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Accreditation ΕΝ ISO 17025-2005 1999 - UKAS ACCREDITATED 2011-ESYD (Hellenic Accreditation Body)

Health Claims Related to “Polyphenols” in Olive (EFSA opinion 2011) Dr. Despina Tsipi Head of Food Safety & Quality Laboratories, A Chemical Division General Chemical State Laboratory Athens 11521, Greece dtsipi@gcsl.gr. Accreditation ΕΝ ISO 17025-2005 1999 - UKAS ACCREDITATED

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Accreditation ΕΝ ISO 17025-2005 1999 - UKAS ACCREDITATED 2011-ESYD (Hellenic Accreditation Body)

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  1. Health Claims Related to “Polyphenols” in Olive (EFSA opinion 2011)Dr. Despina TsipiHead of Food Safety & Quality Laboratories,A Chemical DivisionGeneral Chemical State LaboratoryAthens 11521, Greecedtsipi@gcsl.gr

  2. AccreditationΕΝ ISO 17025-2005 1999 - UKAS ACCREDITATED 2011-ESYD (Hellenic Accreditation Body) IOC Recognition GENERAL CHEMICAL STATE LABORATORY (GCSL) A CHEMICAL SERVICE ATHENS, GREECE EU –NATIONAL REFERENCE LABORATORIES www.gcsl.gr FOOD SAFETY & QUALITY LABORATORIES EURLs-NRLs-OFLs NETWORK

  3. Health Claims ‘Health claim’ means any claim that states, suggests or implies that a relationship exists between a food category or a food or one of its constituents and health.

  4. Nutrition Claims ‘Nutrition claim’ means any claim that states, suggests or implies that a food has particular beneficial nutritional properties due to: • the energy (calorific value) it(i) provides,(ii) provides at a reduced or increased rate, or(iii) does not provide; and/or (b) the nutrients or other substances it(i) contains,(ii) contains in reduced or increased proportions, or(iii) does not contain;

  5. EXAMPLES Nutrition Claims • the energy (calorific value) Low Energy Energy-Reduced Energy-Free (b) the nutrients or other substances (i) High Protein(ii) Low Fat, (iii) “this product is a source of Vitamin E” Health Claims “Vitamin E contributes to the protection of cells from oxidative stress”

  6. Nutrition & Health Claims • REGULATION (EC) No 1924/2006 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCILof 20 December 2006on nutrition and health claims made on foods Amendments: Regulation EU on the provision of food information (2011/1169/EEC)

  7. Health Claims (a) • In EU, health claims made on foods are prohibited unless they are authorized in accordance with Regulation (EC) 1924/2006 and included in a list of permitted claims. • Permitted health claims must be accompanied with all necessary conditions, including restrictions, for their use.

  8. Health Claims (b) • In 2008, EC received more than 44,000 national health claims from Member States (MS) and after their examination a consolidated list of 4,600 claims was established and submitted to EFSA for a scientific advice. www.ec.europa.eu/nuhclaims • The scientific evaluation of EFSA was based on generally accepted scientific evidence and its opinions on health claims were published in EFSA Journal between October 2009 and July 2011. www.efsa.europa.eu/efsajournal .

  9. Health Claims (c) • Health claims that did not receive a favorable assessment on their scientific substantiation, as it was not concluded that a cause and effect relationship had been established between a food or one of its constituents and the claimed effect, were not authorized. • Finally only 2,282 health claims were authorized

  10. AUTHORIZATION–LIST OF PERMITTED HEALTH CLAIMS COMMISSION REGULATION (EU) No 432/2012of 16 May 2012establishing a list of permitted health claims made on foods, other than those referring to thereduction of disease risk and to children’s development and health

  11. Health Claims Related to Polyphenols in Olive • In 2010, EFSA was asked to provide a scientific opinion on a list of health claims related to polyphenols (e.g hydroxytyrosol and oleuropein complex) in olive (olive fruit, olive mill waste waters or olive oil, Oleaeuropaea L. extract and leaf). EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) EFSA Journal 2011;9(4):2033

  12. EFSA-NDA Panel- health claims inrelation to polyphenols in olive (a) • “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),

  13. EFSA-NDA Panel- health claims inrelation to polyphenols in olive (b) • “contributes to the upper respiratory tract health”, (ID 3468), • “can help to maintain a normal function of gastrointestinal tract”(3779) • “contributes to body defenses against external agents” (ID 3467)

  14. EFSA-NDA PanelScientific Assessment • 1. Characterization of food/constituent • 2. Relevance of the claimed effect to human health • 3. Scientific substantiation of the claimed effect • 4. Proposed wording • 5. Conditions and restrictions on use

  15. EFSA-NDA PanelScientific Assessment • 1. Characterization of food/constituent Polyphenols (e.g. hydroxytyrosol and Oleuropein complex) in olive (olive fruit, olive mill waste waters or olive oil, OleaeuropaeaL. extractand leaf)

  16. Polyphenols = Polar Phenolic Compounds in Olives (a) Olea europea 1908, Bourquelot και Vintilesco 1960, Panizzi, Scarpati, Oriente

  17. Polyphenols =Polar Phenolic Compounds in Olives(b) Tyrosol [I] HydroxyTyrosol [II]

  18. EFSA-NDA PanelScientific Assessment • 2. Relevance of the claimed effect to human health “ Protection of Low Density Lipoprotein (LDL) particles from oxidative damage” ID1333, 1638, 1639, 1696, 2865 NDA opinion: may be a beneficial physiological effect

  19. EFSA-NDA PanelScientific Assessment • 2. Relevance of the claimed effect to human health • “maintenance of normal blood HDL-cholesterol concentrations” (ID 1639), • “ maintenance of normal blood pressure” (ID 3781), NDA opinion: may be a beneficial physiological effect

  20. EFSA-NDA Panel -Scientific Assessment • 2. Relevance of the claimed effect to human health: “anti-inflammatory properties”(ID 1882), NDA opinion: this claim refers to diseases such as osteoarthritis or rheumatoid arthritis, in which a reduction of inflammation would be a therapeutic target for the treatment of disease & does not comply the criteria of Regulation

  21. EFSA-NDA Panel -Scientific Assessment • 2. Relevance of the claimed effect to human health: • “contributes to the upper respiratory tract health”, (ID 3468), • “can help to maintain a normal function of gastrointestinal tract”(ID3779) , • “contributes to body defenses against external agents” (ID 3467) NDA opinion : claims are general & no specific as required by Regulation

  22. EFSA-NDA PanelScientific Assessment 3. Scientific substantiation of the claimed effect (a) “ Protection of Low Density Lipoprotein (LDL) particles from oxidative damage”, ID1333, 1638, 1639, 1696, 2865

  23. 3. Scientific substantiation of the claimed effect (b) Oxidative damage to lipids was assessed in a multicenter human intervention study (*) • 200 males subjects for 3 weeks (25ml or 22g/daily) • Low-Polyphenol Olive Oil (2.7 mg/kg) • Medium-Polyphenol Olive Oil (164 mg/kg) • High-Polyphenol Olive Oil (366 mg/kg) • Measurements • Biomarkers: conjugated dienes, hydroxy fatty acids & oxLDL • Hydroxytyrosol & tyrosol in urine Results :A significant linear decrease in biomarkers of lipid peroxidation was observed in association with the increasing phenolic content of the olive oils (*) Covas et.al., 2006, Annals of Internal Medicine,145, 333-341

  24. 3.Scientific substantiation of the claimed effect (c) NDA-panel : Conclusions can be drawn from • the large number of samples, n=200 • the sufficient characterization of polyphenols in the olive oils, SPE-HPLC-DAD, LC-MS/MS (*) • the use of valid biomarkers to assess lipid peroxidation & the peroxidation of LDL • the dose-dependent response observed between the intake of polyphenols in olive oil and the decrease of oxLDL (*) J of Agric. Food Chem. 2005, 53, 4331-4340

  25. 3.Scientific substantiation of the claimed effect (e) NDA-panel : Conclusions can be drawn from • the inverse relationship between the concentration of phenolic metabolites in LDL particles & the degree of LDL peroxidation (*) (*) De la Torre-Carbot et al. 2010, J. of Nutrition, 140, 501-8.

  26. EFSA-NDA PanelScientific Assessment 3. Scientific substantiation of the claimed effect (a) “ Protection of Low Density Lipoprotein (LDL) particles from oxidative damage”, EFSA-NDA opinion: a cause & effect relationship has been established by consumption of olive oil “polyphenols” and “Protection of Low Density Lipoprotein (LDL) particles from oxidative damage”

  27. EFSA-NDA PanelScientific Assessment • 3. Scientific substantiation of the claimed effect • “maintenance of normal blood HDL-cholesterol concentrations” (ID 1639), NDA opinion: the evidence provided is insufficient to establish a cause and effect relationship between the consumption of olive oil “polyphenols” and “maintenance of normal blood HDL-cholesterol concentrations” No authorization (ID 1639)

  28. EFSA-NDA PanelScientific Assessment • 3. Scientific substantiation of the claimed effect “ maintenance of normal blood pressure” (ID 3781), NDA opinion:a cause and effect relationship hasnot been established between the consumption of olive oil “polyphenols” and “ maintenance of normal blood pressure” No authorization (ID 3781)

  29. EFSA-NDA PanelScientific Assessment 4. Proposed wording NDA opinion:“ Consumption of olive oil “polyphenols” contributes to the protection of blood lipids from oxidative stress” Authorization of Health Claim

  30. EFSA-NDA PanelScientific Assessment • 5. Conditions and restrictions on use NDA opinion: 5mg of hydroxytyrosol and its derivatives (e.g. oleuropein complex & tyrosol) in olive oil consumed daily Daily consumption: 20g olive oil Authorization of Health Claim

  31. Health claim NDA opinion EFSA 2011 EU AUTHORIZATION 2012 • REG. EC.432/ 2012 • Daily Intake : • 5 mg / 20 g olive oil • or • 250 mg/ kg olive oil • Exp. Time bottled olive oil = 18 months

  32. EU AUTHORIZATION REG. EC.432/ 2012 • Conditions of use of the claim • The claim may be used only for olive oil containing 5 mg of hydroxytyrosol and its derivatives (e.g. oleuropein complex & tyrosol) per 20 g olive oil (*). In order to bear the claim information shall be given to the consumer that the beneficial effect is obtained by a daily intake of 20 g olive oil • (*)250 mg/ kg olive oil • Exp. Time bottled olive oil = 18 months

  33. Analytical Methods (a) EFSA NDA opinion No analytical method was suggested No list of Target compounds “Polyphenols in olive can be characterized by their content of hydroxytyrosol and its derivatives (e.g. oleuropein complex & tyrosol)” “These polyphenols can be measured by established methods”

  34. Analytical Methods (b) Determination of Total Polyphenols using the Folin-Ciocalteau method based on reducing capacity EFSA NDA opinion: a non specific method, overestimation

  35. Sample (olive oil) Preparation : Phenols isolation Instrumental Analysis, HPLC-DAD Polyphenols Analysis ± Hydrolysis LIQUID LIQUID EXTRACTION, LLE SOLID PHASE EXTRACTION, SPE ± Hydrolysis Calculation of Results Lack of Reference Standards Shimadzu Seminar August 2006

  36. Phenols isolation(*) Sample Preparation LIQUID-LIQUID EXTRACTION, LLE 2 g Olive Oil +IS (Syringic acid ) + 5 ml Methanol –H2O 8:2, LLE Ultrasonic bath Centrifuge External calibration standards: tyrosol and syringic acid (*) INTERNATIONAL OLIVE COUNCIL COI/T.20/Doc No 29 November 2009 “DETERMINATION OF BIOPHENOLS IN OLIVE OILS BY HPLC "

  37. Phenols Analysis(*) HPLC-DAD at 280nm Instrumental Analysis, HPLC-DAD • HPLC: • Column: C18 reverse-phase (4.6 mm x 25 cm), type Spherisorb ODS-2 5mm, 100 A°, • Spectrophotometric UV detector at 280 nm, • Identification : Spectra using a photodiode detector with a range from 200 nm to 400 nm, • Ternary linear elution gradient: H2O 0.2 % H3PO4 (V/V) (A), methanol (B), acetonitrile (C). (*)INTERNATIONAL OLIVE COUNCIL COI/T.20/Doc No 29 November 2009 “DETERMINATION OF BIOPHENOLS IN OLIVE OILS BY HPLC "

  38. HPLC-UV Chromatogram of polar phenolic compounds in olive oil, Method COI/T. 20/Doc. No 29

  39. Method COI/T. 20/Doc. No 29 Table 1. Identification of biophenols peaks.Maximum absorbance (max UV abs) values and relative retention times(RRT)*Peak No Biophenols RRT* Max. UV abs.nm 1 Hydroxytyrosol 0.62 230-280 2 Tyrosol 0.80 230-275 3 Vanillic acid 0.96 260 4 Caffeic acid 0.99 325 5 Syringic acid (internal standard) 1.00 280 6 Vanillin 1.10 310 7 Para-coumaric acid 1.12 310 8 Hydroxytyrosyl acetate 1.20 232-285 9 Ferulic acid 1.26 325 10 Ortho-coumaric acid 1.31 325 11;11a Decarboxymethyl oleuropein aglycone, oxidised dialdehyde form - 235-280 12 Decarboxymethyl oleuropein aglycone, dialdehyde form 1.45 235-280 13 Oleuropein 1.48 230-280 14 Oleuropein aglycone, dialdehyde form 1.52 235-280 15 Tyrosyl acetate 1.54 230-280 16;16a Decarboxymethyl ligstroside aglycone, oxidised dialdehyde form 1.63 235-275

  40. Method COI/T. 20/Doc. No 29 Table 1 (continue). Identification of biophenols peaks.Maximum absorbance (max UV abs) values and relative retention times(RRT)*Peak No Biophenols RRT* Max. UV abs.nm 15 Tyrosyl acetate 1.54 230-280 16;16a Decarboxymethyl ligstroside aglycone, oxidised dialdehyde form 1.63 235-275 17 Decarboxymethyl ligstroside aglycone, dialdehyde form 1.65 235-275 18 Pinoresinol, 1 acetoxy-pinoresinol 1.69 232-280 19 Cinnamic acid 1.73 270 20 Ligstroside aglycone, dialdehyde form 1.74 235-275 21;21a;21b Oleuropein aglycone, oxidised aldehyde and hydroxylic form-235-280 22 Luteolin 1.79 255-350 23 Oleuropein aglycone, aldehyde and hydroxylic form 1.87 235-280 24;24a;24b Ligstroside aglycone, oxidised aldehyde and hydroxylic form-235-275 25 Apigenin 1.98 230-270-340 26 Methyl-luteolin - 255-350 27 Ligstroside aglycone, aldehyde and hydroxylic form 2.03 235-275 (*) The relative retention time is calculated with respect to the retention time of syringic acid. Identification is performed by HPLC-MS

  41. Method COI/T. 20/Doc. No 29 Calculation of the biophenol content (27 compounds) of virgin olive oil (in mg Tyrosol/kg olive oil)

  42. Analytical Method evaluated by EFSA -NDA panel (*) Characterization of the phenolic compounds (23) in olive oils by SPE (diol) , HPLC-DAD (280, 240, 320nm) (*) J of Agric. Food Chem. 2005, 53, 4331-4340 in mg Tyrosol+Oleuropein+Vanillic, p-coumaric acids + Vanillin+luteolin / kg olive oil

  43. Target list of the phenolic compounds (23) in olive oils (*) J of Agric. Food Chem. 2005, 53, 4331-4340 • Phenolic alcohols / expressed as tyrosol (STD) • Phenolic acids (STD: vanillic, p-coumaric) • Vanillin (STD) • Ligstroside derivatives as Oleuropein (STD) • Oleuropein derivatives as Oleuropein (STD) • Flavonoidsexpreessed as luteolin (STD) • in olive oils by SPE (diol) , HPLC-DAD (280, 240, 320nm)

  44. INTERNATIONAL OLIVE COUNCIL MEETINGS OF CHEMISTS TO STUDY METHODS OF ANALYSIS FOR OLIVE OILS AND OLIVE POMACE OILS, At recent IOOC meetings in 2015, the experts placed top priority on revising the current method for the determination of phenols : 1. comparing a colorimetric method ( modified Folin-Ciocalteau method ) with an HPLC method and 2. comparing two methods of extraction (SPE (diol) and liquid/liquid).

  45. Modified LLE-HPLC Method Phenols isolation LIQUID-LIQUID EXTRACTION, LLE 4 g Olive Oil + IS (o-Coumaric acid, p-OH-phenyl acetic acid ) + 2X5 ml Methanol –H2O 8:2, LLE Ultrasonic bath Centrifuge INTERNATIONAL OLIVE OIL COUNCIL , COI, 2015 Restrictive Ring Test “DETERMINATION OF PHENOL COMPOUNDS IN OLIVE OILS "

  46. Modified LLE-HPLC Method Phenols analysis HPLC-DAD (280nm, 335 nm) 280 nm, IS = p-OH-phenyl acetic acid Phenols, Cinnamic acid, Lignans 335nm,IS = o-Coumaric acid (Ferrulic acid, Luteolin, Apigenin) Phenols Calculation, (sum of 17compounds) in mg/kg INTERNATIONAL OLIVE OIL COUNCIL , COI, 2015, Restrictive Ring Test (4Labs) “DETERMINATION OF PHENOL COMPOUNDS IN OLIVE OILS “

  47. GENERAL CHEMICAL STATE LABORATORY • “Mapping of the olive oil market in the primary sector”* Reg (ΕC) 867/2008 • Crop years :2012-13, 2013-14 • 40 olive oil samples were collected from olive mills of seven olive oil producing prefectures of Greece • 20 samples for crop year 2012/2013 • 20 samples for crop year 2013/2014(from the same olive mills) *Project of National Centre for Exports and Olive Oil Promotion of Greece (Acronym: EKEPE in Greek)

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