PL215886B1 - Application of extract from defatted evening primrose seeds - Google Patents

Abstract

The present invention relates to the use of an extract of defatted evening primrose seeds for the production of an agent for preventing or limiting damage to the heart muscle and coronary vessels. Preferably, an extract of defatted evening primrose seeds (Oenothera paradoxa) is used.

Description

Description of the invention

The subject of the invention is a new use of an extract of defatted evening primrose seeds (Oenothera), especially evening primrose (Oenolhera paradoxa).

For many years, the subject of numerous studies has been cardioprotection of the heart muscle, defined as the limitation of damage or prevention of damage to the heart muscle and coronary vessels through endogenous mechanisms or through the action of drugs. One of the problems that has been observed is that after the coronary circulation has been restored to a previously ischemic area, the recovery of cell contraction is markedly delayed. Therefore, ensuring adequate perfusion and oxygenation of the heart muscle cells, despite their viability, may not be sufficient to restore the mechanical efficiency of the heart. Reperfusion, i.e. restoring blood supply to the ischemic area, instead of saving the still living heart cells, can rapidly intensify the process of their damage. The discussed process of ischemic reperfusion injury has been the subject of many studies.

The cardioprotective effect is shown by angiotensin converting enzyme inhibitors (ACE inhibitors) - drugs that are standardly used in arterial hypertension and heart failure. A significant proportion of the cardioprotective effects of ACE inhibitors can be attributed to inhibition of angiotensin II formation. It is a strongly vasoconstrictor and is endowed with a mitogenic effect, which may indicate its important role in the pathogenesis of atherosclerosis. ACE inhibitors, in addition to inhibiting the conversion of angiotensin I to angiotensin II, also inhibit the bradykinin pathway. Bradykinin is a peptide tissue hormone which, by stimulating endothelial receptors, is able to release relaxing substances into the vessel lumen, which lowers blood pressure. Bradykinin also releases other protective substances important for the vessels, e.g. prostanoids and nitric oxide, so its increased concentration is associated with a cardioprotective effect. The beneficial effect on the lipid and carbohydrate metabolism of the body, observed with the chronic administration of ACE inhibitors, is also an element of the cardioprotective effect.

In the search for new cardiovascular therapies, special attention is now paid to compounds that combine the properties of an angiotensin converting enzyme blockade and an inhibitor of neutral endopeptidase that inactivates atrial natriuretic peptide (ANP). These compounds are called vasopeptidases. The benefits of this treatment approach result from the simultaneous inhibition of angiotensin II and the increase in ANP concentration, which exhibits diuretic, natriuretic and antihypertensive properties. The best known of these combinations - omalaprilat - is currently being evaluated in multicentre studies known under the acronym OPERA (Omalaprilal in Persons with Enhanced Risk of Alherosclerolic evenis), involving over 12,500 people in approximately 1,100 clinical centers [Kostis JB, Cobbe S., Johnston C . et al .: Design of the Omalaprilat in Persons with Enhanced Risk of Atherosclerotic events (OPERA) trial. Am. J. Hypertens., 2002; 15: 193-198]. Previous studies have shown high antihypertensive efficacy of this drug and its beneficial effect in patients with heart failure. It should be added, however, that one of the undesirable symptoms was angioedema.

Considering that heart failure is one of the main and increasingly common causes of death and disability, it is important to look for methods that deepen the natural systemic defense processes related to ischemia. Among these methods, those based on the use of natural substances are of particular importance.

One of the widely used plants with medicinal properties is evening primrose (Oenothera). Evening primrose oil, obtained from the seeds of this plant, is characterized by a high content of essential fatty acids, especially γ-linolenic acid. Essential unsaturated fatty acids support the functioning of the cardiovascular system, have a positive effect on the metabolism of cholesterol and triglycerides, and support the body's natural immunity.

In the process of producing evening primrose oil, pomace, i.e. partially defatted seeds, is produced as a by-product. Evening primrose pomace was found to be a valuable source of polyphenols, hence the growing interest in their use in the pharmaceutical industry.

The pro-health effects of polyphenols are known and their particular importance in the prevention of atherosclerosis and heart disease. Polyphenols contained in red wine, the most important of which are quercetin, resveratol, catechin and epigallocatechin, have a wide spectrum of action in the cardiovascular system: they reduce the formation of blood clots, inhibit inflammatory processes in blood vessels, increase their relaxation and improve their relaxation. blood flow and oxygen supply to the heart. At the same time, they inhibit dangerous free radical reactions. Red wine polyphenols can reduce the oxidation of LDL cholesterol and thus prevent adverse atherosclerotic reactions. In turn, hawthorn extracts have an effect similar to phosphodiesterase III inhibitors or cardiac glycosides, relaxant effect on coronary and peripheral flow, but without arrhythmogenic effect. Additionally, they show antioxidant and anti-inflammatory properties. Hawthorn inflorescences, which together with the leaves are a pharmaceutical raw material, contain such polyphenols as flavonoids, as well as oligomeric procyanidins, mainly dimeric B2 procyanidin, trimeric C1 procyanidin and accompanying catechin and epicatechin. The composition of hawthorn (dry) extracts can vary, but most often they contain 2.2% of flavonoids and up to about 19% of procyanidins. A well-known source of plant polyphenols is also the Mediterranean pine bark extract, which contains a whole class of flavonoids, derived from flavan-3-ols. The extract contains catechin, epicatechin, taxifolin as well as procyanidins. Many of the above-mentioned polyphenolic compounds are also found in other plants, such as berries and tea, especially green.

Plant extracts containing polyphenolic compounds may differ significantly from each other due to the different composition of the polyphenol component. They usually contain some basic group of polyphenolic compounds, but there are also important differences between them which are important for the properties of the extracts. The activity of these compounds depends on their bioavailability, and this is closely related to their chemical structure - for example, the antioxidant activity of tea polyphenols is mainly attributed to catechins, and in red wine - to reservatrol. Thus, the proposed uses for polyphenols derived from different plants vary, although uses in the area of cardiovascular disease are generally based on their antioxidant properties.

The antioxidant use of an extract of defatted evening primrose seeds is known from the patent application WO 0018416. From a series of patent applications belonging to the Oriza company, it is known to use the extract as: an agent inducing cell apoptosis (JP 20010105658), an inhibitor of fat absorption (EP 13 12374), an agent against Helicobacter pylori (JP 20030151745), urease inhibitor (JP 20030339266), ulcer treatment agent (JP20040118419). The Polish patent application P-370446 proposes the use of evening primrose polyphenols for the treatment and prevention of osteoporosis. All the above-mentioned applications use the product obtained by extracting defatted evening primrose seeds with aliphatic alcohols, acetone, water or mixtures of these solvents.

Our research has shown that the extract of defatted evening primrose seeds has an ACE inhibitor effect and, at the same time, an inhibitor of neutral endopeptidase.

The essence of the invention is the use of an extract of defatted evening primrose seeds for the production of a composition for the prevention or reduction of damage to the heart muscle and coronary vessels.

The use of evening primrose extract for the preparation of a preparation for preventing or limiting the process of ischemia-reperfusion damage to the heart muscle is particularly advantageous.

Preferably, an extract of defatted evening primrose (Oenothera paradoxa) seeds is used.

Preferably, the total proportion of polyphenolic compounds in the extract is not less than 40%, based on catechin.

Preferably, the total proportion of proanthocyanidins in the extract is at least 30%, based on cyanidinium chloride.

In the use according to the invention, the defatted evening primrose extract is administered in the form of a pharmaceutical preparation or a dietary preparation containing, in addition to the extract, also pharmaceutically acceptable auxiliaries and / or as food additives. The preparation may be in a form suitable for oral administration, in particular in the form of a tablet, pill, dragee, capsule, powder, granules, suspension.

The preparation to be used according to the invention may additionally contain other active substances used in the cardioprotection of the heart muscle and coronary vessels.

Studies on the composition of extracts from defatted evening primrose seeds show that they contain, among others. ellagic acid, gallic acid, catechin, epicatechin and procyanidin gallate (incl.

proanthocyanidin B3), oenotein B and penta-O-galloyl-D-glucose (PGG). PGG, next to procyanidins

With a different degree of polymerization, it is the dominant compound in the extract of defatted evening primrose seeds and plays an important role in its therapeutic activity.

The dual action of evening primrose seed extract as ACE inhibitors and neutral endopeptidase inhibitors increases the levels of natriuretic peptides and vasodilating peptides and, at the same time, inhibits the renin-angiotensin-aldosterone system. Inhibition of bradykinin degradation allows to maintain its concentration at a level that ensures cardioprotective effect on the heart muscle and coronary vessels.

Evening primrose seed extract is obtained from defatted or partially defatted seeds. The degreasing effect is obtained by pressing the ripe seeds in hydraulic presses under the pressure of several hundred atmospheres. In this process, about 60% of the oil is removed and the seed husk is damaged, thus creating favorable conditions for the penetration of the inside of the husk by the solvent in the extraction process. This process takes place at a temperature not exceeding 40 ° C. The defatted seed is then extracted with an aliphatic alcohol, preferably ethanol, preferably in a mixture with water. The obtained extracts are concentrated, filtered and spray dried. A preparation in the form of a powder is obtained.

The extract of defatted evening primrose seeds was tested for activity against the enzymes ACE and neutral endopeptidase NEP. For this purpose, extracts were prepared from the extract, which were then divided into fractions, and compounds were isolated from the most active fractions. Fractions, extracts and compounds were tested. Woes showed a clear inhibition of vasopeptidases in a concentration dependent manner. HPLC studies showed a similar qualitative composition of active extracts, only some quantitative differences in the total of polyphenolic compounds were found. The most active compounds turned out to be oenotein B, procyanidin B3, penta-O-galloyl-3-D-glucose (PGG) and, to a lesser extent, methyl gallate. In one of the most active fractions the presence of procyanidins was found, the monomers of which are catechin and epicatechin gallate. Procyanidin and PGG largely determine the action of the entire extract, and other polyphenolic compounds present may act synergistically.

The subject matter of the invention and the tests carried out confirming the activity of evening primrose extracts in the use according to the invention are presented in more detail in the examples of implementation.

P r z k ł a d 1

Preparation of the raw extract.

Ripe evening primrose seeds are extruded in a hydraulic press at a temperature not exceeding 40 ° C. The defatted seeds are placed in the extractor, and then they are poured over with 60% ethanol at the temperature of 25-30 ° C, in the amount of 2-2.5 volumes of seeds. The content of the extractor is left for 1 hour without stirring, and then the alcoholic extract is drained from the bottom of the apparatus. The first volume of water at 25-30 ° C is poured into the extractor again and left for 1 hour, then the water extract is drained from the bottom of the apparatus. The extraction with water is repeated four times. The aqueous extracts are combined and processed further. The post-extraction mass remaining in the extractor is removed from the apparatus and sent for disposal.

As a result of the extraction process, approximately 1 volume of alcoholic extract and approximately 4 volumes of aqueous extracts are obtained. The next step in the process is to remove the alcohol from the extracts and concentrate the polyphenol solution. This process is carried out in a vacuum evaporator. The alcohol extract is distilled separately without mixing it with the aqueous extracts. The alcohol extract is concentrated twice, while the water extracts are 3-4 times concentrated. The alcohol distillates obtained during the process are standardized and reused for the extraction of a new portion of seeds. The decoctions obtained during the distillation of the extracts are allowed to cool, and then they are cleaned of sediments by centrifugation or filtration. Clear polyphenol solutions are spray dried.

As a result of the described process, a polyphenol extract in the form of a powder is obtained, containing at least 40% polyphenols, calculated as catechin, with the following composition: procyanidin, including procyanidin B3, 1,2, 3, 4, 6-O-penta-O -galoyl-3-D-glucose, catechin, epicatechin, epicatechin gallate, gallic acid, methyl gallate, ellagic acid, caffeic acid, quercetin, quercetin glucuronide, oenotein B. The total polyphenol content was greater than 40% and the total procyanidin content was higher than 30%.

P r z k ł a d 2

Preparation of extracts and fractions.

g of Oenothera paradoxa polyphenol extract in the form of a powder, obtained according to example 1, extracted with: (a) water (2x100 mL), (b) 60% ethanol (v / v, 2x100 mL), (c) isopropaPL 215 886 B1 nol (2x100 mL), (d) 30% isopropanol (v / v, 2x100 mL), for 60 minutes, at room temperature. After drying the isopropanol solution (c) in vacuo at 40 ° C, 2.36 g of a residue was obtained. The aqueous solution (a) and the aqueous residue after evaporation of the solvents in organic solutions (b) and (d)) were lyophilized. 1.93 g of product for solution (a), 2.58 g of product for solution (b) and 2.05 g of product for solution (d) are obtained.

g of a 30% isopropanol solution was dissolved in water (200 mL) and extracted with ethyl acetate (3 × 200 mL). The combined extracts were dried under vacuum at 45 ° C to give a residue of 3.2 g. The remaining aqueous solution was lyophilized to give 16.2 g of product.

2.5 g of the ethyl acetate extract (EA) obtained above was dissolved in 10 mL of methanol, and absorbed onto 5 g of polyamide, and applied to a column (diameter 5 cm, height 8 cm) packed with polyamide 6 (particle size 0.05-0.16 mm, Carl Roth) and eluted with methanol (50 mL fractions collected) and acetone-water solution (7: 3; 50 mL fractions collected). 100 fractions were collected and pooled into major fractions 1-6A according to their polyphenol composition as assessed by TLC thin layer chromatography. All fractions were lyophilized.

7.5 g of the aqueous residue (AR) after extraction with ethyl acetate was dissolved in 40 mL of a methanol-water mixture (7: 3), applied to a column (4 cm diameter and 40 cm high) filled with Sephadex LH-20 (particle size 0.025-0.100 mm) , Pharmacia, Uppsala, Sweden) and eluted with a methanol-water solution (7: 3; 50 mL fractions collected). 40 fractions were collected and pooled into major fractions 1-5B according to their polyphenol composition as assessed by TLC thin layer chromatography. All fractions were lyophilized.

The extracts and fractions were stored at -18 ° C.

The obtained fractions were tested for biological activity. The result is shown in the figure, in which Fig. 1 shows the test of the ethyl acetate solution and fraction 1-6 A, and Fig. 2 shows the test of the aqueous residue and fraction 1-5 B.

P r z k ł a d 3

Determination of the content of polyphenolic compounds and proanthocyanidins in the extracts obtained according to example 2.

The total content of polyphenolic compounds was determined by the Folin-Ciocalteu method in terms of catechin. 0.05 g of each extract was dissolved in 25 mL of a methanol-water solution (7: 3), then 0.2 mL of each extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 10 mL of 10% Na2CO3 solution and made up to 50 mL with water. The mixture was left in the dark for 30 min. Absorbances were measured spectrophotometrically at 700 nm.

The total content of proanthocyanidins was determined in accordance with the 4th European Pharmacopoeia as cyanidinium chloride. 0.2 g of each extract was dissolved in 30 mL of a methanol-water solution (7: 3) and heated to reflux for 80 min with 15 mL of 25% HCl. After cooling, the extracts were filtered and made up to 250 mL with a methanol-water solution (7: 3). 50 mL of each solution was evaporated to approximately 3 mL and 15 mL of water was added. The solution was then extracted with butanol (3 x 15 mL). The organic layers were transferred to a volumetric flask and filled with butanol to 100 mL. Absorbances were measured spectrophotometrically at 545 nm.

The total content of polyphenols and proanthocyanidins in the tested extracts is presented in Table 1.

P r z k ł a d 4

Determination of the angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) activity of the extracts obtained according to example 2.

Measurement of angiotensin-converting enzyme (ACE) activity

The enzymatic reaction consisted in the ACE enzyme cleavage of the added substrate Hip-L-His-L-Leu (hypuryl-histidyl-leucine) and formation of the product (histidyl-leucine), followed by formation of the fluorescing His-Leu complex with aldehyde-o-phthalate o-phthalidyl which is determined spectrofluorimetrically.

50 μ test solution (lyophilized extract / compound solution in phosphate buffer), 30 μΐ phosphate buffer (pH = 8.3), 150 μΐ enzyme solution (1: 300 boar sperm) and 20 μl substrate (Hip-L -His-L-leu 24 mM) The reaction mixture was incubated for 30 min. at 37 ° C, the reactions were stopped by adding 1000 μΙ 0.4M NaOH, then 100 μΙ 2% methanolic o-phthalic-dialdehyde solution was added and left in a dark place for 10 minutes until the formation of a fluorescent complex. Then 300 μl 2M HCl was added and left for for 30 minutes in a dark place.

PL 215 886 B1

The fluorescence of the resulting product was measured spectrofluorimetrically at X _wzb = 365 nm and X _emission = 500 nm at a slit width of 3 nm.

A control test without enzyme was performed to eliminate the fluorescence that the test solution itself may exhibit, as well as a positive control test without the addition of an inhibitor.

Determination of neutral endopeptidase (NEP) activity.

NEP activity was tested in two stages. The enzymatic reaction of the first stage consisted in cleavage of the substrate SAAP-AMC (succinyl-alanyl-alanilphenylalanil-7-amido-4-methylcoumarin) by the enzyme and formation of phenylalanil-7-amido-4-methyl coumarin (P-AMC). In the second reaction step, the added APN (aminopeptidase N) enzyme cuts the phenylalanine releasing the fluorescent compound.

50 μl test solution (lyophilized extract / compound solution in HEPES buffer), 50 μl 8 μ 8 lisinopril (to inhibit ACE activity), 50 μl substrate (400 μΜ), 350 μl HEPES buffer ρΗ = 7.4 and 150 μl of enzyme solution (1: 1000 boar sperm). The reaction mixture was incubated for 1 hour. at 37 ° C, the reactions were stopped by adding 50 µl of phosphoramidone (50 µΜ). Then 20 μl of APN solution (1: 235) was added, the mixture was incubated for 1 hour. at 56 ° C, the reaction was stopped with 800 μl of acetone. The fluorescence of the resulting product was measured spectrofluorimetrically at X _wzb = 367 nm and X _emission = 440 nm with a slit width of 3 nm.

A control without enzyme was performed to eliminate any fluorescence that the test solution may show, and a control to eliminate the inhibitory effect of the inhibitor on APN, and a positive control without the addition of the inhibitor.

Statistical analysis.

The determination for each concentration was performed in duplicate in triplicate, the results were presented as mean ± standard deviation (SD). The IC50 values, i.e. the concentration of the extract at which the enzyme activity is 50% inhibited, were read from the concentration-inhibition percentage curve.

The activity test results are shown in Table 1.

P r z k ł a d 5

Isolation of active compounds

The ethyl acetate fraction 5A (225 mg) was applied to a column (2.5 cm diameter, 35 cm high) packed with Toyopearl HW-40, (Tosoh, Tokyo, Japan) and eluted with acetone-water solution (7: 3) (fractions were collected after 10 mL). Fractions 7-9 (5A) were re-separated on the same column, eluting with an acetone-water solution (4: 6) (10 mL each). 53 g of 1,2,3,4,6-O-penta-O-galloyl-D-glucose are obtained (compound 1).

The aqueous fraction of 2B (70 mg) was applied to a column (2.5 cm diameter, 20 cm high) packed with Toyopearl HW-40, (Tosoh) and eluted with a methanol-water solution (7: 3) (10 mL fractions were collected). 10 mg of methyl gallate (compound 2) are obtained.

The aqueous fraction 4B (150 mg) was applied to a column (2.5 cm diameter, 20 cm high) packed with Toyopearl HW-40, (Tosoh) and eluted with a methanol-water solution (7: 3) (10 mL fractions were collected). 31 mg of (+) - catechin (4a> 8) - (+) - catechin (Procyanidin B3 Compound 3) was obtained.

1,2,3,4,6-O-penta-O-galloyl-pD-glucose (compound 1): off-white powder; UV, Xmax 281 nm; ESI-MS (pos. Ion) m / z 963.1 [M + Na] +; ¹ H NMR (MD3OD) Fragment glucose: δ 6.46 (1H, d, J = 8 Hz, H-1), 6.20 (1H, t, H-3), 5.85 (1H, m, H-4), 4.78 (1 H , m, H-5), 4.51 (1H, m, H-6). Galloyl fragment: δ 7.16, 7.18, 7.23, 7.25, 7.32 (each 2H, s). ¹³ C NMR (MD3OD) glucose fragment: 93.85 (C-1), 74.45 (C-5), 74.13 (C-3), 72.21 (C-4), 69.82 (C-2), 63.15 (C-6) . Galloyl fragment: 167.99, 167.35, 167.08, 166.98, 166.28 (carbonyl group signals), 146.63, 146.53, 146.50, 146.44, 146.34 (C-3, C-5), 141.00, 140.53, 140.48, 140.27, 140.14 (C-4 ), 121.00, 120.31, 120.17, 120.13, 119.62 (C-1), 110.63, 110.48, 110.42, 110.40, 110.35 (C-2, C-6).

Methyl gallate compound 2: light brown powder; UV, Xmax 273 nm; ESI- MS (pos. Ion) m / z 207.0 [M + Na] ⁺ ; ¹ H NMR (MD3OD): δ 6.96 (2H, s, H-2, H-6), 3.75 (3H, s, -OCH3). ¹³ C NMR (MD3OD): 169.2 (-COOH), 144.8 (C-3, C-5), 138.5 (C-4), 120.7 (C-1), 110.0 (C-2, C-6), 52.7 (-OCH3).

(+) - catechin (4a> 8) - (+) - catechin (Procyanidin B3) (compound 3): light brown powder; UV, Xmax 236 nm, 279 nm; ESI-MS (pos. Ion) m / z 601.1 [M + Na] +; thiolysis shows the presence of (+) - catechin and (+) - catechin thioether; ¹³ C NMR (MD3OD): 157.9-156.9 (C-5u, C-5t, C-7u, C-7t), 146.29, 145.96, 145.89, 145.58 (C-3'u, C-3't, C- 4'u, C-4't), 132.28 (C-1'u, C-1't), 120.09 (C-6'u), 119.43 (C-6't), 116.13, 1 15.94, 115.39 , 115.31 (C-2'u, C-2't, C-5'u, C-5't), 110.09 (C-8t), 100.87

PL 215 886 B1 (C-10u, C-10t), 96.35 (C-6t), 95.90 (C-6u), 95.56 (C-8u), 82.91 (C-3u), 77.13 (C-2u), 73.19 (C-2t), 68.86 (C-3t), 37.21 (C-4t), 28.57 (C-4u).

The active compounds were analyzed using the following methods.

Thin layer chromatography (TLC).

TLC thin layer chromatography was performed on silica gel 60 F254 (layer thickness 0.25 mm) and on polyamide 11 F254 (Merck KgaA). Chromatograms were developed in horizontal chambers (Chromdes, Lublin, Poland).

Detection of polyphenols: TLC studies were performed on polyamide using 60% CH3COOH as eluent or on silica gel using CHCl3-EtOAc-HCOOH (5: 4: 1) as eluent. Spots corresponding to the compounds were detected by UV254, 366 light and by spraying the chromatograms with 5% methanolic FeCl3 solution (dark blue spots) or 1% vanillin solution in H2SO4 (pink spots).

High performance liquid chromatography (HPLC).

The composition of polyphenols of individual extracts and fractions was assessed by means of HPLC with a diode detector. Separation was carried out on a Luna C-18, 25x4.6 mm, 5 µm column (Phenomenex, Torrance, CA). The mobile phase was: (A) 2.5% CH3COOH and (B) CH3CN + 2.5% CH3COOH (80:20). The following gradient was used to separate the compounds: 7-20% B (45 '); 20-40% B (70 '); 40-100% B (75 '); 100% B (80 '). The flow rate was 1 mL / min. UV spectra were recorded at wavelengths between 200 and 400 nm, chromatograms were recorded at 280 and 350 nm.

The samples were dissolved in a mixture of MeOH + 2.5% CH3COOH (1: 1) to concentrations of 10; 5 or 2.5 mg mL ^-1 .

Thiolysis.

Procyanidins thiolysis was performed in order to determine the qualitative composition of oligomers and polymers. The test was performed as follows: the samples were dissolved in methanol (50 μΐ) and 50 μΐ 3.3% hydrochloric acid in methanol and 100 μΙ 5% benzyl mercaptan in methanol were added. The mixture was incubated at 40 ° C for 30 min. Monomeric degradation products of procyanidins were investigated by HPLC.

The isolated compounds were tested for the activity of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) by the methods as in Example 4, except that a solution of the compound in phosphate buffer was prepared for the determination of the angiotensin converting enzyme (ACE) activity, and a solution of the compound was prepared for the determination of neutral endopeptidase. in the HEPES buffer. The results are shown in Table 2.

T a b e ΐ a 1

The total content of polyphenols and procyanidins in the tested extracts and their ability to inhibit vasopeptidase activity at a concentration of 25 μg / ml, IC50 values are given in parentheses.

Extracts Whole content polyphenols [mg / g] Whole procjanidin content [mg / g] ACE inhibition (IC50 pg / ml) NEP inhibition (IC50 pg / ml) aqueous 459.9 ± 0.1 443.3 ± 2.7 36.9 ± 1.3 (55) 84.0 ± 2.8 (4.7) 60% ethanol 653.3 ± 0.8 643.4 ± 0.2 33.3 ± 2.3 (37) 84.6 ± 2.5 (4.9) isopropanol 272.1 ± 0.6 402.6 ± 2.5 n.in (130) 70.5 ± 5.4 (15) 30% isopropanol 581.3 ± 1.6 518.9 ± 1.6 44.7 ± 2.7 (26) 90.4 ± 2.5 (6.1) ethyl acetate 860.0 ± 3.0 464.1 ± 2.9 46.4 ± 0.6 (32) 85.0 ± 3.1 (9.5) residual water 552.0 ± 3.1 473.9 ± 3.5 16.7 ± 2.9 (73) 90.6 ± 0.6 (7.0) Lisinopril (-) (-) IC50 0.0045 μg / ml (-) Phosphoramidone (-) (-) (-) IC50 0.0053 μg / ml

e.g. - no inhibition (-) - not tested

PL 215 886 B1

T a b e l a 2

Vaso-peptidase activity inhibition by polyphenols at a concentration of 100 µg / ml, IC50 values are given in parentheses (µΜ).

unions ACE inhibition (IC50 pM) NEP inhibition (IC50 pM) (+) - Catechin 27.2 ± 4.1 (> 350) 12.0 ± 1.0 (> 350) (-) - Epicatechin 47.0 ± 2.2 (> 350) 13.0 ± 2.1 (> 350) (-) - epicatechin gallate 60.8 ± 4.0 (165) 26.4 ± 2.0 (> 350) Gallic acid 37.4 ± 4.8 (> 500) 41.1 ± 6.5 (480) Ellagic acid 57.0 ± 2.0 (400) 26.0 ± 2.0 (> 500) Coffee acid 79.0 ± 2.0 (220) 28.0 ± 6.0 (> 500) Oenotein 27.0 ± 6.0 (250) 70.0 ± 5.0 (20) Quercetin 51.0 ± 4.2 (330) 38.7 ± 1.2 (> 350) Quercetin Glucuronide 54.0 ± 3.0 (200) 50.0 ± 4.1 (250) Pentagaloilo glucose 76.7 ± 0.8 (35) 81.7 ± 2.0 (12.5) Methyl gallate 52.5 ± 2.8 (500) 80.0 ± 0.5 (65) Procyanidin B3 64.1 ± 2.5 (135) 88.0 ± 0.6 (18) Lisinopril IC50 1 nM (-) Phosphoramidone (-) IC 50 9 nM

e.g. - no inhibition (-) - not tested

Claims (5)

1. Use of an extract of defatted evening primrose seeds for the preparation of a composition for the prevention or reduction of damage to the heart muscle and coronary vessels. 2. Application according to exacerbate. The method of claim 1, wherein the myocardial injury is ischemia reperfusion injury. 3. Use according to claim 1 The method of claim 1 or 2, characterized in that the extract of defatted evening primrose (Oenothera paradoxa) seeds is used. 4. Use according to claim 1 The method of claim 1 or 2, characterized in that the total proportion of polyphenolic compounds in the extract is at least 40%, based on catechin. 5. Use according to claim 1 The method of claim 1, characterized in that the total proportion of proanthocyanidins in the extract is at least 30%, based on the cyanidinium chloride.