WO2021240041A1 - Method for producing 3,4-dihydroxyphenylglycol (dhpg) - Google Patents
Method for producing 3,4-dihydroxyphenylglycol (dhpg) Download PDFInfo
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- WO2021240041A1 WO2021240041A1 PCT/ES2021/070384 ES2021070384W WO2021240041A1 WO 2021240041 A1 WO2021240041 A1 WO 2021240041A1 ES 2021070384 W ES2021070384 W ES 2021070384W WO 2021240041 A1 WO2021240041 A1 WO 2021240041A1
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- dhfg
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- ethanol
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- MTVWFVDWRVYDOR-UHFFFAOYSA-N 3,4-Dihydroxyphenylglycol Chemical compound OCC(O)C1=CC=C(O)C(O)=C1 MTVWFVDWRVYDOR-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title 1
- JUUBCHWRXWPFFH-UHFFFAOYSA-N Hydroxytyrosol Chemical compound OCCC1=CC=C(O)C(O)=C1 JUUBCHWRXWPFFH-UHFFFAOYSA-N 0.000 claims abstract description 109
- 235000003248 hydroxytyrosol Nutrition 0.000 claims abstract description 56
- 229940095066 hydroxytyrosol Drugs 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 51
- 240000007817 Olea europaea Species 0.000 claims abstract description 17
- 235000002725 Olea europaea Nutrition 0.000 claims abstract description 16
- 239000000047 product Substances 0.000 claims abstract description 11
- 241000196324 Embryophyta Species 0.000 claims abstract description 10
- 241000013557 Plantaginaceae Species 0.000 claims abstract description 8
- 239000006227 byproduct Substances 0.000 claims abstract description 6
- 241000207965 Acanthaceae Species 0.000 claims abstract description 4
- 241000208838 Asteraceae Species 0.000 claims abstract description 4
- 241000207923 Lamiaceae Species 0.000 claims abstract description 4
- 241000207834 Oleaceae Species 0.000 claims abstract description 4
- 241000308150 Orobanchaceae Species 0.000 claims abstract description 4
- RFWGABANNQMHMZ-UHFFFAOYSA-N 8-acetoxy-7-acetyl-6,7,7a,8-tetrahydro-5H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]quinoline Natural products CC=C1C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O RFWGABANNQMHMZ-UHFFFAOYSA-N 0.000 claims description 35
- HKVGJQVJNQRJPO-UHFFFAOYSA-N Demethyloleuropein Natural products O1C=C(C(O)=O)C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(=CC)C1OC1OC(CO)C(O)C(O)C1O HKVGJQVJNQRJPO-UHFFFAOYSA-N 0.000 claims description 35
- RFWGABANNQMHMZ-HYYSZPHDSA-N Oleuropein Chemical compound O([C@@H]1OC=C([C@H](C1=CC)CC(=O)OCCC=1C=C(O)C(O)=CC=1)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RFWGABANNQMHMZ-HYYSZPHDSA-N 0.000 claims description 35
- 235000011576 oleuropein Nutrition 0.000 claims description 35
- RFWGABANNQMHMZ-CARRXEGNSA-N oleuropein Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)C(=CC)[C@H]1CC(=O)OCCc3ccc(O)c(O)c3 RFWGABANNQMHMZ-CARRXEGNSA-N 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 239000011347 resin Substances 0.000 claims description 21
- 229920005989 resin Polymers 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- 125000000129 anionic group Chemical group 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 12
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- 230000000717 retained effect Effects 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 125000002091 cationic group Chemical group 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000007073 chemical hydrolysis Effects 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- 229930182478 glucoside Natural products 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000001728 nano-filtration Methods 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- FIKLMMHLPVXWJN-WRWORJQWSA-N Elenolic acid Chemical compound COC(=O)C1=CO[C@@H](O)C(=CC)[C@@H]1CC(O)=O FIKLMMHLPVXWJN-WRWORJQWSA-N 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims description 2
- 238000001223 reverse osmosis Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000007669 thermal treatment Methods 0.000 claims description 2
- 150000008131 glucosides Chemical class 0.000 claims 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 7
- 239000004006 olive oil Substances 0.000 abstract description 4
- 235000008390 olive oil Nutrition 0.000 abstract description 4
- HOOWCUZPEFNHDT-UHFFFAOYSA-N DHPG Natural products OC(=O)C(N)C1=CC(O)=CC(O)=C1 HOOWCUZPEFNHDT-UHFFFAOYSA-N 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 150000002989 phenols Chemical class 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- -1 ¡NOS Proteins 0.000 description 4
- 230000009418 agronomic effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000207836 Olea <angiosperm> Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- JAEITFQIDOKJCK-IBGZPJMESA-N CCCCCCCCCCCC(=O)OC(=O)[C@@H](NO)CC1=CC=C(O)C=C1 Chemical compound CCCCCCCCCCCC(=O)OC(=O)[C@@H](NO)CC1=CC=C(O)C=C1 JAEITFQIDOKJCK-IBGZPJMESA-N 0.000 description 1
- 101150071146 COX2 gene Proteins 0.000 description 1
- 101100114534 Caenorhabditis elegans ctc-2 gene Proteins 0.000 description 1
- 241000186427 Cutibacterium acnes Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 244000307700 Fragaria vesca Species 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 101150000187 PTGS2 gene Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 239000002530 phenolic antioxidant Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 229940055019 propionibacterium acne Drugs 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000013520 translational research Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
- C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
- C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
- C07C39/11—Alkylated hydroxy benzenes containing also acyclically bound hydroxy groups, e.g. saligenol
Definitions
- the invention refers to the procedure defined above where the purification (T2) of step (iii) is carried out by chromatography with cationic resins, anionic resins or mixtures thereof to obtain DHFG-P with a degree of purity of up to at least 80% by dry weight of the DHFG-E obtained in the chromatography of step (ii), retaining it on the column.
- the invention refers to the process defined above that also comprises a stage (v) of chemical or enzymatic hydrolysis (T3) of the Oleuro-E extract obtained in stage (iv) or to obtain an extract rich in HT (FIT -AND).
- T3 chemical or enzymatic hydrolysis
- FIT -AND extract rich in HT
- Stage 2 Corresponds to stage (ii) of the process of the invention, where the liquid fraction A1 is passed through an ion exchange column (C1) that does not retain most of the DFIFG, so that the liquid fraction itself fraction A1 forms the first final product, the extract of free or low-HT DFIFG (DFIFG-E).
- C1 ion exchange column
- DFIFG-E extract of free or low-HT DFIFG
- a weak anionic type resin partially activated with an acid solution at pH between 5 and 8 can be used.
- subsequent elutions with water or organic solvents such as ethanol allow the obtaining of a second extract rich in oleuropein (Oleuropein).
- E) (optional step (iv) of the process of the invention).
- Stage 3 Corresponds to optional stage (iii) of the invention, where the DHFG-E fraction can be subjected to filtration through nano and / or ultrafiltration membranes and / or to a chromatographic step with ion exchange resin (T2) or adsorption to finally obtain a product with a higher degree of purity than DHFG-P.
- T2 ion exchange resin
- T3 an extract rich in HT (HT-E) (T3) can be obtained by means of optional chemical hydrolysis stage (v) or enzymatic.
- the liquid is passed through a column of weak anionic type ionic resin (C1), obtaining an unretained fraction (1 L) with a concentration of 0.21 g / L of DFIFG (with a purity of between 5-20% referred to dry weight), traces of HT ( ⁇ 0.01 g / L) and traces of its precursors ( ⁇ 0.05 g / L).
- This fraction can already be used as a rich fraction of DFIFG (DFIFG-E) avoiding the antagonistic effect of HT, and therefore enabling its agronomic use among others.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention relates to a method for extracting the phenol 3,4-dihydroxyphenylglycol (DHPG) present in any part of the plant, products or by-products derived from olive tree or any other plant product of the family Oleaceae, Orobanchaceae, Plantaginaceae, Compositae, Lamiaceae, Acanthaceae and/or Scrophulariaceae, in particular in olive leaf, and in olive oil (OA) with low release of the phenol hydroxytyrosol (HT).
Description
DESCRIPCIÓN DESCRIPTION
Procedimiento de obtención de 3,4-dihidroxifenilqMcol (DHFG) Procedure for obtaining 3,4-dihydroxyphenylqMcol (DHFG)
La presente invención se refiere a un procedimiento de extracción del fenol 3,4- dihidroxifenilglicol (DHFG) a partir de cualquier parte de la planta, productos o subproductos derivados del olivo o cualquier otro producto vegetal de la familia Oleaceae, Orobanchaceae, Plantaginaceae, Compositae, Lamiaceae, Acanthaceae y/o Scrophulariaceae, particularmente en la hoja de olivo, y en el aceite de oliva (OA) con una baja liberación del fenol hidroxitirosol (HT). The present invention refers to a process for the extraction of phenol 3,4-dihydroxyphenylglycol (DHFG) from any part of the plant, products or by-products derived from the olive tree or any other plant product of the Oleaceae, Orobanchaceae, Plantaginaceae, Compositae family. , Lamiaceae, Acanthaceae and / or Scrophulariaceae, particularly in the olive leaf, and in olive oil (OA) with a low release of phenol hydroxytyrosol (HT).
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Uno de los fenoles más estudiados es el hidroxitirosol (HT), que es un fenol simple cuya propiedad más destacada y estudiada es la de su carácter antioxidante en distintos tipos de matrices (Rodríguez., G., Rodríguez., R., Fernández-Bolaños., J., Guillén., R., Jiménez, A. (2007). Antioxidant activity of effluents during the purification of hydroxytyrosol and 3,4-dihydroxyphenyl glycol from olive oil waste. European Food Research and Technology.V 224, Issue 6, pp 733-741 ; Bermudez-Oria, A., Rodríguez- Gutiérrez, G., Rubio-Senet, F., Fernández-Prior, A., Fernández-Bolaños, J, (2018). Effect of edible pectin-fish gelatin films containing the olive antioxidants hydroxytyrosol and 3,4-dihydroxyphenylglycol on beef meat during refrigerated storage. Meat Science. V 148, pp 213-218; y Bermúdez-Oria, A., Rodríguez-Gutiérrez, G., Fernández-Prior, M.A., Vioque, B., Fernández-Bolaños, J. (2019). Strawberry dietary fibre functionalized with phenolic antioxidants from olives. Interactions between polysaccharides and phenolic compounds. Food Chemistry), sin embargo, hay que resaltar muchas otras como potente antimicrobiano de amplio espectro en distintos tipos de cepas bacterianas (Natasa Zoric, Igor Horvat, Nevenka Kopjar, Ante Vucemilovic, Darío Kremer, Sinisa Tomic, Ivan Kosalec., (2013). Hydroxytyrosol Expresses Antifungal Activity in vitro. Current Drug Targets.V 14, Issue 9; y Eilami O., Manuela Oliverio., Saeid Hosseinian., Amin Hossaini Motlagh., Mohsen Naghmachi., (2017). Antimicrobial Effects of Hydroxytyrosol Extracted From Olive Leaves, on Propionibacterium Acnés. Middle East Journal of family Medicine. V 15, Issue 10), antiapoptótico contra células cancerígenas (Burattini, S., Baldassarri, S., Accorsi, A., Piatti, E., Madrona, A., Espartero, J.L., Candiracci, M., Zappia, C., Falcieria, E, (2013). Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate. Food and Chemical Toxicology.V 55, pp 248-256), presenta
alta toxicidad frente al virus VIH (Bedoya et al, 2016) además de poseer efecto antiinflamtorio (Zhang, X., Cao, J., Zhong, L, (2009). Hydroxytyrosol inhibits pro- inflammatory cytokines, ¡NOS, and COX-2 expression in human monocytic cells. Naunyn-Schmiedeberg's Archives of Pharmacology. V379. Issue 6, pp 581-586). One of the most studied phenols is hydroxytyrosol (HT), which is a simple phenol whose most outstanding and studied property is its antioxidant character in different types of matrices (Rodríguez., G., Rodríguez., R., Fernández- Bolaños., J., Guillén., R., Jiménez, A. (2007). Antioxidant activity of effluents during the purification of hydroxytyrosol and 3,4-dihydroxyphenyl glycol from olive oil waste. European Food Research and Technology.V 224, Issue 6, pp 733-741; Bermudez-Oria, A., Rodríguez- Gutiérrez, G., Rubio-Senet, F., Fernández-Prior, A., Fernández-Bolaños, J, (2018). Effect of edible pectin -fish gelatin films containing the olive antioxidants hydroxytyrosol and 3,4-dihydroxyphenylglycol on beef meat during refrigerated storage. Meat Science. V 148, pp 213-218; and Bermúdez-Oria, A., Rodríguez-Gutiérrez, G., Fernández- Prior, MA, Vioque, B., Fernández-Bolaños, J. (2019). Strawberry dietary fiber functionalized with phenolic antioxidants from olives. Interactions between pol ysaccharides and phenolic compounds. Food Chemistry), however, many others should be highlighted as a powerful broad-spectrum antimicrobial in different types of bacterial strains (Natasa Zoric, Igor Horvat, Nevenka Kopjar, Ante Vucemilovic, Darío Kremer, Sinisa Tomic, Ivan Kosalec., (2013) . Hydroxytyrosol Expresses Antifungal Activity in vitro. Current Drug Targets.V 14, Issue 9; and Eilami O., Manuela Oliverio., Saeid Hosseinian., Amin Hossaini Motlagh., Mohsen Naghmachi., (2017). Antimicrobial Effects of Hydroxytyrosol Extracted From Olive Leaves, on Propionibacterium Acnes. Middle East Journal of family Medicine. V 15, Issue 10), antiapoptotic against cancer cells (Burattini, S., Baldassarri, S., Accorsi, A., Piatti, E., Madrona, A. , Espartero, JL, Candiracci, M., Zappia, C., Falcieria, E, (2013). Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate. Food and Chemical Toxicology.V 55, pp 248-256), presents high toxicity against the HIV virus (Bedoya et al, 2016) in addition to having an anti-inflammatory effect (Zhang, X., Cao, J., Zhong, L, (2009). Hydroxytyrosol inhibits pro inflammatory cytokines, ¡NOS, and COX- 2 expression in human monocytic cells. Naunyn-Schmiedeberg's Archives of Pharmacology. V379. Issue 6, pp 581-586).
El fenol 3,4-dihidroxifenilglicol (DHFG) está presente junto con el fenol hidroxitirosol (HT) en el aceite de oliva y las aceitunas de mesas, además, el HT está presente en mayor cantidad que el DHFG. Phenol 3,4-dihydroxyphenylglycol (DHFG) is present together with phenol hydroxytyrosol (HT) in olive oil and table olives, in addition, HT is present in greater amounts than DHFG.
El DHFG se ha descrito como fitorregulador natural (ver Denfeld, Quin E., Habecker, Beth A., Woodward, William R, (2019). Measurement of plasma norepinephrine and 3,4- dihydroxyphenylglycol: Method development for a translational research study. BMC Research Notes), pero este uso lo presenta cuando la cantidad de HT que le acompaña es muy baja o nula. DHFG has been described as a natural phytoregulator (see Denfeld, Quin E., Habecker, Beth A., Woodward, William R, (2019). Measurement of plasma norepinephrine and 3,4-dihydroxyphenylglycol: Method development for a translational research study. BMC Research Notes), but this use is presented when the amount of HT that accompanies it is very low or null.
La dificultad de separar ambos fenoles conlleva a sistemas que encarecen su purificación, no permitiendo su desarrollo a nivel agronómico y dificultando su desarrollo a otros niveles como la alimentación o la cosmética, entre otros. The difficulty of separating both phenols leads to systems that make their purification more expensive, not allowing their development at the agronomic level and hindering their development at other levels such as food or cosmetics, among others.
En el estado de la técnica se ha descrito un procedimiento de purificación de DHFG a través de su retención y desorción en resinas iónicas (ver ES2341526B1 ). En dicho proceso se utilizan combinaciones de resinas de intercambio iónico para poder separar el DHFG del HT, y en todos los casos se absorbe y se eluye el DHFG para su purificación. Al absorberse completamente el DHFG dificulta su separación del HT, ya que al ser químicamente muy parecidos se eluyen prácticamente al mismo tiempo. Por lo tanto, supone por un lado el empleo de más de un tipo de resina y columna y por otro una gran pérdida de DHFG y una alta degradación. Todo ello se traduce en elevados costes para la purificación del DHFG y la obtención de extractos en donde el HT está también presente en altas concentraciones. In the state of the art, a procedure for the purification of DHFG has been described through its retention and desorption in ionic resins (see ES2341526B1). In this process, combinations of ion exchange resins are used to separate DHFG from HT, and in all cases DHFG is absorbed and eluted for purification. As DHFG is completely absorbed, it makes it difficult to separate it from HT, since being chemically very similar, they elute practically at the same time. Therefore, it involves, on the one hand, the use of more than one type of resin and column and, on the other, a great loss of DHFG and high degradation. All this translates into high costs for the purification of DHFG and the obtaining of extracts where HT is also present in high concentrations.
Así pues, los procedimientos de extracción de DHFG descritos en el estado de la técnica tienen la desventaja de que la concentración de HT presente en el extracto es muy alta o el DHFG se degrada en cierta proporción. Thus, the DHFG extraction procedures described in the state of the art have the disadvantage that the HT concentration present in the extract is very high or the DHFG degrades to a certain extent.
Por tanto, sería deseable disponer de un procedimiento que permitiese extraer DHFG libre de HT y que, en consecuencia, sea más económico que los descritos hasta la fecha
y permita que el DHFG de alta pureza así obtenido se pueda utilizar en el campo de la agronómica. Therefore, it would be desirable to have a procedure that allows the extraction of HT-free DHFG and that, consequently, is cheaper than those described to date. and allow the high purity DHFG thus obtained to be used in the field of agronomics.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
En un primer aspecto, la presente invención se refiere a un procedimiento de obtención de 3,4-dihidroxifenilglicol (DHFG) que comprende las etapas de: i) tratamiento térmico (T1) a una temperatura de entre 25 eC y 140 eC de la fuente de DHFG (F0) disuelta en agua o en una disolución de etanol en agua con una concentración de etanol en agua de entre 10% y 50% (v/v) , durante un tiempo de entre 20 minutos y 5 h, con una rampa de temperatura inferior a 10 minutos para liberar una fracción líquida (A1) que contiene: entre 0,01 g/L y 2,5 g/L de DHFG (DHFG-E), entre 0,001 g/L y 0,25 g/L de HT, y entre 0,03 y 15 g/L de formas conjugadas de HT, usando relaciones de la fuente de DHFG (F0) y el agua o en una disolución de etanol en agua de entre 5:1 (kg:L) y 1 :20 (kg:L); y ii) cromatografía de la fracción líquida (A1 ) obtenida tras el tratamiento térmico de la etapa (i) utilizando una resina aniónica (C1) a un pH de entre 5 y 8 para retener las formas conjugadas de HT y más del 70% del HT libre y para obtener una fracción líquida no retenida en la columna cromatográfica de DHFG (DHFG-E) con una concentración de entre 0,005 g/L y 2,5 g/L de DHFG de concentración usando relaciones de la fuente de DHFG (F0) y el agua o en una disolución de etanol en agua de entre 5:1 (kg:L) y 1 :20 (kg:L).. In a first aspect, the present invention relates to a process for obtaining 3,4-dihydroxyphenylglycol (DHFG) comprising the steps of: i) heat treatment (T1) at a temperature of between 25 and 140 C and C the DHFG source (F0) dissolved in water or in a solution of ethanol in water with a concentration of ethanol in water between 10% and 50% (v / v), for a time between 20 minutes and 5 h, with a temperature ramp of less than 10 minutes to release a liquid fraction (A1) containing: between 0.01 g / L and 2.5 g / L of DHFG (DHFG-E), between 0.001 g / L and 0.25 g / L of HT, and between 0.03 and 15 g / L of conjugated forms of HT, using ratios of the source of DHFG (F0) and water or in a solution of ethanol in water of between 5: 1 (kg : L) and 1:20 (kg: L); and ii) chromatography of the liquid fraction (A1) obtained after the thermal treatment of step (i) using an anionic resin (C1) at a pH between 5 and 8 to retain the conjugated forms of HT and more than 70% of the Free HT and to obtain a liquid fraction not retained on the DHFG column chromatography (DHFG-E) with a concentration of between 0.005 g / L and 2.5 g / L of DHFG concentration using DHFG source ratios (F0 ) and water or in a solution of ethanol in water between 5: 1 (kg: L) and 1:20 (kg: L).
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde el tratamiento térmico (T1) de la etapa (i) se lleva a cabo entre 90 eC y 140 eC, preferiblemente entre 90 eC y 130 eC, y más preferiblemente a 120 eC. In another embodiment the invention relates to the process described above, wherein the heat treatment (T1) of step (i) is carried out between 90 and C 140 and C, preferably between 90 and C 130 and C, and preferably at 120 e C.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde el tratamiento térmico (T1) de la etapa (i) se lleva a cabo a una temperatura de entre 90 eC y 140 eC, preferiblemente entre 90 eC y 130 eC, y más preferiblemente a 120 eC, durante un tiempo de entre 10 minutos y 1 ,5 h con una rampa de temperatura inferior a 5 minutos. In another embodiment the invention relates to the process described above, wherein the heat treatment (T1) of step (i) is carried out at a temperature of between 90 and C 140 and C, preferably between 90 and C 130 and C, and more preferably to 120 and C, for a time of between 10 minutes and 1, 5 h with a ramp temperature below 5 minutes.
Es importante que la fuente de DHFG (F0) sea rica en este compuesto y, también, que tenga una concentración de HT libre por debajo del 10% de la concentración de DHFG.It is important that the source of DHFG (F0) is rich in this compound and, also, that it has a free HT concentration below 10% of the DHFG concentration.
Así, una de las fuentes preferidas de la invención es la hoja de olivo.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la fuente de DHFG (F0) es cualquier parte de la planta, productos o subproductos derivados del olivo o cualquier otro producto vegetal de la familia Oleaceae, Orobanchaceae, Plantaginaceae, Compositae, Lamiaceae, Acanthaceae y/o Scrophulariaceae, preferiblemente cualquier planta, productos o subproductos derivados del olivo, y más preferiblemente la hoja de olivo. Thus, one of the preferred sources of the invention is the olive leaf. In another embodiment, the invention refers to the process described above, where the source of DHFG (F0) is any part of the plant, products or by-products derived from the olive tree or any other plant product of the Oleaceae, Orobanchaceae, Plantaginaceae, Compositae, Lamiaceae family. , Acanthaceae and / or Scrophulariaceae, preferably any plants, products or by-products derived from the olive tree, and more preferably the olive leaf.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde las formas conjugadas de HT son oleuropeína, glucósido del HT y sus derivados secoiridoides, y preferiblemente las formas conjugadas de HT son oleuropeína. In another embodiment the invention relates to the process described above, where the conjugated forms of HT are oleuropein, HT glucoside and their secoiridoid derivatives, and preferably the conjugated forms of HT are oleuropein.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la fracción líquida A1 obtenida en la etapa (i) se somete a una etapa de centrifugación, filtración y/o sedimentación antes de la etapa (ii). In another embodiment, the invention refers to the process described above, where the liquid fraction A1 obtained in stage (i) is subjected to a centrifugation, filtration and / or sedimentation stage before stage (ii).
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la fuente de DFIFG (F0) está disuelta en agua. En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la fuente de DFIFG (F0) es hoja de olivo y está disuelta en agua. In another embodiment the invention refers to the process described above, where the DFIFG source (F0) is dissolved in water. In another embodiment the invention refers to the process described above, where the source of DFIFG (F0) is olive leaf and is dissolved in water.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la resina aniónica (C1 ) de la etapa (ii) es una resina aniónica débil. In another embodiment, the invention refers to the process described above, where the anionic resin (C1) of step (ii) is a weak anionic resin.
En otra realización la invención se refiere al procedimiento descrito anteriormente, donde la etapa (ii) se lleva a cabo a un pH de 6,5. In another embodiment the invention refers to the process described above, where step (ii) is carried out at a pH of 6.5.
Así pues, el procedimiento de obtención de DFIFG sin hidroxitirosol (HT) de la presente invención se basa en obtener una fuente sin o con muy baja concentración de HT libre. Es por ello por lo que los ensayos se han realizado con hoja de olivo en donde hay una importante concentración de DFIFG y el HT está conjugado formando la oleuropeína. Thus, the process for obtaining DFIFG without hydroxytyrosol (HT) of the present invention is based on obtaining a source without or with a very low concentration of free HT. That is why the tests have been carried out with olive leaf where there is a significant concentration of DFIFG and the HT is conjugated to form oleuropein.
En la primera etapa del procedimiento (etapa (i)) se aplica un tratamiento que solubilice el DFIFG de la fuente, particularmente de la hoja de olivo, sin que el HT se llegue a liberar, permaneciendo en forma conjugada.
Tratamientos térmicos por encima de 140 eC para todos los tiempos ensayados producen una liberación y cierta degradación del DHFG seguida de una importante hidrólisis de las formas conjugadas de HT, y en particular de la oleuropeína. Tratamientos en donde la muestra haya sufrido una rampa de calentamiento superior a 10 minutos han supuesto una importante hidrólisis de las formas conjugadas de HT, en particular de la oleuropeína, a pesar de una buena solubilización del DHFG, por lo que se ha generado mucho HT libre, lo cual es no deseable en este proceso. La primera etapa de tratamiento térmico del procedimiento de extracción de DHFG permite obtener una fuente líquida rica en DHFG que, a continuación, habrá que se separar de las formas conjugadas de HT, fundamentalmente la oleuropeína, el glucósido del HT y sus derivados secoiridoides. La segunda etapa del procedimiento es la aplicación de un sistema cromatográfico a la fuente líquida rica en DHFG obtenida en la etapa anterior de tratamiento, en donde no se retiene el DHFG y sí se retienen las formas conjugadas de HT tales como la oleuropeína y los demás precursores del HT. Dicha cromatografía se lleva a cabo con resinas de tipo iónico. In the first stage of the procedure (stage (i)) a treatment is applied that solubilizes the DFIFG from the source, particularly from the olive leaf, without the HT being released, remaining in conjugated form. Heat treatments above 140 and C for all times tested and produce a release of the DHFG some degradation followed by extensive hydrolysis of conjugated forms HT, particularly oleuropein. Treatments in which the sample has undergone a heating ramp greater than 10 minutes have involved significant hydrolysis of the conjugated forms of HT, particularly oleuropein, despite a good solubilization of DHFG, which is why a lot of HT has been generated. free, which is undesirable in this process. The first stage of heat treatment of the DHFG extraction procedure makes it possible to obtain a liquid source rich in DHFG that will then have to be separated from the conjugated forms of HT, mainly oleuropein, HT glucoside and their secoiridoid derivatives. The second stage of the procedure is the application of a chromatographic system to the liquid source rich in DHFG obtained in the previous stage of treatment, where DHFG is not retained and the conjugated forms of HT such as oleuropein and others are retained. precursors of HT. Said chromatography is carried out with ionic type resins.
En otra realización la invención se refiere al procedimiento definido anteriormente, que además comprende una etapa (iii) de purificación (T2) de la fracción DHFG-E obtenida en la etapa (ii) para obtener DHFG con un grado de pureza mayor (DHFG-P) tras su retención en la columna (C1 ). In another embodiment, the invention refers to the process defined above, which also comprises a step (iii) of purification (T2) of the DHFG-E fraction obtained in step (ii) to obtain DHFG with a higher degree of purity (DHFG- P) after retention in column (C1).
En otra realización, la invención se refiere al procedimiento definido anteriormente donde la purificación (T2) de la etapa (iii) se lleva a cabo por cromatografía con resinas catiónicas, resinas aniónicas o mezclas de las mismas para obtener DHFG-P con un grado de pureza de hasta al menos un 80% en peso seco del DHFG-E obtenido en la cromatografía de la etapa (ii) reteniéndolo en la columna. In another embodiment, the invention refers to the procedure defined above where the purification (T2) of step (iii) is carried out by chromatography with cationic resins, anionic resins or mixtures thereof to obtain DHFG-P with a degree of purity of up to at least 80% by dry weight of the DHFG-E obtained in the chromatography of step (ii), retaining it on the column.
En otra realización, la invención se refiere al procedimiento definido anteriormente donde la purificación (T2) de la etapa (iii) se lleva a cabo por cromatografía con resinas de adsorción y elución con agua y/o etanol para obtener DHFG-P con un grado de pureza de hasta un 99,6% en peso seco del DHFG-E obtenido en la cromatografía de la etapa (ii) reteniéndolo en la columna.
En otra realización, la invención se refiere al procedimiento definido anteriormente donde la purificación (T2) de la etapa (iii) se lleva a cabo por filtración con membranas de filtración, nanofiltración o de ultrafiltración para aumentar el grado de pureza de entre un 20% y un 60% en peso seco del DHFG obtenido en la cromatografía de la etapa (ii). In another embodiment, the invention refers to the procedure defined above where the purification (T2) of step (iii) is carried out by chromatography with adsorption resins and elution with water and / or ethanol to obtain DHFG-P with a degree purity of up to 99.6% by dry weight of the DHFG-E obtained in the chromatography of step (ii), retaining it on the column. In another embodiment, the invention refers to the procedure defined above where the purification (T2) of step (iii) is carried out by filtration with filtration, nanofiltration or ultrafiltration membranes to increase the degree of purity between 20% and 60% by dry weight of the DHFG obtained in the chromatography of step (ii).
En otra realización, la invención se refiere al procedimiento definido anteriormente donde la purificación (T2) de la etapa (iii) se lleva a cabo por osmosis inversa para eliminar parte del contenido en agua sin variar el grado de pureza de DHFG-P. In another embodiment, the invention refers to the process defined above where the purification (T2) of step (iii) is carried out by reverse osmosis to remove part of the water content without varying the degree of purity of DHFG-P.
En otra realización la invención se refiere al procedimiento definido anteriormente, que además comprende una etapa (iv) de elución con agua, etanol o sus mezclas de los conjugados de HT retenidos en la columna C1 de la etapa (ii), preferiblemente de oleuropeína, para obtener un extracto rico en oleuropeína (Oleuro-E), las moléculas que componen el Oleuro-E y/o otros derivados secoiridoides, preferiblemente para obtener un extracto rico en oleuropeína (Oleuro-E), hidroxitirosol (HT-E), ácido elenólico y/o sus derivados, más preferiblemente para obtener un extracto rico en oleuropeína (Oleuro- E), hidroxitirosol (FIT-E) y/o ácido elenólico, y aún más preferiblemente para obtener un extracto rico en oleuropeína (Oleuro-E). In another embodiment, the invention refers to the process defined above, which further comprises a step (iv) of elution with water, ethanol or their mixtures of the HT conjugates retained in column C1 of step (ii), preferably oleuropein, to obtain an extract rich in oleuropein (Oleuro-E), the molecules that make up Oleuro-E and / or other secoiridoid derivatives, preferably to obtain an extract rich in oleuropein (Oleuro-E), hydroxytyrosol (HT-E), acid elenolic and / or its derivatives, more preferably to obtain an extract rich in oleuropein (Oleuro-E), hydroxytyrosol (FIT-E) and / or elenolic acid, and even more preferably to obtain an extract rich in oleuropein (Oleuro-E) .
En otra realización la invención se refiere al procedimiento definido anteriormente que además comprende una etapa (v) de hidrólisis química o enzimática (T3) del extracto de Oleuro-E obtenido en la etapa (iv) o para obtener un extracto rico en HT (FIT-E). Así pues, la presente invención permite disponer de un tratamiento efectivo de extracción de DFIFG en el que no se libere el HT y en el que no se degrade el DGFFI. In another embodiment, the invention refers to the process defined above that also comprises a stage (v) of chemical or enzymatic hydrolysis (T3) of the Oleuro-E extract obtained in stage (iv) or to obtain an extract rich in HT (FIT -AND). Thus, the present invention makes it possible to have an effective DFIFG extraction treatment in which HT is not released and in which DGFFI is not degraded.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
BREVE DESCRIPCIÓN DE LAS FIGURAS
Fig. 1 muestra el esquema básico del procedimiento: F0 es la fuente de DHFG, T1 es el tratamiento térmico, A1 es la fracción líquida, C1 es la columna de intercambio iónico, T2 resina iónica o mezcla, resina de absorción o filtración por membrana y T3 es una hidrólisis ácida o enzimática. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the basic scheme of the procedure: F0 is the source of DHFG, T1 is the heat treatment, A1 is the liquid fraction, C1 is the ion exchange column, T2 ionic resin or mixture, absorption resin or membrane filtration and T3 is acidic or enzymatic hydrolysis.
EJEMPLOS EXAMPLES
A continuación, se ilustrará la invención mediante unos ensayos realizados por los inventores que pone de manifiesto la efectividad del procedimiento de la invención. The invention will be illustrated below by means of tests carried out by the inventors that demonstrate the effectiveness of the process of the invention.
En la figura 1 se muestra el esquema básico del procedimiento: Figure 1 shows the basic outline of the procedure:
- Etapa 1 : Corresponde a la etapa (i) de procedimiento de la invención. En esta etapa la fuente de DFIFG (F0), caracterizada por que posee poca concentración de HT libre, se somete a un tratamiento térmico (T1) en el que logre solubilizar al DFIFG evitando la hidrólisis de los precursores de HT, y con ello evitando la formación de HT libre. En el caso de tratarse de una fuente sólida o semisólida será necesaria la adición de agua (o una disolución de etanol en agua) dependiendo del grado de humedad de dicha fuente. Finalmente, el líquido (A1) se obtiene por centrifugación, filtración y/o sedimentación. - Stage 1: Corresponds to stage (i) of the process of the invention. In this stage the DFIFG source (F0), characterized by having a low concentration of free HT, is subjected to a heat treatment (T1) in which it manages to solubilize the DFIFG avoiding the hydrolysis of the HT precursors, and thus avoiding the formation of free HT. In the case of a solid or semi-solid source, the addition of water (or a solution of ethanol in water) will be necessary depending on the degree of humidity of said source. Finally, the liquid (A1) is obtained by centrifugation, filtration and / or sedimentation.
- Etapa 2: Corresponde a la etapa (ii) del procedimiento de la invención, donde la fracción líquida A1 se hace pasar a través de una columna de intercambio iónico (C1) que no retenga la mayor parte del DFIFG, de forma que la propia fracción A1 forme el primer producto final, el extracto de DFIFG libre o con bajo contenido en HT (DFIFG-E). Para ello se puede utilizar una resina de tipo aniónica débil activada parcialmente con una disolución ácida a pH entre 5 y 8. Opcionalmente, posteriores eluciones con agua o con disolventes orgánicos como el etanol permiten la obtención de un segundo extracto rico en oleuropeína (Oleuro-E) (etapa (iv) opcional del procedimiento de la invención). - Stage 2: Corresponds to stage (ii) of the process of the invention, where the liquid fraction A1 is passed through an ion exchange column (C1) that does not retain most of the DFIFG, so that the liquid fraction itself fraction A1 forms the first final product, the extract of free or low-HT DFIFG (DFIFG-E). For this, a weak anionic type resin partially activated with an acid solution at pH between 5 and 8 can be used. Optionally, subsequent elutions with water or organic solvents such as ethanol allow the obtaining of a second extract rich in oleuropein (Oleuropein). E) (optional step (iv) of the process of the invention).
- Etapa 3: Corresponde a la etapa (iii) opcional de la invención, donde la fracción DHFG- E puede ser sometida a filtración por membranas de nano y/o ultrafiltración y/o a un paso cromatográfico con resina de intercambio iónico (T2) o de adsorción para obtener finalmente un producto con un mayor grado de pureza de DHFG-P. Por otra parte, a partir del extracto Oleuro-E obtenido en la etapa (iv) opcional, se puede obtener un extracto rico en HT (HT-E) (T3) mediante la etapa (v) opcional de hidrólisis química o
enzimática. - Stage 3: Corresponds to optional stage (iii) of the invention, where the DHFG-E fraction can be subjected to filtration through nano and / or ultrafiltration membranes and / or to a chromatographic step with ion exchange resin (T2) or adsorption to finally obtain a product with a higher degree of purity than DHFG-P. On the other hand, from the Oleuro-E extract obtained in optional stage (iv), an extract rich in HT (HT-E) (T3) can be obtained by means of optional chemical hydrolysis stage (v) or enzymatic.
Ejemplo 1 : Experimento de laboratorio: El ejemplo consiste en el tratamiento térmico a 120 eC en un sistema de autoclave de la hoja de olivo en agua durante 30 minutos usando 200 g de la hoja parcialmente molida (F0) para aumentar la superficie de extracción. Se obtiene un litro de un líquido (A1 ) tras filtrar con una tela filtrante con una concentración de 0,25 g/L de DHFG, 0,02 g / de HT y de 0,7 g/L de oleuropeína entre otros fenoles. Example 1: Laboratory experiment: The example consists of the heat treatment at 120 e C in an autoclave system of the olive leaf in water for 30 minutes using 200 g of the partially ground leaf (F0) to increase the extraction surface . One liter of a liquid (A1) is obtained after filtering with a filter cloth with a concentration of 0.25 g / L of DHFG, 0.02 g / of HT and 0.7 g / L of oleuropein among other phenols.
El líquido se hace pasar por una columna de resina iónica tipo aniónica débil (C1), obteniéndose una fracción no retenida (1 L) con una concentración de 0,21 g/L de DFIFG (con una pureza de entre el 5-20 % referido a peso seco), trazas de HT (< 0,01 g/L) y trazas de sus precursores (< 0,05 g/L). Esta fracción puede ser ya usada como fracción rica de DFIFG (DFIFG-E) evitando el efecto antagónico del HT, y por tanto posibilitando su uso agronómico entre otros. The liquid is passed through a column of weak anionic type ionic resin (C1), obtaining an unretained fraction (1 L) with a concentration of 0.21 g / L of DFIFG (with a purity of between 5-20% referred to dry weight), traces of HT (<0.01 g / L) and traces of its precursors (<0.05 g / L). This fraction can already be used as a rich fraction of DFIFG (DFIFG-E) avoiding the antagonistic effect of HT, and therefore enabling its agronomic use among others.
Por otro lado, al continuar la elución de la columna con agua se obtiene un licor de 3 L con una concentración de oleuropeína de 0,15 g/L y una pureza en peso seco de entre 10-60% (Oleuro-E). On the other hand, by continuing the elution of the column with water, a 3 L liquor is obtained with an oleuropein concentration of 0.15 g / L and a dry weight purity of between 10-60% (Oleuro-E).
Ejemplo 2: Experimento en planta piloto. Example 2: Experiment in a pilot plant.
Se trataron 10 kg de hoja de olivo parcialmente trituradas (F0) en un reactor con calentamiento directo e indirecto con vapor de agua a 130 eC durante 30 minutos, evitando rampas de temperatura. Se obtuvieron 10 L de líquido (A1 ) una vez separados en una centrífuga de maya, con una concentración de 0,30 g/L de DFIFG, 0,07 g/L de HT y 0,5 g/L de oleuropeína. Los 10 L se pasaron a través de una columna con 30 L de resina aniónica débil (C1), obteniendo los 10 L pasados con una concentración de 0,25 g/L de DFIFG y por debajo de los 0,001 g/L de HT y trazas de oleuropeína (DHFG-E). 10 kg were treated olive leaf partially crushed (F0) in a reactor with direct and indirect heating with steam at 130 C for 30 minutes and avoiding temperature ramps. 10 L of liquid (A1) were obtained once separated in a maya centrifuge, with a concentration of 0.30 g / L of DFIFG, 0.07 g / L of HT and 0.5 g / L of oleuropein. The 10 L were passed through a column with 30 L of weak anionic resin (C1), obtaining the 10 L passed with a concentration of 0.25 g / L of DFIFG and below 0.001 g / L of HT and traces of oleuropein (DHFG-E).
A continuación, la columna C1 se eluyó con agua hasta obtener 50 L con una concentración de oleuropeína de 0,08 g/L y una pureza en peso de 10-30 %. Partiendo del extracto DHFG-E se usaron 20 L de una resina mixta catiónica fuerte con aniónica (T2) en donde se retuvo el 95 % del DHFG, que fue finalmente eluida con agua
obteniendo un producto final con una pureza por encima del 85 % de DHFG (DHFG-P). Subsequently, column C1 was eluted with water until obtaining 50 L with an oleuropein concentration of 0.08 g / L and a purity by weight of 10-30%. Starting from the DHFG-E extract, 20 L of a strong cationic mixed resin with anionic (T2) was used, where 95% of the DHFG was retained, which was finally eluted with water. obtaining a final product with a purity above 85% of DHFG (DHFG-P).
Partiendo del extracto de Oleuro-E se realizó una hidrólisis ácida con ácido clorhídrico 3N a 100 eC durante 15 minutos, resultado la hidrólisis total de la oleuropeína alcanzando los 30 mg/L de HT (HT-E).
From the extract Oleuro-E acid hydrolysis was performed with 3N hydrochloric acid and 100 C for 15 minutes, resulted complete hydrolysis of oleuropein reaching 30 mg / L of HT (HT-E).
Claims
1. Procedimiento de obtención de 3,4-dihidroxifenilglicol (DHFG) que comprende las etapas de: i) tratamiento térmico (T1 ) a un temperatura de entre 90 eC y 140 eC de la fuente de DHFG (F0) disuelta en agua o en una disolución de etanol en agua con una concentración de etanol en agua de entre 10% y 50% (v/v), durante un tiempo de entre 10 minutos y 1 ,5 h con una rampa de temperatura inferior a 5 minutos para liberar una fracción líquida (A1) que contiene: entre 0,01 g/L y 2,5 g/L de DHFG (DHFG-E), entre 0,001 g/L y 0,25 g/L de HT, y entre 0,03 y 15 g/L de formas conjugadas de HT, usando relaciones de la fuente de DHFG (F0) y el agua o en una disolución de etanol en aguade entre 5:1 (kg:L) y 1 :20 (kg:L); y ii) cromatografía de la fracción líquida (A1) obtenida tras el tratamiento térmico de la etapa (i) utilizando una resina aniónica (C1) a un pH de entre 5 y 8 para retener las formas conjugadas de HT y más del 70% del HT libre y para obtener una fracción líquida no retenida en la columna cromatográfica de DHFG (DHFG-E) con una concentración de entre 0,005 g/L y 2,5 g/L de DHFG de concentración usando relaciones de la fuente de DHFG (F0) y el agua o en una disolución de etanol en agua de entre 5:1 (kg:L) y 1 :20 (kg:L). 1. A process for obtaining 3,4-dihydroxyphenylglycol (DHFG) comprising the steps of: i) heat treatment (T1) to a temperature between 90 and 140 C and C source DHFG (F0) dissolved in water or in a solution of ethanol in water with a concentration of ethanol in water between 10% and 50% (v / v), for a time between 10 minutes and 1.5 h with a temperature ramp of less than 5 minutes to release a liquid fraction (A1) containing: between 0.01 g / L and 2.5 g / L of DHFG (DHFG-E), between 0.001 g / L and 0.25 g / L of HT, and between 0 , 03 and 15 g / L of conjugated forms of HT, using ratios of the source of DHFG (F0) and water or in a solution of ethanol in water of between 5: 1 (kg: L) and 1: 20 (kg: L); and ii) chromatography of the liquid fraction (A1) obtained after the thermal treatment of step (i) using an anionic resin (C1) at a pH between 5 and 8 to retain the conjugated forms of HT and more than 70% of the Free HT and to obtain a liquid fraction not retained on the DHFG column chromatography (DHFG-E) with a concentration of between 0.005 g / L and 2.5 g / L of DHFG concentration using DHFG source ratios (F0 ) and water or in a solution of ethanol in water between 5: 1 (kg: L) and 1:20 (kg: L).
2. El procedimiento según la reivindicación 1 , donde el tratamiento térmico (T1) de la etapa (i) se lleva a cabo entre 90 eC y 130 eC. 2. The method of claim 1 wherein the heat treatment (T1) of step (i) is carried out between 90 and 130 C and C.
3. El procedimiento según cualquiera de las reivindicaciones 1 o 2, donde la fuente de DHFG (F0) es cualquier parte de la planta, productos o subproductos derivados del olivo o cualquier otro producto vegetal de la familia Oleaceae, Orobanchaceae, Plantaginaceae, Compositae, Lamiaceae, Acanthaceae y/o Scrophulariaceae. 3. The process according to any of claims 1 or 2, where the source of DHFG (F0) is any part of the plant, products or by-products derived from the olive tree or any other plant product of the Oleaceae, Orobanchaceae, Plantaginaceae, Compositae family, Lamiaceae, Acanthaceae and / or Scrophulariaceae.
4. El procedimiento según la reivindicación 3, donde la fuente de DHFG (F0) es cualquier planta, productos o subproductos derivados del olivo. 4. The process according to claim 3, wherein the source of DHFG (F0) is any plant, products or by-products derived from the olive tree.
5. El procedimiento según la reivindicación 4, donde la fuente de DHFG (F0) es la hoja de olivo. 5. The process according to claim 4, wherein the source of DHFG (F0) is olive leaf.
6. El procedimiento según cualquiera de las reivindicaciones 1 a 5, donde las formas conjugadas de HT son oleuropeína, glucósido del HT y sus derivados secoiridoides.
6. The process according to any of claims 1 to 5, wherein the conjugated forms of HT are oleuropein, HT glucoside and their secoiridoid derivatives.
7. El procedimiento según la reivindicación 6, donde las formas conjugadas de HT son oleuropeína. 7. The process according to claim 6, wherein the conjugated forms of HT are oleuropein.
8. El procedimiento según cualquiera de las reivindicaciones 1 a 7, donde la fracción líquida A1 obtenida en la etapa (i) se somete a una etapa de centrifugación, filtración y/o sedimentación antes de la etapa (ii). 8. The process according to any of claims 1 to 7, wherein the liquid fraction A1 obtained in step (i) is subjected to a centrifugation, filtration and / or sedimentation step before step (ii).
9. El procedimiento según cualquiera de las reivindicaciones 1 a 8, donde la fuente de DHFG (FO) está disuelta en agua. The process according to any one of claims 1 to 8, wherein the source of DHFG (FO) is dissolved in water.
10. El procedimiento según cualquiera de las reivindicaciones 1 a 9, donde la resina aniónica (C1) de la etapa (ii) es una resina aniónica débil. The process according to any one of claims 1 to 9, wherein the anionic resin (C1) of step (ii) is a weak anionic resin.
11 . El procedimiento según cualquiera de las reivindicaciones 1 a 10, donde la etapa (ii) se lleva a cabo a un pH de 6,5. eleven . The process according to any of claims 1 to 10, wherein step (ii) is carried out at a pH of 6.5.
12. El procedimiento según cualquiera de las reivindicaciones 1 a 11 , que además comprende una etapa (iii) de purificación (T2) de la fracción DFIFG-E obtenida en la etapa (ii) para obtener DFIFG con un grado de pureza mayor (DFIFG-P) tras su retención en la columna (C1 ). 12. The process according to any of claims 1 to 11, further comprising a step (iii) of purification (T2) of the DFIFG-E fraction obtained in step (ii) to obtain DFIFG with a higher degree of purity (DFIFG -P) after retention in column (C1).
13. El procedimiento según la reivindicación 12, donde la purificación (T2) de la etapa (iii) se lleva a cabo por cromatografía con resinas catiónicas, resinas aniónicas o mezclas de las mismas, por cromatografía con resinas de adsorción y elución con agua y/o etanol, cabo por filtración con membranas de filtración, nanofiltración o de ultrafiltración o por osmosis inversa para eliminar parte del contenido en agua. The process according to claim 12, wherein the purification (T2) of step (iii) is carried out by chromatography with cationic resins, anionic resins or mixtures thereof, by chromatography with adsorption resins and elution with water and / or ethanol, carried out by filtration with filtration membranes, nanofiltration or ultrafiltration or by reverse osmosis to remove part of the water content.
14. El procedimiento según cualquiera de las reivindicaciones 1 a 13, que además comprende una etapa (iv) de elución con agua, etanol o sus mezclas de los conjugados de HT retenidos en la columna C1 de la etapa (ii), preferiblemente de oleuropeína, para obtener un extracto rico en oleuropeína (Oleuro-E), hidroxitirosol (HT-E), ácido elenólico y/o sus derivados. The process according to any of claims 1 to 13, further comprising a step (iv) of elution with water, ethanol or their mixtures of the HT conjugates retained in column C1 of step (ii), preferably oleuropein , to obtain an extract rich in oleuropein (Oleuro-E), hydroxytyrosol (HT-E), elenolic acid and / or its derivatives.
15. El procedimiento según cualquiera de las reivindicaciones 1 a 14, que además comprende una etapa (v) de hidrólisis química o enzimática (T3) del extracto de
Oleuro-E obtenido en la etapa (iv) o para obtener un extracto rico en HT (HT-E).
The process according to any of claims 1 to 14, further comprising a step (v) of chemical or enzymatic hydrolysis (T3) of the extract of Oleuro-E obtained in step (iv) or to obtain an extract rich in HT (HT-E).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ESP202030495 | 2020-05-28 | ||
| ES202030495A ES2881826B2 (en) | 2020-05-28 | 2020-05-28 | PROCEDURE FOR OBTAINING 3,4-DIHYDROXYPHENYLGLYCOL (DHFG) |
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| WO2021240041A1 true WO2021240041A1 (en) | 2021-12-02 |
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| PCT/ES2021/070384 WO2021240041A1 (en) | 2020-05-28 | 2021-05-28 | Method for producing 3,4-dihydroxyphenylglycol (dhpg) |
Country Status (3)
| Country | Link |
|---|---|
| ES (1) | ES2881826B2 (en) |
| PT (1) | PT2021240041B (en) |
| WO (1) | WO2021240041A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2377510A1 (en) * | 2008-12-19 | 2011-10-19 | Consejo Superior de Investigaciones Científicas (CSIC) | Method for purifying 3,4-dihydroxyphenylglycol (dhpg) from plant products |
| ES2395032A1 (en) * | 2011-06-21 | 2013-02-07 | Consejo Superior De Investigaciones Científicas (Csic) | Phenol extract from heat-treated olive subproducts |
| EP2743248A1 (en) * | 2011-07-08 | 2014-06-18 | Consejo Superior De Investigaciones Científicas (CSIC) | Method for obtaining hydroxytyrosol extract, mixture of hydroxytyrosol and 3,4-dihydroxyphenylglycol extract, and hydroxytyrosyl acetate extract, from by-products of the olive tree, and the purification thereof |
-
2020
- 2020-05-28 ES ES202030495A patent/ES2881826B2/en active Active
-
2021
- 2021-05-28 WO PCT/ES2021/070384 patent/WO2021240041A1/en active Application Filing
- 2021-05-28 PT PT2021070384A patent/PT2021240041B/en active IP Right Grant
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2377510A1 (en) * | 2008-12-19 | 2011-10-19 | Consejo Superior de Investigaciones Científicas (CSIC) | Method for purifying 3,4-dihydroxyphenylglycol (dhpg) from plant products |
| ES2395032A1 (en) * | 2011-06-21 | 2013-02-07 | Consejo Superior De Investigaciones Científicas (Csic) | Phenol extract from heat-treated olive subproducts |
| EP2743248A1 (en) * | 2011-07-08 | 2014-06-18 | Consejo Superior De Investigaciones Científicas (CSIC) | Method for obtaining hydroxytyrosol extract, mixture of hydroxytyrosol and 3,4-dihydroxyphenylglycol extract, and hydroxytyrosyl acetate extract, from by-products of the olive tree, and the purification thereof |
Non-Patent Citations (2)
| Title |
|---|
| GUILLERMO RODRIGUEZ ET AL.: "Antioxidant activity of effluents during the purification of hydroxytyrosol and 3,4-dihydroxyphenyl glycol from olive oil waste", EUROPEAN FOOD RESEARCH AND TECHNOLOGY, vol. 224, no. 6, 30 May 2006 (2006-05-30), Berlin Heidelberg, pages 733 - 741, XP019488860, ISSN: 1438-2385 * |
| RODRIGUEZ GUILLERMO ET AL.: "3,4-Dihydroxyphenylglycol (DHPG): An Important Phenolic Compound Present in Natural Table Olives", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 57, no. 14, 22 July 2009 (2009-07-22), pages 6298 - 6304, XP055877524, ISSN: 0021-8561, DOI: 10.1021/jf803512r * |
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| Publication number | Publication date |
|---|---|
| ES2881826B2 (en) | 2022-09-14 |
| ES2881826A1 (en) | 2021-11-30 |
| PT2021240041B (en) | 2024-05-14 |
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