ water.
Procedure – Extraction of phenolics
Weigh 50 ∓ 1 mg or 500 ∓ 1 mg of lyophilized leaf powder into a plastic screw cap tube and record the exact sample weight. Add 5 ml of extraction solvent consisting of 50% v/v AR acetone in MilliQ water using a bottle top dispenser and then sonicate the samples for 30 min at 50 Hz and 4∘ C in an ultrasonic bath (model FX12P, Unisonics, Australia). To collect solid leaf material at the bottom of the tubes, centrifuge the extracts for 2 min at 900 rpm and 4∘ C (Jouan centrifuge CR4.12 refrigerated; sequencer lab). After transferring the supernatant to 20 ml glass containers with plastic screw caps repeat the extraction twice and store at 4∘ C until further processing.
Preparation of internal tannin
Authors often cite their results in Quebracho equivalents per mass of plant tissue. It may sometimes be more appropriate to give the concentration in units that represent the material under study. For example, in a study of blue gum (Eucalyptus globulus) chemistry, we prepared a bulk tannin standard from all of the samples and used this as a reference.
Materials/Equipment – Preparation of internal tannin
0.1 M ytterbium acetate solution containing 422.2 mg of 99.9% ytterbium (III) acetate tetrahydrate (Yb(C2H302).4H20) (Sigma) in 10 ml of distilled water
Procedure – Preparation of internal tannin
Prepare an internal tannin of proanthocyanidins using trivalent ytterbium (GinerChavez et al., 1997). Take 1 ml aliquots from all of the extractions prepared above and pool them together in a 200 ml beaker. Transfer the contents of the beaker to a separating funnel and wash them three times with an equal volume (ca 100 ml) of AR petroleum spirit (Ajax Finechem) to remove chlorophyll. Then evaporate the traces of petroleum spirit with a short centrifugation step in a SpeedVac centrifuge. Repeat the process using AR ethyl acetate (Ajax Finechem) as the washing solvent to remove monomeric phenolic compounds and evaporate the remaining traces of ethyl acetate with a short centrifugation step. Transfer the remaining aqueous solution of condensed tannins (ca 50 ml) to a Falcon tube and add 2 ml of 0.1 M ytterbium acetate solution. Store the solution overnight at 4∘ C and then centrifuge it at 3000 x g for 10 min at 4∘ C. Wash the ytterbium-precipitate (Yb-ppt) pellet that forms in the bottom of the tube with 50 % aqueous acetone and centrifuge. Repeat the process and then repeat again with acetone. Dry the pellet in an oven at 50∘ C and then pulverize it in liquid nitrogen with a mortar and pestle. Store the Yb-ppt -20∘ C in the dark for later use as an internal tannin. Later, prepare an internal tannin solution (5 mg.ml-1) by dissolving 100 mg Yb-ppt in 20 ml of 50% aqueous acetone containing 0.5% of AR concentrated hydrochloric acid (HCl conc.) (Ajax Finechem).
Prepare a series of standards using quebracho and internal tannin. We find that a series of standards up to 1 mg/ml covers the range expected. Note that a bulk eucalypt internal tannin would dissolve easily only in acidified aqueous acetone.
Acid butanol assay for proanthocyanidins
Materials/Equipment – Acid butanol assay for proanthocyanidins
Acid butanol (n-butanol HCl, 95:5) containing 50.0 ml AR HCl conc (Ajax Finechem) 950 ml AR n-butanol (May and Baker, Australia)
Iron reagent (2% in 2N HCl) containing 1 g ACS ammonium iron (III) sulfate dodecahydrate (NH4Fe(SO4)2.12H2O)(Sigma) in 41.25 ml distilled water and 8.75 ml AR HCl conc. (Ajax Finechem)
Procedure – Acid butanol assay for proanthocyanidins
Determine the amount of proanthocyanidins in the crude extracts using the method described by (Porter et al., 1986) and Hagerman (2002), but see the note below about dilutions and appropriate extract volumes. An autodispensing system makes analyzing many samples simple. In our laboratory we use the two high precision syringes (1 ml and 25 ml, Hamilton, USA) attached to the Microlab 500 dispenser (Hamilton) to add 6 ml aliquots of acid butanol and 0.2 ml of iron reagent to Pyrex Teflon-lined screw cap culture tubes (ca 10 ml). Using a calibrated micropipette (1000 μl, Eppendorf, Germany), add 1 ml of extract and then mix the tube thoroughly. Zero the spectrophotometer (in our case a Beckman DU-640) using a blank containing one millilitre of extraction solvent (50% acidified aqueous acetone), and then take “pre-heating” measurements from all samples by reading the absorbance at 550 nm. Afterwards, cap the tubes tightly and place them in a water bath at 95∘ C for one hour. After cooling, read the absorbance again at 550 nm, subtracting the measurements taken before you heated the sample. Prepare standard curves using the internal tannin, prepared earlier and quebracho tannin. Calculate the concentrations with reference to the different standard curves using regression analysis, and express the results as “equivalents per extracted dry weight of leaves” (mg.g-1 DW).
Notes and troubleshooting tips
Getting an appropriate reading (i.e., absorbance units between 0 and 1) on a spectrophotometer is tricky and depends on the concentration of tannins. We found that adding 1 ml of the extract prepared above gives absorbance readings that are several times the limits of a linear response for many eucalypt samples. Diluting such samples 1:6 gives sensible readings. Thus, we suggest adding only 100 l of extract for eucalypt assays and even this amount still requires diluting some samples. Clearly, some trial and error is necessary to reach appropriate dilutions.
Links to resources and suppliers
Cork, S.J., Krockenberger, A.K., 1991. Methods and pitfalls of extracting condensed tannins and other phenolics from plants – insights from investigations on Eucalyptus leaves. J. Chem. Ecol. 17, 123-134.
DeGabriel, J.L., Wallis, I.R., Moore, B.D., Foley, W.J., 2008. A simple, integrative assay to quantify nutritional quality of browses for herbivores. Oecologia 156, 107-116.
Giner-Chavez, B.I., Van Soest, P.J., Robertson, J.B., Lascano, C., Reed, J.D., Pell, A.N., 1997. A method for isolating condensed tannins from crude plant extracts with trivalent ytterbium. J. Sci. Food. Agric. 74, 359-368.
Hagerman (2002) ‘The Tannin Handbook’ https://www.academia.edu/25605508/The_Tannin_Handbook
Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25, 223-230.
AOAC – Association of Official Agricultural Chemists (The AOAC publishes a journal on official methods of chemical analysis)
Health, safety & hazardous waste disposal considerations
SAFETY: Before starting, read all of the related Material Safety Data Sheets (MSDS) and make sure you understand any dangers in the assay. Familiarise yourself with local regulations for the disposal of chemicals.
Search terms and classification
Tannins, proanthocyanidins, phenolic, defence, herbivory, NIRS available nitrogen