Method for obtaining oleacein and oleomissionary type secoiridoids and for producing respective pharmaceutical preparations

Abstract

The present invention relates to a method for obtaining oleacein and oleomissional substances, as well as certain derivatives and bioactive diol forms thereof, from olive fruits and leaves, in pure form or as mixtures, by selective extraction of specially selected leaves or fruits with water or organic solvents. The invention also relates to pharmaceutical preparations containing the above-mentioned substances in various combinations, and the therapeutic properties of these preparations for the treatment of cancer, degenerative diseases of the central nervous system, diabetes, hyperlipidemia, inflammatory diseases, and the prevention of atherosclerotic plaque and thrombus formation.

Description

[Technical field]

[0001] The present application relates to a method for obtaining pure oleacein and oleomissional from olive fruits or leaves, pharmaceutical preparations containing certain of these, and the therapeutic properties of these preparations. The present application relates to the fields of medicine, pharmacy and food science. [Background technology]

[0002] The common types are: [ka] (where: [ka] R1=H or COOCH3, and R2=CHO. The secoiridoids are important components of olive oil. In particular, S-(ε)-oleacein (1) and S-(ε)-oleomissional (2) are phenolic substances present in most olive oil varieties at concentrations between 0 and 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 dialdehyde forms (2a, b). For all of these substances, there are multiple published methods for isolation from natural sources or chemical synthesis, and for all of them, there are numerous published biological and pharmaceutical properties (anti-inflammatory, anti-cancer, anti-diabetic, neuroactive, antioxidant). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Journal of Agricultural and Food Chemistry 2014, 62(3), 600-607 [Non-Patent Document 2] OLIVAE 2015, 122, 22-35 [Non-Patent Document 3] J. Chem. Soc., Perkin trans. 1995, 1, 1519-1523 [Non-Patent Document 4] Food Res. Intl. 2000, 33: 475-485 [Non-Patent Document 5] Phytochemistry Letters 8 (2014) 163-170 [Non-Patent Document 6] J. Agric. Food Chem. 2015, 66, 6053-6063 [Non-Patent Document 7] Journal of Ethnopharmacology 120 (2008) 220-225 [Non-Patent Document 8] Procopio et al. J. Agric. Food. Chem. 2009, 57, 11161-11167 [Non-Patent Document 9] Conde et al., Food Chem. 2008, 114, 806- 812 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the inventors recognize based on their own experiments that these substances are not truly bioavailable active forms. In fact, when substances (1) and (2) come into contact with human body fluids, they chemically react with water at relatively slow rates that differ for each substance, and gradually transform into the active forms 5S-(E)-oleaceindiol (3) and 5S-(E)-oleomissionadiol (4), which show improved activity and significantly improved water solubility compared to substances (1) and (2). [Means for solving the problem]

[0005] It is therefore of great importance to develop methods that allow the production of substances 3, 4 in pure form, as well as pharmaceutical preparations or nutraceuticals or cosmetic products that contain said bioavailable forms 3, 4, either separately or as a mixture, and therefore do not need to be activated when they enter the human body. At the same time, it is of great importance to find new methods that lead to the production of substances 1, 2 with higher yields than existing methods and without the need for purification by liquid chromatography.

[0006] To date, the structure of substance (3) has never been completely and accurately elucidated, and no industrially applicable method for the preparation of substances (3) and (4) selectively or in the form of a mixture has been described. Spectroscopic data of substance (4) after enzymatic hydrolysis of pure oleuropein has been described (J. Chem. Soc., Perkin trans. 1995, 1, 1519-1523), and substance (4) has been prepared in very low yields from olive fruit extracts as a mixture with other unavailable substances (Food Res. Intl. 2000, 33: 475-485).

[0007] It should be mentioned that there are references to the use of plants of the species Phillyrea, used to produce the S-(Z)-oleaceindiol isomers in very low yields as a mixture with other non-utilizable substances (Phytochemistry Letters 8 (2014) 163-170). Furthermore, the isomers S-(Z)-oleaceindiol and 5S-(Z)-oleomissionadiol appear to be produced in very low yields as a mixture with other non-utilizable substances during the extraction of olive oil with a mixture of water and methanol (J. Agric. Food Chem. 2015, 66, 6053-6063).

[0008] It is very important to note that substances (1), (2) have different functional groups and in particular two aldehyde groups that can react with water in different ways and under different conditions (pH, temperature, time), and therefore the association of a particular structure (3), (4) with a particular therapeutic action is unclear and of an inventive nature.

[0009] The key element of the innovation of the present invention is the use of polymeric hydrophilic alcohols as solubilizing agents. The proposed alcohols are biocompatible and capable of completely solubilizing the substances (1), (2) and at the same time releasing their active forms (3), (4) when they come into contact with water.

[0010] It should be mentioned that there are references to the use of plants of the Ligustrum species used to produce oleacein (1) in very low yields, as a mixture with other substances and using complex purification procedures (Journal of Ethnopharmacology 120 (2008) 220-225).

[0011] The chemical structure of the substance according to the invention is as follows: [ka] [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a spectroscopic study of solution C according to the present invention in heavy water (DO) using NMR spectroscopy. [Diagram 2] FIG. 2 is a spectroscopic examination diagram using NMR spectroscopy of solution D according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1. Principle of the process for obtaining oleacein diol (3) and oleacein (1) Oleacein diol (3) is produced by the reaction of oleacein (1) with water. Oleacein is produced biosynthetically from the precursor oleuropein substance under the action of β-glucosidase followed by demethylation and decarboxylation reactions. The inventors have found that the richest source of oleuropein is fresh olive leaves (Olea europaea L. and all subspecies and cultivars) or partially dried leaves, where the activity of glycosidases and demethylation enzymes is maintained. These enzymes are kept in separate cellular compartments from the precursors, so the reactions do not occur when the leaves are intact.

[0014] When the plant enzymes come into contact with the precursors in the presence of water during cutting of the leaves, the conversion of oleuropein to oleacein is achieved in yields that can reach 1% by weight of the fresh leaves. When the leaves are cut in the absence of water, the yield of this reaction is very low (<0.1%) and has no practical utility. Also, the reaction does not occur when the water temperature is other than 15-40°C. Previous studies carried out, for example by extraction of olive leaves at high temperatures above 100°C (Procopio et al. J. Agric. Food. Chem. 2009, 57, 11161-11167 or Conde et al., Food Chem. 2008, 114, 806-812), have shown that the extract obtained contains only oleuropein or its degradation products, such as hydroxytyrosol, but not oleacein, due to the fact that glucosidases are inactivated at high temperatures and are therefore unable to react with oleuropein.

[0015] The inventors further discovered, based on their own experiments, that oleacein diol is quantitatively and selectively extracted in the organic phase when it comes into contact with a non-polar organic solvent such as dichloromethane or ethyl acetate, releasing water molecules and returning to its lipophilic form of aldehyde. Utilizing all the above findings from their own experiments, the inventors developed the following method for producing a solution of pure oleacein diol:

[0016] 1a(i). Fresh olive leaves (or leaves with a moisture content of 0.1-10%) of the species Olea europaea L. (including all subspecies and cultivars) are cut in the presence of water in a ratio of 1:1 to 1:20 (leaf:water, wt / wt) at 15-40°C and left in contact for a period of 30 minutes to 12 hours (mixture A).

[0017] 1a(ii). Alternatively, intact fresh or frozen fruits of Olea europaea L. (including all subspecies and cultivars) are contacted with water in a ratio of 1:1 to 1:20 at 15-40° C. for a period of 30 minutes to 12 hours. The yield is higher when the fruits are frozen because the cells are disrupted during the thawing process, achieving instantaneous contact of the substrate (oleuropein) with the deglycosylating and demethylating enzymes.

[0018] 1b. The aqueous mixture resulting from 1a(i) or 1a(ii) is filtered to obtain an aqueous phase. This solution contains oleacein diol mixed with sugars and other water soluble phenols.

[0019] 1c. Extract the aqueous solution from step 1b with a water immiscible organic solvent (e.g. dichloromethane or ethyl acetate or supercritical carbon dioxide) in a ratio of 1:10 to 10:1 to obtain an organic phase.

[0020] 1d. The organic phase from step 1c is evaporated to give product B, which contains 95% pure oleacein without the need for further chromatographic purification, in contrast to all methods known to date for the production of oleacein.

[0021] 1e. Product B (oleacein) is mixed with deionized distilled water under vigorous stirring at a temperature between 15 and 40 °C for a period of 5 minutes to 24 hours, which during the chemical reaction with water is converted into a solution of oleacein diol (solution C) with a content of up to 2%. Particular attention should be paid to the pH of the water, which should be neutral (6.9-7.1).

[0022] 1f. Alternatively, product B (oleacein from step 1d) can be mixed with PEG 200-400 polyethylene glycol, which can be converted to stable polyethylene glycol hemiacetals (5) with chain lengths n=4-9 as shown in the formula (Solution F).

[0023] 1g. Solution F can be diluted with water in a ratio of PEG 200 (or 400):water such as 1:10 to 1:20 or 1:100 to obtain solution G, which also converts the polyethylene glycol hemiacetal of oleacein (5) to oleacein diol (3). Solution G can be used for direct administration to humans orally (syrup, solution, suspension), by injection, or transdermally (incorporated into a cream or gel).

[0024] 1h. Using solution C or F or G in the appropriate concentration, suppositories can be prepared after mixing with a suitable base (suppository base) or tablets can be prepared after mixing with excipients (e.g. lactose), fluid bed drying and compression. Note that in the preparation of tablets, the substance can remain in the diol form by using suitable excipients and adjusting the moisture level during drying. This is confirmed by the immediate dissolution of the tablet in water, which would not occur if the substance was in the lipophilic aldehyde form.

[0025] Spectroscopic examination of solution C using NMR spectroscopy in deuterium oxide (D2O) (Figure 1) shows that this is a true solution of the diol form and not an emulsion of the dialdehyde form in the aqueous phase, with only very small amounts present.

[0026] Pharmaceutical properties of oleacein diol: 1i. Solutions C or F or G exhibited strong antioxidant activity by inhibiting lipid peroxidation at a concentration of 1 μM. 1k. Studies of the cytotoxic activity of solutions C or F or G showed that a concentration of 5 μM oleacein diol could lead to 50% death of HeLa and MCF-7 cancer cells, therefore solutions C or F or G and any pharmaceutical preparations derived therefrom can be used for the treatment of cancer. 11. Solution C or F or G with 50 μM concentration of oleacein diol showed 65% inhibition of COX-2 enzyme, therefore solution C or F or G and any pharmaceutical preparation derived therefrom can be used for the treatment of inflammatory diseases. 1m. By extension of cases 1i-1l, solutions C or F or G have identical therapeutic properties (neuroprotective, antidiabetic, antithrombotic) as all cases in which oleacein is at least partially converted to oleacein diol in the aqueous medium in which the experiments to measure its biological activity are carried out. EXAMPLES

[0027] Example 1 Dried olive leaves (10 kg) of the Kalamata variety with a moisture content of 1% are cut in the presence of water (100 L) at 25°C and left in contact for 30 minutes. The mixture is filtered to obtain the aqueous phase. The aqueous solution is extracted with dichloromethane (80 L) to obtain the dichloromethane layer, which is evaporated under vacuum at 30°C. The residue (100 g) consists of oleacein with a purity of 95%. The oleacein is converted into a pure oleacein diol solution by mixing with water (10 L) under vigorous stirring for 30 minutes. Alternatively, oleacein (1 g) is mixed with PEG 200 or PEG 400 (5 mL) to convert it into a stable polyethylene glycol hemiacetal. This solution is stable for 12 months at room temperature. The solution can be diluted to 100 mL with a saturated sugar solution to provide a syrup containing 1% oleacein diol, which is stable for use as an anti-inflammatory agent for 15 to 30 days.

[0028] Example 2 Intact olive fruits (200 g) are placed in a freezer (-20°C) for 24 hours and then placed in contact with water (500 mL) at 25°C for 24 hours. The mixture is filtered to obtain the aqueous phase. The aqueous solution is extracted with dichloromethane (250 mL) and the dichloromethane layer is evaporated under vacuum. The residue (1 g) consists of oleacein with a purity of 95%. This oleacein is converted into a solution of pure oleacein diol by mixing with water (100 mL) under stirring for 30 minutes. Alternatively, oleacein (1 g) is mixed with PEG 200 or PEG 400 (5 mL) to convert it into a stable polyethylene glycol hemiacetal. 100 μL of this polyethylene glycol is mixed with the dissolved suppository base (2 g) and cooled to obtain suppositories of 100 mg.

[0029] 2. Principle of the method for obtaining oleomissionary diol (4) and oleomissional (2) The inventors have discovered, based on their own experiments, that when olive leaves or intact unripe green olive fruits are contacted with an organic lipophilic catalyst without grinding the plant material, only the first step, deglycosylation, occurs, but not the second step, demethylation, thereby selectively converting the precursor oleuropein substance to the oleomissional.

[0030] After removing the organic solvent, the material can be contacted with an aqueous solution and reacted very slowly with water to convert it into water-soluble oleomissionariol (4), which can then be separated from the water-immiscible terpenes extracted in the first step by the organic solvent. Filtration gives a pure solution of oleomissionariol.

[0031] Evaporation of the aqueous solution allows dehydration reactions to gradually restore the aldehyde / enol forms to give pure oleomissional (2), which at this stage coexists with the two stereoisomeric oleuropein dials (2a, b).

[0032] Using the above findings derived from our own experiments, we have developed the following method to obtain a solution of pure oleomissionary diol:

[0033] 2a. Uncrushed fresh olive leaves or fresh, intact olive fruits are contacted with a water-immiscible organic solvent (e.g. dichloromethane or ethyl acetate or supercritical carbon dioxide) in a ratio of 1:1 to 1:20 at 15-40° C. and left in contact for a period of 30 minutes to 12 hours (mixture A).

[0034] 2b. The mixture is filtered to obtain the organic phase. This solution contains oleomissionals mixed with terpenes (mainly oleanolic acid and maslinic acid).

[0035] 2c. Evaporate the solution under reduced pressure at 30° C. to give a residue.

[0036] 2d. The residue is dissolved in water (pH 7 or less) with stirring at 15-30°C for 1-24 hours, during which the oleomissional (and its isomers) are converted into a solution of oleomissionary diol during the chemical reaction with water, while all other components remain undissolved. These undissolved components are removed by filtration to obtain an aqueous solution D containing pure oleomissionary diol (4).

[0037] 2e. The solution is evaporated to obtain Product E, which is in the form of a viscous liquid and contains oleomissionals with an overall purity of greater than 95% (in equilibrium with its isomers).

[0038] 2f. Product E is dissolved in PEG 200 or 400 and converted to solution H, which contains the stable oleomissional polyethylene glycol hemiacetal (6).

[0039] 2g. Solution H can be diluted with water in a ratio of PEG200 (or 400):water=1:10 or 1:20 or 1:100, etc., to obtain Solution J, in which oleomissionary diol polyethylene glycol hemiacetal (6) is converted to oleomissionary diol (4). Solution J can be used for direct administration to humans orally (syrup, solution, suspension), by injection, or transdermally (in a cream or gel).

[0040] 2h. Solutions D or H or J can be used in appropriate concentrations 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. Note that in the preparation of tablets, the material can remain in the diol form by using suitable excipients and adjusting the moisture level during drying.

[0041] As can be seen from spectroscopic examination of solution D using NMR spectroscopy (Figure 2), this is a true solution of the diol form present in two diastereomeric forms and not an emulsion of the dialdehyde form.

[0042] Pharmaceutical properties of oleomissionadiol: 2i. Solutions D or H or J exhibited strong antioxidant activity by inhibiting lipid peroxidation at a concentration of 1 μM. 2j. Cytotoxic activity studies of solutions D or H or J showed that oleomissionadiol at a concentration of 50 μM can lead to 50% cell death in HeLa and MCF-7 cancer cells, therefore solutions D or H or J and any pharmaceutical preparations derived therefrom can be used for the treatment of cancer. 2k. Solution D or H or J having 40 μM concentration of oleacein diol showed 62% inhibition of COX-2 enzyme, therefore solution D or H or J and any pharmaceutical preparation derived therefrom can be used for the treatment of inflammatory diseases.

[0043] Example 3 Fresh olive leaves (100 g) of the Koroneiki variety are contacted with dichloromethane (500 g) for 30 minutes at 25°C. The mixture is filtered and the organic phase is evaporated under vacuum. The residue is dissolved in deionized distilled water (100 mL) at pH=6 with stirring for 24 hours and filtered to obtain a solution of pure oleomissional. The solution is evaporated or freeze-dried to obtain oleomissional (160 mg) with a purity of more than 95%. Alternatively, oleomissional (160 mg) is mixed with PEG200 or PEG400 (5 mL) to convert it into a stable polyethylene glycol hemiacetal. The resulting solution is diluted to 100 mL with a saturated sugar solution to provide a syrup containing 0.16% oleomissional, which is stable for use as an anti-inflammatory agent for 15 to 30 days.

[0044] Example 4 Fresh, unblemished olives of the Koroneiki variety (200 g) are contacted with dichloromethane (500 g) at room temperature for 30 minutes. The mixture is filtered and the organic phase is evaporated under vacuum. The residue is then dissolved in deionized distilled water (100 mL) at pH=6 with stirring for 24 hours and filtered to obtain a solution of pure oleomissionaryol. The solution is evaporated or freeze-dried to obtain oleomissionary (200 mg) with a purity of >95%. Alternatively, oleomissionary (1 g) is mixed with PEG200 or PEG400 (5 mL) to convert it into a stable polyethylene glycol hemiacetal of oleomissionary.

Claims

1. The following types: 【Chemistry 1】 (however: 【Chemistry 2】 and R 1 =H or COOCH 3 and 【Chemistry 4】 A method for obtaining a secoiridoid of the formula contacting plant material of the species Olea europaea L (including all subspecies and cultivars) with a solvent in a ratio of 1:1 to 1:20 at a temperature of 15 to 40°C and leaving the contact for a period of 30 minutes to 12 hours, to activate the reaction of the enzymes present in the plant material with oleuropein at said temperature to produce said mixture of secoiridoids (A), wherein the plant material is fresh or dried leaves with a moisture content of 0.1% to 10% that have been previously ground in an aqueous solution, or fresh intact fruit or fruit that has been chilled (at 2-8°C) or frozen (at -18 to -24°C) for at least 24 hours, and the solvent is water, or the plant material is comprised of intact fresh leaves or fruits, and the solvent is a water-immiscible organic solvent selected from the group of solvents consisting of dichloromethane, ethyl acetate, and supercritical carbon dioxide; A method for obtaining said secoiridoid.

2. the plant material is fresh or dried leaves with a moisture content of 0.1% to 10% that have been previously ground in an aqueous solution, or fresh intact fruit or fruit that has been chilled (at 2-8° C.) or frozen (at −18 to −24° C.) for at least 24 hours, and the solvent is water; The method further comprises: filtering the mixture (A); Obtaining an aqueous phase; extracting the aqueous phase with a water-immiscible organic solvent selected from the group of solvents consisting of dichloromethane, ethyl acetate, and supercritical carbon dioxide in a ratio of 10:1 to 1:10; obtaining and vaporizing an organic phase; Obtaining a product (B), said product (B) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =H, R 2 A method for obtaining the secoiridoid of claim 1, comprising the step of:

3. mixing said product (B) with deionized distilled water at a pH of 6.9-7.1, in a ratio of up to 2% w / w and at a temperature of 15-40° C., for a period of 5 minutes to 24 hours; Obtaining a solution (C), said solution (C) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =H, R 2 =CH(OH) 2 3. A method for obtaining a secoiridoid according to claim 2, further comprising the step of:

4. The plant material consists of fresh, intact leaves or fruits; the solvent is a water-immiscible organic solvent selected from the group of solvents consisting of dichloromethane, ethyl acetate, and supercritical carbon dioxide; The method further comprises: filtering the mixture (A); Obtaining an organic phase; vaporizing the organic phase; Obtaining a residue; mixing the residue with deionized distilled water at pH=<7 in a ratio of up to 2% w / w and at a temperature of 15-35° C. for a period of 1-24 hours under stirring; removing insoluble components by filtration; and Obtaining a solution (D), the solution (D) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =COOCH 3 and R 2 =CH(OH) 2 2. A method for obtaining a secoiridoid according to claim 1, comprising the step of:

5. The method further comprises the step of evaporating the solution (D) under reduced pressure and at a temperature of less than 40° C., The vaporizing step provides a product (E), which is: 【Chemistry 7】 Where: 【Chemistry 2】 and R 1 =COOCH 3 and R 2 A method for obtaining a secoiridoid according to claim 4, containing a substance where =CHO.

6. The method further comprises the step of mixing the product (B) with polyethylene glycol having an average molecular weight of 200 to 400 (PEG 200-400); The mixing step provides a solution (F), the solution (F) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =H, 【Chemistry 6】 3. A method for obtaining a secoiridoid according to claim 2, comprising the step of:

7. The method further comprises the step of mixing the product (E) with polyethylene glycol having an average molecular weight of 200 to 400 (PEG 200-400); The mixing step provides a solution (H), the solution (H) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =COOCH 3 and 【Chemistry 6】 6. A method for obtaining a secoiridoid according to claim 5, comprising the step of:

8. The method further comprises the step of diluting the solution (F) with water in a ratio of polyethylene glycol:water=1:10, 1:20, or 1:100; The diluting step provides a solution (G), the solution (G) comprising: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =H, R 2 =CH(OH) 2 7. A method for obtaining a secoiridoid according to claim 6, comprising the step of:

9. The method further comprises the step of diluting the solution (H) with water in a ratio of polyethylene glycol:water=1:10, 1:20, or 1:100; The diluting step provides a solution (J), the solution (J) having: 【Chemistry 7】 Where: 【Chemistry 5】 and R 1 =COOCH 3 and R 2 =CH(OH) 2 8. A method for obtaining a secoiridoid according to claim 7, comprising the step of:

10. A method according to any one of claims 1 to 9, comprising the steps of: 【Chemistry 1】 (however, 【Chemistry 5】 and R 1 =H or COOCH 3 and 【Chemistry 6】 Secoiridoid.

11. A method according to any one of claims 1 to 9, comprising the steps of: 【Chemistry 1】 (however, 【Chemistry 5】 and R 1 =H, R 2 =CH(OH) 2 is) Secoiridoid.

12. 12. Use of a secoiridoid according to claim 10 or 11, mixed with suitable excipients to prepare a pharmaceutical preparation for administration either orally, transdermally, by injection or, after drying and compression, in the form of a suppository or tablet.

13. 12. Use of a secoiridoid according to claim 10 or 11 mixed with suitable excipients to produce a dietary supplement.

14. The use of a secoiridoid according to claim 12, wherein the pharmaceutical preparation is for the treatment of cancer, neurodegenerative diseases, diabetes, hyperlipidemia or inflammatory diseases, or for the prevention of atherosclerotic plaque and thrombus formation.

Images