Content of polyphenolic acids in organically and conventionally grown crops.

Ľubomír Daško, Alain Maquet, Elke Anklam

Abstract – Secondary plants metabolites – polyphenolic acids - play important role in plant defence system. Polyphenolic acids content determination is used with the aim to discriminate organically and conventionally produced plants. The procedure suitable for determination of twelve polyphenolic acids is described. Applying gradient separation in High Performance Liquid Chromatography and Diode Array Detector for their content estimation suitable baseline separation was achieved. Results of free, glycosidically bound and esterified polyphenolic acids are presented. Model plant products are carrot and winter wheat produced under organic and conventional agricultural system.

Introduction
Organic market is becoming more and more important particularly in Europe. A review on growing organic food market with its pros and cons was recently published (Siderer et al., 2005). But the organic market is based on the confidence consumers have to organic food products. Today many claims on the food labels that relate to the perceived added value are rarely supported by analytical data and evidence, leaving regulators to rely solely on paper auditing procedures to monitor compliance. It is important to stress that there is not yet available suitable method or a group of methods which clearly discriminate organic and conventional food. 
Determination of the content of some plants secondary metabolites is of increasing interest with the aim to discriminate organic versus conventional plants products (Young et al., 2005; Dimberg et al., 2005; Hajšlová et al. 2005). There are reported  trends in higher content of chlorogenic acid in organically grown potatoes versus conventional ones (Hajšlová et al. 2005). Contrary to these results there is no difference in content of secondary metabolites in oat grains (Dimberg et al., 2005) as well as vegetables (Young et al., 2005).
	Secondary metabolites mainly polyphenols are of great interest because of their possible positive human health impact (Willcox et al., 2004; Le Corre, et al. 2005; Lule and Xia, 2005). They play also important role in plants defence system (Lister et al., 1994; Fallico et al., 1996; Kawano et al., 2004) especially when bacterial and fungal pathogens invasions (Waldron et al., 1996) were confirmed.
In conventional agriculture pesticides are used to protect plants against bacterial and fungal pathogens. That is why it is probable that there could be different content of polyphenolic acids in organically grown plants versus conventional ones. 
   Polyphenolic acids can be found in plants in various forms. They could be detected as free molecules, bound as glycosides or esters, binary molecular bounding was also reported (Waldron et al., 1996) as well as binary bound polyphenolic acid   glycosides (Allerdings et al., 2005). To determine the “true” content of polyphenolic acids is in this time opened question. There are many different procedures used for polyphenolic acids determination, reviews on these topics were recently published (Tura and Robarts, 2002; Robards, 2003; Tsao and Deng, 2004).    
We selected 12 polyphenolic acids in our study. Three different extraction methods for their determination were applied. One separation procedure was suggested for the all three different extraction procedures. Developed method was used with the aim to discriminate organically and conventionally produced carrot and winter wheat.       

Experimental
All twelve polyphenolic acids were purchased from Sigma-Aldrich – gallic acid, 3.4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, Salicylic acid, trans-cinamic acid. 
   Free polyphenolic acids determination
    Freeze-dried material was used for analysis. Sample amount was 0.5 g. Extraction with 8 ml of mixture of methanol : water (85 : 15) was performed for  30 minutes in ultrasonic bath. tert-Butyl-4-Hydroxyanisole (BHA) was added into extraction mixture in amount 1mg/ml. After centrifugation and filtration the samples were ready for injection.
     Glycosidically bound polyphenolic acids 
	As in previous case freeze-dried material was used for extraction. Extraction mixture was identical, except of hydrochloric acid was added in to have at the end 1 M solution of HCl. The samples stayed over night, centrifuged and filtrated. Filtrate was ready for injection. 
Esterified polyphenolivc acids.
 	This procedure was similar to those of acidic hydrolysis. In this case esters were hydrolysed with sodium hydroxide which was added into extraction mixture (methanol : water, 85 : 15, and BHA 1 mg/ml) to receive final concentration 1 M NaOH. Hydrolysis was done overnight. Afterwards pH of extraction mixture was reduced to 2 with HCl and samples were centrifuged, filtrated and ready for injection. 
Chromatography separation  
   Gradient elution of standard (mixture of twelve polyphenolic acids) as well as the samples was performed applying Zorbax SB C18 column (Agilent technologies). Chromatography equipment was from Bio-Tec with Diode Array Detector (DAD), measuring was done at the wavelength 280 nm. 
  
Results
Suitable baseline separation of all twelve polyphenolic acids was achieved by gradient elution within 57 minutes – Fig. 1. 

  Figure 1. Gradient separation of polyphenolic acids: 1-gallic acid, 2- 3.4-dihydroxybenzoic acid, 3- 4-hydroxybenzoic acid, 4- chlorogenic acid, 5- vanillic acid, 6- caffeic acid, 7- syringic acid, 8- p-coumaric acid, 9- ferulic acid, 10-sinapic acid, 11- salicylic acid, 12- trans-cinamic acid               

Different extracting procedures were compared having in mind that polyphenolic acids can be present in plants in various forms as was already mentioned in the Introduction. The suitable fit-for-purpose extraction procedures were selected. Recovery of free polyphenolic acids extraction was tested by spiking carrot.samples. The values of recovery were within the range from 81 to 108 %. Detection level is within 10 mg/kg to 5 mg/kg. This is sufficient because the content of polyphenolic acids is at the levels more than 10 mg/kg. Relative standard deviation is dependent on the concentration; close to the detection level is within the range 20 – 15 % and at levels above detection level drop under 10 %. 
Results of analysis of carrot and winter wheat (free, glycosidically bound and esterified polyphenolic acids) grown under organic and conventional conditions will be presented. Observed variance in the amount of polyphenolic acids between plants within the same agricultural system could be due to the role of polyphenolic acids in plants defence system. The pathogens attack of each plant is not the same within the field. This is probably the source  of the variability of the content of polyphenolic acids in plants from the same agricultural system.  

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