Letter pubs.acs.org/JAFC
Addressing Analytical Requirements To Support Health Claims on “Olive Oil Polyphenols” (EC Regulation 432/2012) ABSTRACT: A health claim on “olive oil polyphenols”, namely, hydroxytyrosol and its derivatives, was recently approved by EC Regulation 432/2012. As no official method exists so far for the complete separation of all forms of hydroxytyrosol and tyrosol present in the oil, it is of utmost importance to find a simple, reproducible, and undisputable protocol to protect consumers and avoid unfair competition. In this study two hydrolysis protocols for the complex forms either in the polar fraction of the oil (protocol 1) or directly in the oil (protocol 2) were comparatively applied to a series of extra virgin olive oils. Protocol 1 gave at least 50% higher levels of total hydroxytyrosol and tyrosol. Nevertheless, the minimum amount of 5 mg/20 g oil of phenols was fulfilled only when suitable correction factors were introduced in the calculations to account for the differences in the mass between simple and complex phenols. Changes in the terminology used in the health claim are also proposed. KEYWORDS: total hydroxytyrosol and tyrosol content determination, “olive oil polyphenols” health claim, virgin olive oil
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INTRODUCTION Consumers are cautious about the nutrition and health claims provided on food labeling, which are expected to assist them in making sensible choices. To increase confidence in the market and ensure a high level of consumer protection, the European Food Safety Authority (EFSA) works for the approval of clear, accurate, and corroborated nutrition and health claims as well as of any other type of food labeling.1,2 Substantiation of a nutrition or health claim is a tedious, time-demanding procedure that encompasses several evaluation steps and eventually ends, when it is approved, in the relevant community register.3 Following the above-mentioned procedure a health claim on “olive oil polyphenols” was made only very recently after many years of discussion.4 The health claim was spelled out as: “Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress.” The claim may be used only for olive oil, which contains at least 5 mg of hydroxytyrosol and its derivatives (e.g., oleuropein complex and tyrosol) per 20 g of olive oil. Furthermore, information is given to the consumer that “the beneficial effect is obtained with a daily intake of 20 g of olive oil”. This health claim presents some weaknesses regarding terminology and analytical methodology. The term “olive oil polyphenols” is not entirely clear and accurate, taking into account that only fresh virgin olive oil of high quality contains considerable amounts of oleuropein/ligstroside aglycons and derivatives.5 “Olive oil” is a generic term for the type of oil (f rom olives) and does not correspond to any of the edible categories authorized by the European Union, the United States, or elsewhere. “Virgin olive oil” (VOO) is the appropriate term. Moreover, the term “polyphenols”, probably deriving decades ago from wine phenolic compound terminology, does not coincide with the basic structure of the secoiridoids present in VOO for which the claim was assigned (i.e., hydroxytyrosol and its derivative, e.g. oleuropein complex and tyrosol). “Virgin olive oil bioactive phenols” (i.e., hydroxytyrosol/tyrosol and their bound forms) better expresses the scientific content of the health claim. Beyond concerns for inaccurate terminology, there is a serious lack of a standardized analytical method that will allow quantitative determination of unequivocally identified individual phenolic compounds belonging to the group of © 2014 American Chemical Society
hydroxytyrosol/tyrosol and its derivatives. The latter comprises more than 10 identified compounds.5 This lack has an impact on the soundness of the lower limit set (5 mg/20 g oil) for the health claim. The LC-MS protocols cited in the relevant EFSA opinion6 result in not well-resolved peaks of individual phenols present in the polar extract of virgin olive oil so that MS becomes a mandatory identification tool. A candidate protocol could be the one recommended by the International Olive Council.7 However, difficulties in complete separation of all types of phenolic compounds in one chromatographic run and limitations in the choice of standards for accurate quantification in the UV region or using other detection means, repeatedly discussed over the past 20 years,5 do not support its adoption for standardization. To address such a challenge, any experienced analytical chemist would reach the decision of simplifying the analytical protocol. Simplification in this case study would involve hydrolysis of the bound forms of hydroxytyrosol and tyrosol and quantification of their total free forms. Aiming at determination of antioxidant potential, Mulinacci et al.8 examined both acidic and alkaline hydrolysis of the socalled polar fraction of VOO that contains the bioactive phenolic compounds under discussion. They found that only acidic conditions liberate hydroxytyrosol (Htyr) and tyrosol (Tyr) without destroying them. More recently, Romero and Brenes9 applied a hydrolytic reaction directly to oil to ease the determination of total Htyr and Tyr ignoring, though, the efforts made by the previous authors. In the present paper, we provide evidence about the superiority of determining total Htyr and Tyr content in the hydrolysate of the polar fraction and address complications in its calculation.
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MATERIALS AND METHODS
Chemicals and Samples. Tyr (98%) and caffeic acid (98%) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany); Htyr (≥98%) and oleuropein (98%) were from Extrasynthèse (Geney, France). Other reagents and solvents of appropriate grade were Received: Revised: Accepted: Published: 2459
December 4, 2013 February 26, 2014 February 27, 2014 February 27, 2014 dx.doi.org/10.1021/jf5005918 | J. Agric. Food Chem. 2014, 62, 2459−2461
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Letter
purchased from various producers. A total of 10 samples of extra VOOs, coded Fv, FN, F1, Zv, Z1, Z2, Cv, C1, Cha1, and Cha2, were chosen among a sample collection of the laboratory. The samples were kept at −23 °C in dark glass bottles with minimum headspace flushed with N2 until analysis. Polar Fraction Preparation. The polar fraction was extracted from 2.5 g of VOO dissolved in 5 mL of hexane using an equal volume of methanol/water (60:40 v/v). The mixture was vortexed for 2 min and centrifuged for 10 min at 3500 rpm. The isolated polar fraction was then used for the determination of total polar phenol content (TPP) colorimetrically10 and RP-HPLC separation of phenolic compounds prior to and after acidic hydrolysis.8,9 A second extraction was not necessary as >95% of TPP was obtained in the first extract (n = 5). Repeatability of the process was adequate (CV% = 4.7 for TPP = 7.67 mg caffeic acid/20 g oil, n = 5). Acidic Hydrolysis according to Mulinacci et al.8 (Protocol 1). Briefly, an aliquot (200 μL) from the polar fraction was mixed with 200 μL of a 1 M H2SO4 solution. The mixture was maintained in a water bath at 80 °C for 2 h. The procedure was carried out in triplicate. Each hydrolysate was then diluted with 200 μL of acetonitrile/water 50:50, v/v. The three replicates were combined to obtain a representative hydrolysate. The latter was filtered through a 0.45 μm pore size regenerated cellulose membrane (Schleicher and Schuell, MicroScience GmbH, Dassel, Germany) before injection into the chromatograph. Method intra- and interday repeatabilities (in two different days) were examined for a VOO sample (n = 5), and the respective CV% values obtained for Htyr and Tyr were 0.9/2.4 and 1.5/2.2. Samples were then analyzed in triplicate. Acidic Hydrolysis according to Romero and Brenes 9 (Protocol 2). Fifty milliliters of 2 M HCl was added to 2.5 g of VOO in a 100 mL glass bottle that was closed with a polypropylene cap. The mixture was vigorously agitated at 250 rpm in a shaker incubator (model MkX, Stoke Poges, UK) at 25 °C for 6 h in triplicate for each sample. Finally, 2 mL of the aqueous phase was removed by a plastic pipet from each replicate, mixed to form a representative hydrolysate, and filtered through a 0.45 μm regenerated cellulose membrane before injection into the chromatograph. Samples were analyzed in triplicate. RP-HPLC Analysis. The elution system used for the chromatographic separation of polar phenols on a Nucleosil C18 (250 × 4.6 mm, 5 μm) column (Macherey-Nägel, Düren, Germany) consisted of 1% aqueous acetic acid (solvent A) and acetonitrile (solvent B). The gradient was as follows: 0−2 min, 5% B; 2−15 min, 25% B; 15−22 min, 25% B; 22−30 min, 40% B; 30−40 min, 60% B; 40−50 min, 95% B; 50−52 min, 95% B; 52−54 min, 100% B; 54−60 min, 5% B, at a flow rate of 0.5 mL/min and an injection volume of 20 μL. Diode array (UV 6000 LP model, cell volume = 10 μL, Thermo Separation Products, San Jose, CA, USA) and fluorescence (SSI 502 model, cell volume = 8 μL, Scientific Systems Inc., State College, PA, USA) detectors were used in line. Calibration curves were constructed for Htyr and Tyr at appropriate wavelengths5 [280 nm; exc 280 nm/em 320 nm] (Supporting Information Figure S1). The limit of detection (LOD) and the limit of quantification (LOQ) were estimated for both Htyr and Tyr at 280 nm using the following equations:11 LOD = 3.3 × σ/S′ and LOQ = 10 × σ/S′ (where σ is the standard error of the y intercept and S′ the slope of the calibration curve). The values obtained were 0.7 and 2.0 ng/μL (Htyr) and 1.9 and 5.9 ng/μL (Tyr), respectively.
be critical (Table 1). Nevertheless, the yields derived for Htyr and Tyr were half of those obtained using protocol 1, as shown in the same table. Table 1. Effect of Agitation on the Yield of Htyr, Tyr, and Their Sum according to Protocol 2 hydrolysis conditions, 2 M HCl, 6 h
Htyra (mg/20 g oil)
Tyra (mg/20 g oil)
Htyr + Tyr (mg/20 g oil)
400 rpmb 200 rpmb 250 rpmc
0.66 a ± 0.01 0.69 ab ± 0.02 0.72 b ± 0.01
0.31 a ± 0.01 0.33 ab ± 0.02 0.35 b ± 0.01
0.97 1.02 1.07
isolation of polar fraction/ 1 M H2SO4, 2 h, 80 °Cd
1.63 c ± 0.03
0.69 c ± 0.02
2.32
a Data are the mean ± standard deviation (n = 3). Different letters in the columns represent significant differences in concentration (p < 0.05). bBench-top yellow-line orbital shaker (IKA-Werke GmbH & Co. KG). cShaker incubator (model MkX, Stoke Poges, UK). d Protocol 1.
On the basis of the above findings the two protocols were applied to a series of extra VOOs varied in TPP levels (Table 2). The superiority of protocol 1 was evident in all cases (2−7Table 2. Effect of Acidic Hydrolysis Protocols on the Total Htyr and Tyr Content in Extra VOOs of Various TPP Levels Htyr + Tyra,b (mg/20 g oil) TPPc sample (mg caffeic acid/20 g oil) Zv Z1 Z2 Cv C1 Fv FN F1 Cha1 Cha2
6.42 a 5.55 b 8.39 c 7.95 cd 7.80 ed 9.07 ed 7.96 cd 2.03 f 4.20 g 7.67 h
± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.1 0.1 0.1 0.4 0.5 0.1 0.3 0.5
protocol 1
protocol 2
protocol 1/ protocol2
2.34 3.16 3.10 1.76 2.92 2.28 2.91 3.73 3.11 2.57
0.88 0.94 1.08 0.76 0.70 0.77 0.84 0.53 0.51 1.08
2.7 3.4 2.9 2.3 4.2 3.0 3.5 7.0 6.1 2.4
a
Quantification at 280 nm. bHtyr and Tyr were quantified using respective external calibration curves. Each [Htyr + Tyr] value is the sum of mean values of individual phenols measured in triplicate. cData are the mean ± standard deviation (n = 3). Different letters in the same column represent significant differences in concentration (p < 0.05).
fold higher levels). The trend observed for the total content was also reflected in the ratios of individual phenol levels (data not shown). Subsequent discussion was, consequently, based on the results obtained using protocol 1. None of the extra VOO samples contained the minimum required level of total Htyr and Tyr content (5 mg/20 g oil), although some of them (Z2, Fv, FN, Cv) presented a rather high TPP content, unusual for commercial products. Using one standard curve for quantification of both phenols, it was evident that the use of Tyr resulted in higher levels in comparison to those using Htyr due to differences in ε values (see Figure S1A in the Supporting Information). Even so, the required limit was not achieved for any of the VOO samples. Fluorescence detection, which is more sensitive than UV absorption, gave comparable
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RESULTS AND DISCUSSION First, the stability of Htyr and Tyr and the completeness of oleuropein hydrolysis claimed by Mulinacci et al.8 were verified. Both compounds were found to be stable, and estimated losses for a solution of 250 mg/L accounted for approximately 6.1% (n = 3) for Htyr and 3% (n = 3) for Tyr using the respective calibration curves. Hydrolysis of an oleuropein solution (250 mg/L) was complete, leading to the sole formation of Htyr. Similarly, the parameter “agitation” of protocol 2 was tested for one VOO sample using two shaker types and was not found to 2460
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(4) European Community. Council Regulation No. 432/2012 of 16 May 2012 establishing a list of permitted health claims made on foods, other than those referring to the reduction of disease risk and to children’s development and health. Off. J. Eur. Communities 2012, L 136, 1−40. (5) Tsimidou, M. Z. Analytical methodologies: phenolic compounds related to olive oil taste issues. In Handbook of Olive Oil: Analysis and Properties, 2nd ed.; Aparicio, R., Harwood, J., Eds.; Springer Science + Business Media: New York, 2013; pp 311−333. (6) EFSA Panel on Dietetic Products Nutrition and Allergens. Scientific Opinion on the substantiation of health claims related to polyphenols in olive and protection of LDL particles from oxidative damage (ID 1333, 1638 1639, 1696 2865), maintenance of normal blood HDL cholesterol concentrations (ID 1639), maintenance of normal blood pressure (ID 3781), “anti-inflammatory properties” (ID 1882), “contributes to the upper respiratory tract health” (ID 3468), “can help to maintain a normal function of gastrointestinal tract” (3779), and “contributes to body defences against external agents” (ID 3467) pursuant to Article 13(1) of Regulation (EC) No. 1924/2006. EFSA J. 2011, 9, 2033−2058. (7) International Olive Council (IOC). Determination of biophenols in olive oil by HPLC. COI/T.20/Doc. no. 29, November 2009. (8) Mulinacci, N.; Giaccherini, C.; Ieri, F.; Innocenti, M.; Romani, A.; Vincieri, F. F. Evaluation of lignans and free and linked hydroxytyrosol and tyrosol in extra virgin olive oil after hydrolysis processes. J. Sci. Food Agric. 2006, 86, 757−764. (9) Romero, C.; Brenes, M. Analysis of total contents of hydroxytyrosol and tyrosol in olive oils. J. Agric. Food Chem. 2012, 60, 9017−9022. (10) Blekas, G.; Psomiadou, E.; Tsimidou, M.; Boskou, D. On the importance of total polar phenols to monitor the stability of Greek virgin olive oil. Eur. J. Lipid Sci. Technol. 2002, 104, 340−346. (11) Snyder, L. R.; Kirkland, J. J.; Dolan, J. W. Introduction to Modern Liquid Chromatography; Wiley: Hoboken, NJ, USA, 2010; pp 510− 514. (12) Nenadis, N.; Wang, L. F.; Tsimidou, M. Z.; Zhang, H. Y. Radical scavenging potential of phenolic compounds encountered in O. europaea products as indicated by calculation of bond dissociation enthalpy and ionization potential values. J. Agric. Food Chem. 2005, 53, 295−299.
estimations of the total content of Htyr and Tyr (∼1.2−1.4-fold higher) by means of the two calibration curves (see Table S1 in the Supporting Information). Using one standard curve it was shown that Htyr resulted in higher levels of total phenols in comparison to those using Tyr due to differences in their fluorescence response (see Figure S1B in the Supporting Information). Some of the samples reached the required limit (see Table S1 in the Supporting Information). From the TPP content, RP-HPLC profiles, and levels of Htyr and Tyr of the polar fraction prior to hydrolysis, it was observed that all of the oils were rich in complex forms of Htyr and Tyr. Indeed, the respective ranges prior to hydrolysis were extremely low (Htyr, 0.03−0.5 mg/20 g oil; Tyr, nd−0.43 mg/20 g oil), as expected for fresh oils. At this point we have to stress that calculation on a mass basis introduces a systematic error because the bound forms have a much higher molecular mass compared to those of simple phenols. The mean MW of the 10 most known bound forms of Htyr and Tyr12 is ∼346 amu. If a correction factor is introduced in the quantification using both standards (Htyr, 2.2; Tyr, 2.5) to account for the differences in mass between Htyr, Tyr, and their complex forms, respectively, then 9 of 10 VOO samples satisfied the health claim using UV detection at 280 nm (5.2 −8.9 mg/20 g oil), and all of them satisfied the health claim when fluorescence was used (5.5−10.6 mg/20 g oil). In conclusion, after many years of consultation a health claim was finally approved for VOO bioactive phenols, all of them derivatives of oleuropein and ligstroside. Addressing analytical requirements to support this health claim is imperative to ensure consumer protection and avoid unfair competition. Determination of free forms of Htyr and Tyr in the VOO polar fraction hydrolysate and introduction of a correction factor in quantification are needed to have a simple, reproducible, and undisputable analytical protocol.
Aspasia Mastralexi Nikolaos Nenadis Maria Z. Tsimidou*
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Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
ASSOCIATED CONTENT
S Supporting Information *
Table S1 and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.Z.T.) Phone: +30 2310 997796 . Fax: +30 2310 997847. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) European Community. Council Regulation No. 1924/2006 of 20 December 2006 on nutrition and health claims made on foods. Off. J. Eur. Communities 2006, L 404, 9−25. (2) European Community. Council Regulation No. 1169/2011 of 25 October 2011 on the provision of food information to consumers. Off. J. Eur. Communities 2011, L 304, 18−63. (3) EU Register of nutrition and health claims made on foods, http://ec.europa.eu/nuhclaims (accessed Nov 24, 2013). 2461
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