Protocol

Please note

This protocol was contributed by Robin Martin in conjunction with the Carnegie Institution for Science. Please visit the Spectranomics website at https://asnerlab.org/projects/spectranomics/ for more information and spectranomics tools.

Background

This protocol is modified from Ainsworth and Gillespie (2007) for Phenols; Toth and Pavia (2001) and Makaar (2007) for Tannins.

Materials/Equipment

Equipment

• Sartorius balance (0.0001 g)
• Talboys high-throughput homogenizer
• Mini centrifuge
• Vortex mixer
• Orbital shaker
• Microplate reader

Consumable Materials

• Gallic acid monohydrate
• Methanol NF-grade, 99.5%
• Polyvinylpyrrolidone (PVP)
• Folin-ciocalteau phenol TS (F-C reagent)
• Anhydrous sodium carbonate
• 5/8″ stainless steel threaded lugs ¼” width and 1/8″ stainless steel coneballs
• 2 ml threaded and snap-cap microcentrifuge tubes
• 96-well polystyrene clear flat bottom plates

Procedure

Extract Preparation

• Weigh frozen leaf disc (0.385 cm²) and transfer into a grinding tube with ball bearings and lugs.
• Add 0.75ml 95% methanol in water (vol/vol) and in homogenize for two minutes at high intensity.
• Add 0.75ml 95% MeOH to sample. Shake to homogenize.
• Aliquot off exactly 0.75 ml extract to a new 2 ml centrifuge tube containing another 0.75ml 95% MeOH.
• Aliquot 0.5 ml portion of this extract to a new 2 ml centrifuge tube for tannin analysis (detailed below).
• Dilute remaining extract 0.5 ml 95% MeOH to a total volume of 1.5 ml. This will be the -Phenol Extract.’
• Place the phenol extract on an orbital shaker and incubate at room temperature in the dark for 48 hrs before phenol analysis (detailed below)

Tannin Precipitation and Analysis

• Add 0.5 ml methanol to previously labeled 2 ml centrifuge tubes containing 10 mg PVP and place on ice.
• For each sample, transfer 0.5 ml of each tannin extract (referenced above) to a prepared tube, vortex for ten seconds, and incubate on ice for 10 min.
• Prepare two test blanks with 1 ml methanol and 10 mg PVP. These blanks are essential for removing PVP contribution to the absorbance values.
• Centrifuge samples and test blanks for 3 min.
• Transfer 0.75 ml supernatant into a new tube with 10 mg PVP for a second precipitation step.
• Vortex for 10 seconds and incubate on ice for 10 minutes.
• Centrifuge samples and, for each sample, transfer exactly 0.2 ml supernatant into new 2 ml centrifuge tube for “Folin C Assay” (detailed below).

Phenol Preparation and Analysis

• After a 48-hour incubation period, remove phenol extract tubes from shaker.
• Centrifuge samples and, for each sample, transfer exactly 0.2 ml supernatant into new 2 ml centrifuge tube for “Folin C Assay” (detailed below).

Folin C Assay

• Prepare Standard Assay tubes with 0.1ml previously made working standards (refer to Standard Preparation Procedure below).
• Add 0.2 ml 10% Folin-Ciocalteau Phenol reagent in water (vol/vol) to each assay tube prepared either during the tannin or phenol assay (above) and vortex for 10 sec.
• Incubate samples for 30-60 minutes at room temperature in the dark.
• Add 0.8 ml 700 mM sodium carbonate solution in water and incubate at room temp. in the dark for 2 hours.
• Centrifuge samples for 3 minutes.
• Transfer exactly 0.25 ml sample (in triplicate) into 96 well plates.
• Immediately scan in SAFIRE at a fixed wavelength of 735 nm.

Measurement Procedure

• Prepare 7 working standards (25-200 mg/L) of Gallic Acid in 95% methanol in water and standard blanks are prepared for this assay.
• Measure absorbance at 735 nm of triplicate samples on a microplate reader (Tecan SAFIRE). This measurement includes 2×2 multiple reads per well to reduce potential variability caused by the well.
• Transfer all absorbance values to a central phenol and tannin determination sheet where estimates of both secondary metabolites are expressed as gallic acid equivalents (GAE) relative to the standard curve (refer to Data Preparation and Finalization below).

Standard Preparation Procedure

• Prepare fresh working standards from 5 mg/ml stock solution of Gallic acid in 95% methanol solution in water every week and assay with each batch for phenols and tannins.
• Use 10 ml volumetric flasks to prepare standards and store in subdued light at 4^∘C.

 

Working Standard Concentration	Stock Volume (ml) Gallic Acid (5mg/ml)
V25 25 mg/L	0.05 ml
V50 50 mg/L	0.100 ml
V75 75 mg/L	0.150 ml
V100 100 mg/L	0.200 ml
V125 125 mg/L	0.250 ml
V150 150 mg/L	0.300 ml
V175 175 mg/L	0.350 ml
V200 200 mg/L	0.400 ml

Data Preparation and Finalization

Phenol and tannin concentrations are calculated in terms of gallic acid equivalents (GAE) mass basis and corrected for standardization blanks, PVPP test blanks, and dilution factor using the following equations:

• Phenol Concentration in solution after F-C Assay (GAE mg/L) = [(A735sample-A735blank)-Intercept GA standard curve]/slope GA standard curve

 

• Total Phenols (GAE mg/L) = Phenol Conc in the solution that was incubated for 48 hrs.

 

• Tannins = Total Phenols – Phenol Conc in the solution analysed after PVP precipitation.

 

• GAE (mg) = GAE (mg/L) x Total extract volume (ml)/1000 (ml/L)

 

• GAE mg/g = GAE (mg) / Disk dry weight (g)

 

• Disk dry weight is calculated as [Disk wet weight (g) – (disk wet weight (g) *%H2O from total leaf)]