"METHOD FOR OBTAINING OLEACEIN AND OLEOMISSIONAL TYPE SECOIRIDOIDS AND FOR PRODUCING RESPECTIVE PHARMACEUTICAL PREPARATIONS"

FIELD OF ART The application relates to a method for obtaining pure oleacein and oleomissional from olive fruits or leaves as well as certain derivatives thereof, to pharmaceutical preparations containing certain of them and the therapeutic properties of these preparations. The application relates to the field of medicine, pharmacy and food science.

STATE OF THE ART

The secoiridoids of the following general type

and R1 = H or COOCH ₃ , R2 = CHO are important ingredients of olive oil. Specifically, S-(£)-oleacein (1) and S-(£)-oleomissional (2) are phenolic substances present in most olive oil varieties at concentrations ranging from 0 to 1000 mg/Kg (Journal of Agricultural and Food Chemistry 2014, 62(3), 600-607, OLIVAE 2015, 122, 22-35). Substance (2) is in equilibrium with the corresponding dialdehydic forms (2a, b). For all those substances there are several published methods regarding their isolation from natural sources or their chemical synthesis and additionally for all of them there are numerous published biological and pharmaceutical properties (anti-inflammatory, anticancer, antidiabetic, neuroptotective, antioxidant). However, the inventors, based on experiments they have carried out, are aware that these substances are not the true bioavailabie active forms. In fact, substances (1), (2), when they come into contact with human biological fluids, they react chemically with water at a relatively slow rate, different for each substance, and are gradually converted to the active forms of 5S-(£) - oleaceinediol (3), 5S-(£)-oleomissionadiol (4) which exhibit increased activity and greatly increased water solubility with respect to substances (1), (2).

It is therefore very important to develop methods allowing the production of substances (3), (4) in pure form and pharmaceutical preparations or nutritional supplements or cosmetics which will contain the bioavailabie forms (3), (4) individuallly or in mixture, thus not needing to be activated upon entering the human body. At the same time, it is very important to find new methods that will lead to the production of substances (1), (2) at a higher yield than the existing methods and without using liquid chromatography to purify them.

To date, the structure of the substance (3) has never been fully and correctly elucidated, nor has an industrially utilizable method of either selective production or in the form of a mixture of substances (3) and (4) been described. The spectroscopic data of the substance (4) has been described (J. Chem. Soc., Perkin trans. 1995, 1, 1519-1523) after enzymatic hydrolysis of pure oleuropein while the substance (4) has been produced in very low yields, in mixtures with other substances that cannot be utilized, from olive fruit extracts (Food Res. Inti. 2000, 33: 475-485).

It should be mentioned that there are references in the literature regarding the use of plants of the Phillyrea species, which have been used to produce the S-(Z)-oleaceinediol isomer, in mixture with other non-utilizable substances in very low yield (Phytochemistry Letters 8 (2014) 163-170). In addition, the isomeric substances S-(Z)-oleaceinedio! and 5S-(Z)-oleomissionadiol appear to be produced in very low yields, in mixtures with other substances which cannot be utilized, when extracting olive oil with a mixture of water and methanol. (J. Agric. Food Chem. 2015, 66, 6053- 6063.)

It is very important to note that substances (1), (2) have different functional groups and in particular two aldehyde groups that that each one of them can react with water in a different way and under different conditions (pH, temperature, time), the association of the specific sructures (3), (4) with specific therapeutic actions being therefore not obvious and having an inventive nature.

An important element of innovation in the present invention is the use of polymeric hydrophilic alcohols as solubilizing agents. The proposed alcohols are biocompatible and can completely solubilize the substances (1), (2) and at the same time when they come in contact with water, they can release the active forms (3), (4).

It should be noted that there are references in the literature regarding the use of plants of Ligustrum species that have been used for the production of oleacein (1) in very low yield, in mixtures with other substances and with complex purification procedures (Journal of Ethnopharmacology 120 (2008) 220-225).

The chemical structures of the substances described in the present invention are the following:

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

1. Principle of the method for obtaining oleaceinediol (3) and oleacein (1):

Oleaceinediol (3) is produced by the reaction of oleacein (1) with water. Oleacein is biosynthetically produced from the precursor oleuropein substance under the action of b- glucosidase followed by a demethylation and decarboxylation reaction. The inventors know that the most abundant source of oleuropein is either fresh olive leaves (Olea europaea L. and all subspecies and cultivars) or partially dried leaves in which glycosidase and demethylase activity is maintained. The enzymes are kept in separate cell compartments from the precursor substance so that no reaction takes place when the leaf is intact.

If the plant enzymes come into contact with the precursor in the presence of water during leaf shredding then conversion of oleuropein to oleacein is achieved at a yield that can reach 1% by weight of fresh leaf. It is specifically pointed out that if the leaves are cut in the absence of water then the yield of this reaction is very low (<0.1%) having no practical usefulness. Also, the reaction does not occur if the water temperature is other than 15-40 °C. Previous studies performed by extraction of olive leaves with water at a higher temperature of e.g. 100 °C or even higher (Procopio et al. J. Agric. Food. Chem. 2009, 57, 11161-11167 or Conde et al., Food Chem. 2008, 114, 806- 812) indicate that the extracts obtained contain only oleuropein or decomposition products thereof such as hydroxytyrosol but not oleacein due to the fact that at higher temperatures glucosidase is deactivated and therefore cannot react with oleuropein.

In addition, the inventors based on experiments they have performed have found that oleaceinediol when it comes into contact with a non-polar organic solvent such as dichloromethane or ethyl acetate is extracted quantitatively and selectively in the organic phase releasing a molecule of water and returning to the aldehydic lipophilic form. The inventors, utilizing all the above knowledge that has been gained from their experiments, developed the following method of producing a solution of pure oleaceinediol: lai. Fresh olive leaves of the Olea europaea L. species, including all subspecies and cultivars, (or leaves with a moisture content of 0.1-10%) are cut in the presence of water at a ratio (leaves: water w / w) from 1: 1 to 1: 20 at 15-40 °C and left into contact for a time period ranging from 30 minutes to 12 hours (mixture A). laii. Alternatively, intact fresh fruits of Olea europaea L, including all subspecies and cultivars, or frozen fruits come into contact with water at a ratio ranging from 1: 1 to 1:20 at 15-40 ° C for a period of time ranging from 30 minutes to 12 hours. The yield is higher if the fruit is frozen as during the thawing process the cells rupture and instant contact of the substrate (oleuropein) with the deglycosylation and demethylation enzymes is achieved. lb. The aqueous mixture A resulting from lai or laii is filtered and the aqueous phase is obtained. This solution contains oleaceinediol mixed with sugars and other water-soluble phenols. lc. The aqueous solution from step lb is extracted with a water immiscible organic solvent in a ratio ranging from 1:10 to 10: 1 (e.g. dichloromethane or ethyl acetate or supercritical carbon dioxide) and the organic phase is obtained.

Ld. The organic phase from step lc is evaporated and the product B is obtained, containing oleacein of 95% purity, without the need for further chromatographic purification, in contrast to all methods known to date for the production of oleacein. le. Product B (oleacein) is mixed with deionized and distilled water, under vigorous stirring, at a temperature of 15-40 °C for a period ranging from 5 minutes to 24 hours, and during the chemical reaction with water it is converted to a solution of oleacinediol with a content of up to 2% (solution C). Special attention shall be paid to the pH of the water which should be neutral (6.9- 7.1)

Lf. Alternatively, product B (oleacein from step Id) can be mixed with PEG200 to 400 (solution F) polyethylene glycol and converted to a stable polyethylene glycol hemiacetal (5) with a chain size, as shown in the formula, being n = 4-9. Ig. Solution F can be diluted with water at a ratio PEG200 (or 400): water ranging from 1:10 to 1:20 or 1: 100 etc, yielding the Solution G and converting the polyethylene glycol hemiacetal of oleacein (5) to oleaceinediol (3). Solution G can be used for direct administration to humans per os (syrups, solutions, suspensions), injectable or transdermal (incorporated in creams or gels). lh. Solution C or F or G can be used at an appropriate concentration to prepare suppositories after mixing with a suitable base (suppository base) or to prepare tablets after mixing with excipients (e.g. lactose), fluid bed drying and compression. It should be noted that in the preparation of tablets, using a suitable excipient and adjusting the moisture levels during drying allow the substance to remain in diol form. This is confirmed by the instant dissolution of the tablet in water which would not have happened if the substance was in the lipophilic aldehydic form.

As can be seen from the spectroscopic examination of solution C using NMR spectroscopy in deuterated water (D2O) (Figure 1), this is a true solution of the diol form and not an emulsion of the dialdehydic form in the aqueous phase, which is present in a very small amount.

Pharmaceutical properties of oleaceinediol: li. Solution C or F or G showed a potent antioxidant activity by inhibiting lipid peroxidation at a concentration of 1 mM. Lk. A study of the cytotoxic activity of solution C or F or G showed that oleaceinediol at a concentration of 5 mM could lead to death 50% of HeLa and MCF-7 cancer cells and therefore solution C or F or G and any pharmaceutical preparations derived therefrom can be used to treat cancer.

LL. Solution C or F or G at a concentration of 50 mM of oleaceinediol showed 65% inhibition of the COX-2 enzyme and therefore solution C or F or G and any pharmaceutical preparations derived therefrom can be used to treat inflammatory diseases. lm. By extension of Cases li-ll, solutions C or F or G have the same therapeutic properties (neuroprotective, anti-diabetic, antithrombotic) that are known for oleacein as in all cases oleacein is at least partially converted to oleaceinediol in the aqueous medium in which experiments to measure its biological activities are conducted.

Example 1

Dried olive leaves of Kalamata variety (10 kg) with 1% moisture are cut in the presence of water (100 L) at 25 °C and left in contact for 30 min. The mixture is filtered and the aqueous phase is obtained. The aqueous solution is extracted with dichloromethane (80 L) and the dichloromethane layer is obtained and evaporated under vacuum at 30 °C. The residue (100 g) is made up of oleacein with a purity of 95%. The oleacein is mixed with water (10 L) under vigorous stirring for 30 min and transformed into a pure oleaceinediol solution. Alternatively, oleacein (1 g) is mixed with PEG200 or PEG 400 (5 ml) and converted into the stable polyethylene glycol hemiacetal. This solution is stable at room temperature for 12 months. This solution can be diluted to 100 ml with a saturated sugar solution affording a syrup containing 1% oleaceinediol, stable for 15-30 days with anti-inflammatory use.

Example 2

Intact olive fruits (200 g) are placed in the freezer (-20 ° C) for 24 hours and then left for 24 hours in contact with water (500 ml) at 25 °C. The mixture is filtered and the aqueous phase is obtained. The aqueous solution is extracted with dichloromethane (250 ml) and the dichloromethane layer is evaporated under vacuum. The residue (1 g) is made up of oleacein with a purity of 95%. The oleacein is mixed with water (100 ml) under stirring for 30 min and converted to a solution of pure oleaceinediol. Alternatively, oleacein (1 g) is mixed with PEG200 or PEG 400 (5 ml) and converted into stable polyethylene glycol hemiacetal. 100 mI_ of the polyethylene glycol solution are mixed with melted suppository base (2g) and allowed to cool to yield suppositories of 100 mg. 2. Principle of method for obtaining oleomissionadiol (4) and oleomissional (2)

The inventors based on experiments they have performed, they have found that if the olive leaves or the intact unripe green olive fruits come into contact with an organic lipophilic solvent without the plant material being comminuted then only the first stage of deglycosylation takes place, not occurring the second stage of demethylation. In this way, the precursor oleuropein substance, is selectively converted to oleomissional.

This substance, after removal of the organic solvent, may come into contact with an aqueous solution and very slowly react with water and be converted to water-soluble oleomissionadiol (4). In this way it can be separated from the water-insoluble terpenes that are extracted in the first step with the organic solvent. The pure solution of oleomissionadiol is obtained by filtration.

Evaporation of the aqueous solution can provide pure oleomissional (2) as a dehydration reaction occurs and the aldehyde / enol form is gradually restored. At this stage, oleomissional coexists with the two stereoisomeric oleuropeinedials (2a, b).

The inventors, utilizing all of the above knowledge, derived from their experiments, developed the following method of obtaining a solution of pure oleomissionadiol:

2a. Fresh olive leaves or fresh intact olive fruits without being comminuted are brought in contact with a water immiscible organic solvent (e.g. dichloromethane or ethyl acetate or supercritical carbon dioxide) at a ratio ranging from 1: 1 to 1:20 at 15-40 °C and left in contact for a time period ranging from 30 minutes to 12 hours (mixture A).

2b. The mixture is filtered and the organic phase is obtained. This solution contains oleomissional in mixture with terpenes (mainly oleanolic and maslinic acid).

2c. The solution is evaporated under reduced pressure and at 30 °C and the residue is obtained.

2d. The residue is dissolved in water (with a pH of not more than 7) under stirring for 1 to 24 hours at 15-35 ° C and during the chemical reaction with water oleomissional (and its isomers) is converted to a solution of oleomissionadiol, while all other ingredients remain insoluble. The insoluble ingredients are removed by filtration, and the aqueous solution D containing pure oleomissionadiol (4) is obtained.

2e. The solution is evaporated and the product E is obtained, containing oleomissional (in equilibrium with its isomers) with a total purity of> 95% in the form of a viscous liquid.

2f. The product E is dissolved in PEG200 or 400 and converted to a solution H containing the stable oleomissional polyethylene glycol hemiacetal (6). 2g. Solution H can be diluted with water at a ratio of PEG200 (or 400): water = 1:10 or 1:20 or 1: lOOetc, yielding a solution J in which the oleomissionadiol polyethylene glycol hemiacetal (6) is converted to oleomissionadiol (4). Solution J can be used for direct administration to humans per os (syrups, solutions, suspensions), injectable or transdermal (incorporated in creams or gels).

2h. Solution D or H or J may be used at an appropriate concentration to prepare suppositories after mixing with a suitable base (suppository base) or to prepare tablets after mixing with excipients (e.g. lactose), fluid bed drying and compression. It should be noted that in the preparation of the tablets, using a suitable excipient and adjusting the moisture levels during drying allow the substance to remain in the diol form.

As can be seen from the spectroscopic examination of solution D using NMR spectroscopy (Figure 2), this is a true solution of the diol form which exists in two diastereomeric forms and not an emulsion of the dialdehydic form.

Pharmaceutical properties of oleomissionadiol:

2h. Solution D or H or J showed potent antioxidant activity by inhibiting lipid peroxidation at a concentration of 1 mM.

2i. A study of the cytotoxic activity of solution D or H or J showed that oleomissionadiol at a concentration of 50 mM could lead to cell death 50% of HeLa and MCF-7 cancer cells and therefore solution D or H or J and any derived pharmaceutical preparations therefrom can be used to treat cancer.

2j. Solution D or H or J at a concentration of oleaceinediol at 40 mM showed a 62% inhibition of the COX-2 enzyme and therefore Solutions D or H or J and any derived pharmaceutical preparations therefrom can be used to treat inflammatory diseases.

Example 3

Fresh olive leaves of Koroneiki variety (100 g) are left in contact with dichloromethane (500 g) at 25 ° C for 30 min. The mixture is filtered and the organic phase is evaporated under vacuum. The residue is dissolved in deionized and distilled water at pH = 6 (100 ml) under stirring for 24 hours, filtered and a solution of pure oleomissional is obtained. The solution is evaporated or lyophilized and oleomissional (160 mg) is obtained with a purity of> 95%. Alternatively, oleomissional (160 mg) is mixed with PEG200 or PEG 400 (5 ml) and converted to the stable polyethylene glycol hemiacetal. The resulting solution can be diluted to 100 ml with a saturated sugar solution affording a syrup containing 0.16% of oleomissional stable for 15-30 days with anti-inflammatory activity.

Example 4

Fresh intact olives of the Koroneiki variety (200 g) are left in contact with dichloromethane (500 g) at room temperature for 30 min. The mixture is filtered and the organic phase is evaporated under vacuum. The residue is then dissolved in deionized and distilled water at pH = 6 (100 ml) under stirring for 24 hours, filtered and a solution of pure oleomissionadiol is obtained. The solution is evaporated or lyophilized and olieomissional (200 mg) is obtained with a purity of> 95%. Alternatively, oleomissional (1 g) is mixed with PEG200 or PEG 400 (5 ml) and converted to the stable polyethylene glycol hemiacetal of oleomissional.