ES2813774B2 - PROCEDURE FOR THE OLIVE LEAF EXTRACTION OF OLEUROPEIN IN A PHENOLIC FRACTION, FUNCTIONAL FOOD COMPOSITION AND USE - Google Patents

Description

PROCEDURE FOR OLIVE LEAF EXTRACTION OF

OLEUROPEIN IN A PHENOLIC FRACTION, FOOD COMPOSITION

FUNCTIONAL AND USE

Technical sector

The present invention is applicable in the food, cosmetic and pharmaceutical industries. In the food industry as a functional food made up of polyphenols and sugars; in the cosmetic industry in anti-aging creams for its ability to capture free radicals; and in the pharmaceutical industry in drugs against oxidative stress or for the prevention of cardiovascular and degenerative diseases such as cancer and Alzheimer's.

Background of the invention

Eating habits in the Mediterranean basin abundant in fruits, vegetables and extra virgin olive oil, are rich in polyphenols. This is the main reason for the low incidence of various degenerative pathologies; for example, the reduction of cardiovascular risk and the decrease in the risk of developing tumors. Patent documents such as WO 2000038541 A1 describe a healthy diet in which foods have been enriched with antioxidants, mainly phenolic compounds from olive trees.

The main phenolic compound in olive fruit and leaves is oleuropein, with concentrations of 2 to 134 mg g-1 of dry matter. Numerous studies demonstrate the important biological activity of oleuropein. It is an important antioxidant and antibacterial, the main responsible for the anti-inflammatory effect of olive leaf extracts, it shows antiproliferative activity on breast and liver cell lines, acts against diabetes, prevents obesity since it has shown hypocholesterolemic effects in rats, and protects against osteoporosis and osteoarthritis. In addition, it helps prevent neurodegenerative diseases such as Parkinson's and Alzheimer's due to its neuroprotective properties and minimizes the progression of pulmonary fibrosis.

In recent years, high pressure, supercritical fluid or molecular distillation technologies have been applied to extract these components and include them in food matrices. The interest in functional foods, fortified foods and current food supplements is to offer protective effects against high-incidence diseases, such as obesity, cardiovascular disorders and cancer, among others.

Thus, application WO 2017032722 A1 describes a drink made from an extract of olive leaves with secoiridoid derivatives obtained by heating in water and enzymatic additives; and JP 2016079127 A, describes an antioxidant composed of dihydroquercetin and olive leaf extract containing oleuropein and hydroxytyrosol, with application as a food additive.

WO 2008108817 A1 describes a healthy nutritional supplement in which the bitter taste of oleuropein is masked by forming a complex with cyclodextrins aided by glycerin monoesters. According to US 20090061031 A1, oleuropein and its major metabolites, including oleuropein aglycon, hydroxytyrosol, and elenolic acid, are useful for treating obesity, as well as for inhibiting HIV infectivity.

Patent ES 2288123 B1 protects a functional food composition based on olive and citrus extracts for the prevention or treatment of problems related to excess cholesterol and triglycerides in the blood. The extractions are carried out by hydroalcoholic mixtures.

All these oleuropein extraction methods require organic solvents (MeOH/H ² O, EtOH/H ² O or MeCN/H ² O) that are toxic and must be eliminated in the production of a food product.

Faced with this drawback, natural deep eutectic solvents (NADES) avoid the need to reduce or eliminate the extractant. NADES are of great interest as they are a green alternative to toxic conventional solvents.

NADES based on glycerol and glycine, that is, on non-sugar compounds, have been used to extract polyphenols from olive leaves (Athanasiadis, V. et al. Waste Biomass Valor., 2018 , 9, 1985-1992); the document does not suggest the use of sugars as an extractant. The role of methyl p-cyclodextrin as an adjuvant in the extraction of polyphenols from olive leaves with deep eutectic solvents has also been described (Athanasiadis, V. Biomass Convers. Biorefin., 2018 , 8, 345-355), and the use of choline-based deep eutectic solvents in microwave-assisted extraction of phenolic compounds from olive leaf (Alañón, ME et al. Arab. J. Chem., 2018 , in press, https://doi.org/10.1016 /i.arabic.2018.01.003); where together with choline as a hydrogen bond acceptor, acetic acid, oxalic acid, acid tartaric acid, butane-1,4-diol, ethylene glycol, xylitol, propane-1,2-diol, maltose or urea as hydrogen bond donor.

In summary, there is no prior art in the use of sugars as the sole components of a NADES to extract polyphenols from olive leaves.

The problem of the technique is to achieve a simpler extraction procedure for oleuropein from the olive leaf and avoiding toxic solvents. The solution proposed by the present invention is a procedure that replaces the typical organic solvents with sugars or honey acting as NADES.

Description of the invention

The present invention is a procedure for the extraction of a phenolic fraction containing oleuropein from olive leaves, which comprises heating a mixture comprising dried olive leaf pulverized in a deep eutectic solvent of edible natural sugars and water, and filtering to obtain a precipitate and an aqueous phase where the aqueous phase contains the phenolic fraction with oleuropein.

In the scope of the present invention, "pulverized" dry leaf is understood as the dry leaf subjected to a grinding process with a particle size of 0.05 to 1.0 mm.

Another aspect of the present invention is a process consisting of the above steps. Yet another aspect is that the mixture consists of dried pulverized olive leaf and a deep eutectic solvent.

Another preferable aspect of the invention is that said NADES forming sugars are glucose, fructose and sucrose. In this case, the heating occurs at a temperature between 30 and 120°C, preferably between 50% and 100°C because above there is a certain degree of decomposition of the oleuropein and below 50°C the yield is lower, and even more preferably 80°C.

In an even more preferred aspect the glucose, fructose and sucrose are in a molar ratio in water of 1:1:1:11.

A highly preferable aspect of the present invention is that said deep eutectic solvent consisting of edible natural sugars is honey. In this case, heating should be at a temperature between 35 and 50°C so as not to denature the honey, preferably 40°C.

The mixture of sugars will mask the bitterness of oleuropein in the food product in which it is incorporated.

Another preferable aspect of the invention is that said heating occurs for a time between 20 and 240 min, preferably between 50 and 90 min to avoid some decomposition of the oleuropein especially at high times and temperatures, and even more preferably 60 min.

A preferred aspect of the invention comprises a dilution with water prior to the filtration step to improve the performance of the filtrate.

Filtration can be done under vacuum on a paper filter with a pore size between 2.5-25 pm. The liquid fraction is taken, which can then be extracted with an organic phase, preferably ethyl acetate, and concentrated on a rotary evaporator.

The present invention describes the extraction of oleuropein from olive leaves by an environmentally friendly method framed within green chemistry, in which the extractant formed by a mixture of sugars or honey allows a functional food to be directly obtained.

The main advantage of the process of the invention is that it avoids the use of conventional organic solvents, which are toxic and involve purification steps that complicate the process.

Oleuropein, as a phenolic compound with significant bioactivity, is a powerful antioxidant, helps preserve food, preventing rancidity, loss of color and deterioration of aroma and flavor; In addition, it has interesting anti-inflammatory, anti-atherogenic, anti-cancer, hypolipidemic and hypoglycemic pharmacological properties.

The composition obtained can help defend cells against oxidative damage, preventing cardiovascular and neurodegenerative diseases and cancer.

Thus, a very preferable aspect of the invention is a functional food composition comprising a phenolic extract with oleuropein in a percentage of between 80% - 85% by weight with respect to the weight of the total phenolic extract.

Another preferable aspect is the use of the composition of the invention as an ingredient in food products such as candies or jellies, carbonated drinks, juices, bakery products, energy or dairy products.

Another preferable aspect is the use of the composition of the invention as a dietary supplement, even more preferably an antioxidant and antiaging dietary supplement.

In the cosmetic industry, honey enriched in olive polyphenols could be used in the manufacture of anti-aging creams due to its ability to capture free radicals; and in the pharmaceutical industry, the polyphenol content would allow its use against oxidative stress and for the prevention of cardiovascular and degenerative diseases such as cancer and Alzheimer's.

So that another preferable aspect of the invention is the use of the composition in the prevention or treatment of a tumor, neurodegenerative, infectious, inflammatory or cardiovascular disease, or any other for which the oleuropein or the phenolic extract of the olive leaf be beneficial.

Description of the figures

Figure 1 shows the chromatogram obtained after the extraction of the phenolic fraction of the olive leaf with NADES according to Example 1. It shows the presence of oleuropein with a purity of 85% accompanied by hydroxyoleuropein, ligustroside and two isomers of oleuropein ( deduced from UHPLC-MS), as minor compounds. The values are multiplied by a factor of 1000.

examples

Example 1:

Ingredients:

Water................................................. .............................27.2g

Glucose................................................. ......................2.0g

Fructose................................................. ......................2.0g

Saccharose................................................. ...........................3.8g

Dried and pulverized olive leaf....................................10.0 g

10 g of dried olive leaf powdered with NADES (10 g, 2.0 g glucose, 2.0 g fructose, 3.8 g sucrose, 2.2 g water) were heated at 80 °C for a hour. Past that time it was diluted with 25 g of water and filtered on a Büchner funnel under vacuum through a paper filter. The liquid fraction obtained was then extracted with 25 mL of ethyl acetate and then this liquid fraction was concentrated in a rotary evaporator.

The 1H NMR of the extract shows that oleuropein was obtained as the main product. The chromatogram obtained from the phenolic fraction is shown in Figure 1.

The extract quantified by HPLC contained 90 mg of a phenolic fraction, of which 85% is oleuropein.

Example 2:

Ingredients:

Water................................................. ......................... 25.0 g Honey.................... ........................................................... .....10.0g

Dried and pulverized olive leaf....................................10.0 g

10 g of dried olive leaf pulverized with natural honey (10 g) were heated at 40 °C for one hour. After that time, it was diluted with 25 g of water and filtered using a Büchner funnel under vacuum through a paper filter.

The extract quantified by HPLC contained 45 mg of a phenolic fraction, of which oleuropein constitutes 83%.

Claims (9)

1. Procedure for the extraction in olive leaf of a phenolic fraction containing oleuropein, characterized in that said procedure comprises: - heating a mixture comprising dried pulverized olive leaf and a solvent, in which said solvent is a deep eutectic solvent consisting of glucose at 20% by weight, fructose at 20% by weight and sucrose at 38% by weight, with three parts of water (w/v), all with respect to one part by weight of olive leaf, at a temperature between 50°C and 100°C for a time of 50 to 90 min; either heating a mixture comprising dried pulverized olive leaf and a deep eutectic solvent consisting of one part of honey (w/w) with three parts of water (w/v), both relative to one part by weight of olive leaf, at a temperature between 35° and 50°C for 50 to 90 min, - dilute with water, and - filter to obtain a precipitate and an aqueous phase, wherein said aqueous phase contains the phenolic fraction and oleuropein. 2. A process according to claim 1, characterized in that said temperature with the eutectic solvent consisting of glucose, fructose and sucrose is 80°C. 3. A process according to claim 1, characterized in that said temperature with honey as deep eutectic solvent is 40°C. 4. A process according to any of claims 1 to 3, characterized in that said heating time is 60 min. 5. Functional food composition obtained by the method of any of claims 1 to 4, characterized in that it comprises a phenolic extract with oleuropein, in which said oleuropein is present in a proportion of 80% - 85% by weight with respect to the weight of the total phenolic extract. 6. Use of the composition of claim 5, as an ingredient in a food product, wherein said food product is selected from the group consisting of a candy or gummy bear, a carbonated drink, a juice, a bakery product, a product energy or dairy. 7. Use according to claim 6, characterized in that said food product is a dietary supplement. Use according to claim 7, characterized in that said dietary supplement is an antioxidant or antiaging dietary supplement. 9. Use of the composition of claim 5, in the preparation of a medicament for the prevention or treatment of a tumor, neurodegenerative, infectious or inflammatory disease.