EXTRACTION OF POLYPHENOLS FROM OLIVE LEAVES AND HYDROLYSIS OF OLEUROPEIN FOR THE PRODUCTION OF 3-HYDROXYTYROSOL G.K. LAMPROU , A. VLYSIDIS, A.G. VLYSSIDES LABORATORY OF ORGANIC CHEMICAL TECHNOLOGY SCHOOL OF CHEMICAL ENGINEERING NATIONAL TECHNICAL UNIVERSITY OF ATHENS
SCOPES OF OUR RESEARCH Development of a feasible and cost effective methodology for the recovery of olive leaves phenolic compounds. Determination of the optimum controlled conditions, for the production of 3-hydroxytyrosol with an easy-to- apply method. Study and development on novel applications of phenolic compounds in non-food , non-drug and non- cosmetic applications. Integrated management of wastes with the parallel production of high value products. Establishment of environmental friendly techniques for the separation of high value products from agricultural wastes.
STATE OF THE ART Phenolic compounds, known as “polyphenols”, have attracted the attention of research community due to their beneficial action in human health. Their physical role is to provide protection in plants against pathogens, predators or/and stress caused by abiotic factors such as such as hydric deficiency, salinity, low fertilization and climatic conditions. These molecules have been recognized for their anti-carcinogenic, anti-inflammatory, and antimicrobial actions. In 2012, EU Committee established a list of permitted health claims made on foods, based on scientific research made by EFSA. This was a basic factor, in order to become extra virgin oil for its health benefits. In 2017, EU published Regulation 2017/2373, through which authorized the placing on the market of hydroxytyrosol as a novel food ingredient intended for the general population, excluding children under the age of three years, pregnant women, and lactating women. (hydroxytyrosol (HT) as a food additive to fish and vegetable oils up to 215 mg/kg and to margarines up to 175 mg/kg). HT, may be found in many plants. Olea europeaea has a high concentration of this molecule. Olive oil, olive mill waste and leaves are the main sources for the extraction this compound. Although HT is expensive, there is no industrial activity in this field.
Recovery of polyphenols from olive leaves is an important challenge for food and pharmaceutical industrial applications. Oleuropein is the major phenolic compound in olive leaves, and large amounts of HT can be obtained using hydrolysis of oleuropein. HT production requires enzymatic or chemical hydrolysis. The natural mechanism that occurs when the olive tree forms free HT is enzymatic hydrolysis, and specific native β -glycosidase and esterase are implicated. On the contrary, acid hydrolysis is the more used mechanism in the laboratory and industrial processes. ACID HYDROLYSIS OF OLEUROPEIN
METHODOLOGY
EXPERIMENTAL METHODOLOGY FOR EXTRACTING THE PHENOLIC COMPOUNDS Acid hydrolysis process (extraction time 300 mins) Lab scale experiments with initial amount of olive leafs 10 g (14.2% moisture) Design of a 3 3 factorial experiment in order to measure the effect of three important process parameters T emperature (X1) Quantity of the strong acid (X2) Dilution of Olive Leafs (X3) At the end of each run the aqueous phase was T emperature o C H 2 SO 4 % L/S (w/v) Level (X1) (v/w) (X2) (X3) separated and analyzed: -1.682 20.0 2.0% 10.0 - TPC (Folin method) -1 24.1 3.6% 14.1 - Concentration of 3-Hydroxytyrosol, 0 30.0 6.0% 20,0 Tyrosol, Oleuropein (HPLC) 1 36.0 8.4% 26.0 1.682 40.0 10.0% 30.0
EXPERIMENTAL RESULTS OF THE FACTORIAL EXPERIMENT L/S TPC Oleuropein Hydroxytyrosol Tyrosol RUNS Temperature (oC) H2SO4 % (v/w) (v/w) (mg/g db) (mg/g db) (mg/g db) (mg/g db) 1 36.0 (1) 8.4 (1) 26.0 (1) 80,20 22,96 7,84 1,58 2 36.0 (1) 8.4 (1) 14.1(-1) 63,42 9,84 8,33 1,52 3 36.0 (1) 3.6 (-1) 26.0 (1) 75,92 42,15 3,92 2,30 4 36.0 (1) 3.6 (-1) 14.1(-1) 45,76 23,17 2,33 1,22 5 24.1 (-1) 8.4 (1) 26.0 (1) 80,04 30,63 4,83 2,32 6 24.1 (-1) 8.4 (1) 14.1(-1) 58,99 15,75 6,08 1,51 7 24.1 (-1) 3.6 (-1) 26.0 (1) 62,78 43,20 2,06 2,17 8 24.1 (-1) 3.6 (-1) 14.1(-1) 72,85 38,14 2,57 0,79 9 40.0 (1,682) 6.0 (0) 20.0(0) 72,38 30,03 0,71 1,09 10 20.0 (-1,682) 6.0 (0) 20.0(0) 59,37 24,81 0,33 1,09 11 30.0 (0) 10.0 (1,682) 20.0(0) 69,13 11,05 6,51 1,13 12 30.0 (0) 2.0 (-1,682) 20.0(0) 84,99 35,10 0,00 1,31 13 30.0 (0) 6.0 (0) 30.0 (1,682) 80,51 31,78 0,00 1,87 14 30.0 (0) 6.0 (0) 10.0 (-1,682) 65,88 11,46 5,92 0,59 15 30.0 (0) 6.0 (0) 20.0(0) 82,55 24,62 0,00 1,37 16 30.0 (0) 6.0 (0) 20.0(0) 74,82 25,51 0,00 1,37 17 30.0 (0) 6.0 (0) 20.0(0) 83,36 30,94 0,00 1,42 18 30.0 (0) 6.0 (0) 20.0(0) 84,17 30,05 0,00 1,10
MODELLING RESULTS OF THE FACTORIAL DESIGN Regarding the TPC, the significance of the model was adequate R 2 between the experimental and model predictions is 0.83 Important parameters are the square of the T emperature and the L/S ratio Υ TPC = β 0 + β 1 Χ 1 + β 2 Χ 2 + β 3 Χ 3 + β 4 Χ 1 Χ 2 + β 5 Χ 1 Χ 3 + β 6 Χ 2 Χ 3 + β 7 Χ 1 X1 + β 8 Χ 2 X2 + β 9 Χ 3 X3 + β 10 Χ 1 X2 Χ 3 Parameter Estimate t Ratio 83.556 β 0 0.9167 0.47 β 1 -0.09762 -0.05 β 2 6.0427 3.09 β 3 2.3175 0.91 β 4 4.495 1.76 β 5 2.2175 0.87 β 6 -7.021455 -3.45 β 7 -3.066961 -1.51 β 8 -4.433445 -2.18 β 9 -5.5625 -2.17 β 10 Experimental and modelling values for the TPC (mg/g)
MODELLING RESULTS OF THE FACTORIAL DESIGN (CONT’) Regarding the OLEUROPEIN, the significance of the model was also adequate R 2 between the experimental and model predictions is 0.89 Important parameters are the amount of the sulphuric acid and the L/S ratio Υ Oleuropein = β 0 + β 1 Χ 1 + β 2 Χ 2 + β 3 Χ 3 + β 4 Χ 1 Χ 2 + β 5 Χ 1 Χ 3 + β 6 Χ 2 Χ 3 + β 7 Χ 1 X1 + β 8 Χ 2 X2 + β 9 Χ 3 X3 + β 10 Χ 1 X2 Χ 3 Parameter Estimate t Ratio β 0 27.5662 β 1 ‐ 1.524585 ‐ 1.08 β 2 ‐ 7.90278 ‐ 5.6 β 3 6.3128762 4.48 β 4 0.305 0.17 β 5 1.52 0.82 β 6 0.495 0.27 β 7 0.8294436 0.57 β 8 ‐ 0.706746 ‐ 0.48 β 9 ‐ 1.221166 ‐ 0.83 β 10 ‐ 1.96 ‐ 1.06 Experimental and modelling values for the OLEUROPEIN (mg/g)
OPTIMUM EXTRACTION CONDITIONS AND VALIDATION ON THE OPTIMUM VALUE Optimum conditions For X1 was 0.305 (31.8 o C) For X2 was 0.168 (6.40 v/w) For X3 was 0.840 (25.0 v/w) Y TPC = 86.43 mg/g HYDROXYTYROSOL TYROSOL OLEUROPEIN TPC mg/g db mg/g db mg/g db mg/g db 5.69 1.65 7.53 86.4
Optimization per T otal Phenolic Content 1. Theoretical optimum TPC 86.43 mg/g db. Experimental result 86.39 mg/g db Factor Description Level Value Water and Leaves pH 4.27 pH 1.12 After the acidification 2. X1 T 0 C 0,30456 31,81 3. Total Phenolic Content is enhanced by the presence of sulfuric acid. X2 H 2 SO 4 % (v/w) 0,16772 6,40 4. Concentration of Tyrosol is not affected by pH X3 L/S 0,84015 24,99 5. Concentration of Oleuropein in positively affected by H2SO4 6. Hydroxytyrosol is strongly dependent on the pH Tyrosol Total Phenolic Content 2,00 100 80 1,50 60 Blank (mg/g d.b.) 1,00 40 Blank (mg/g d.b.) H2SO4 (mg/g d.b.) 0,50 20 H2SO4 (mg/g d.b.) 0 0,00 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Hydroxytyrosol Oleuropein 6,00 10,00 5,00 4,00 5,00 3,00 2,00 1,00 0,00 0,00 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Blank (mg/g d.b.) H2SO4 (mg/g d.b.) Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
Optimization per Oleuropein 1. Concentration and time of extraction for Oleuropein in positively Factor Description Level Value affected by H2SO4 T 0 C X1 1,6818 40 2. Tyrosol highest concentration raised at 95min. H 2 SO 4 % (v/w) -1,6818 2.0 X2 Water and Leaves pH 4,01 pH 1,90 After the acidification 3. X3 L/S 0,84015 30 4. There was no 3-hydroxytyrosol produced in this pH Oleuropein Tyrosol 10,00 1,85 1,80 8,00 1,75 6,00 1,70 1,65 4,00 1,60 2,00 1,55 1,50 0,00 1,45 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Blank (mg/g d.b.) H2SO4 (mg/g d.b.) Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
CONCLUSIONS We illustrated an extraction process of the phenolic compounds from olive leaves using acid hydrolysis The Factorial Experiment gave us an overall extraction of Hydroxytyrosol 8.33 mg/g of dry OMW and a TPC of 85.33 mg/g of dry base Water can be used as a solvent with satisfying results. The extraction of the polyphenols is increased in low pH. Concentration of Oleuropein, Hydroxytyrosol & Tyrosol is increased under the presence of sulfuric acid, under certain circumstances.
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