Use of the extract of defatted seeds of primrose

The invention concerns the new use of an extract of defatted seeds of primrose {Oenothera), especially of evening primrose (Oenothera par adoxά).

For many years the subject of numerous research is the cardioprotection of myocardium defined as the limitation or prevention of myocardium and coronary vessels damage by endogenous mechanisms or medicament action. One of the observed problems is the fact that after restoration of the coronary circulation to the previously ischemic area the return of contractile activity of cells occurs with distinct delay. Therefore the assurance of suitable perfusion and oxygenation of myocardium cells, despite retaining their vitality may not be sufficient to reach again the mechanical efficiency of the heart. Reperfusion or restoring the inflow to ischemic area, instead of saving still alive cells, can violently intensify the process of their damage. The described process of ischemic-reperfusion damage was and is the subject of numerous research.

The cardioprotective activity is performed by angiotensin-converting enzyme inhibitors (ACE inhibitory) - medicaments conventionally used in arterial hypertension and heart failure. The important role of the cardioprotective activity of ACE inhibitors may be ascribed to the inhibition of angiotensin II formation. It is a potent vascular contractile substance with mitogenic activity which may speak for its important role in the pathogenesis of atherosclerosis. ACE inhibitors, besides of the mechanism consisting in inhibition of the conversion of angiotensin and into angiotensin II demonstrate also the activity consisting in inhibition of bradykinin pathway. Bradykinin is a peptide tissue hormone which by excitation of endothelial receptors demonstrates also an ability to release relaxing substances into vessel lumen which lowers the blood pressure. Moreover, bradykinin releases also other protective substances important for vessels such as prostanoids and nitrogen oxide and therefore its increased level is connected with cardioprotective effect. The advantageous effect on lipid and carbohydrate metabolism of the organism, observed in prolonged administration of ACE inhibitors also forms an

element of cardioprotective activity.

In searching the novel therapies for cardiovascular system currently the specific attention is paid to compounds combining the properties of blocking the angiotensin- converting enzyme and neutral endopeptidase inhibitor, deactivating atrial natriuretic enzyme (ANP). Such compounds are named vasopeptidases. The advantages of such treatment direction result from simultaneous inhibition of angiotensin II and increase of ANP concentration, having diuretic, natriuretic and hypotensive properties. The most known of these combinations - omalaprilat - is currently evaluated in multicenter research known under acronym OPERA {Omalaprilat in Persons with Enhanced Risk of Atherosclerotic events), including over 12,500 persons in ca. 1100 clinical centers [Kostis J.B., Cobbe S., Johnston C. et ah: Design of the Omalaprilat in Persons with Enhanced Risk of Atherosclerotic events (OPERA) trial. Am. J. Hypertens., 2002; 15: 193-198]. The up to now results demonstrated the high hypotensive effectiveness of that medicament and its advantageous influence in patients with cardiac insufficiency. It is however to add that the vasomotoric edema was one of undesirable symptoms.

Taking into account that the cardiac insufficiency is one of main as well as more and more frequent cause of deaths and disabilities, it is very important to search the methods of increasing the natural systemic defensive processes connected with ischemia. Of special importance among such methods are these which are based on the use of native substances.

One of widely used plants with medical properties is primrose {Oenothera). Primrose oil, obtained from seeds of that plant characterizes with high contents of indispensable unsaturated fatty acids, especially γ-linoleic acid. The indispensable unsaturated fatty acids aid in functioning the cardiovascular system, advantageously influence the cholesterol and triglyceride balance as well as strengthen the natural organism immunity.

In the process of manufacturing the oil from primrose seeds oil cake is obtained as a by-product, namely partly defatted seeds. It was found that oil cake of primrose is a valuable source of polyphenols, that results in increased interest of their use in pharmaceutical industry.

Health-promoting activity of polyphenols and their special role in preventing atherosclerosis and heart diseases is well known. Polyphenols contained in red wine, with dominating role of quercetin, resveratol, catechin and epigallocatechin demonstrate broad spectrum of activity in the area of cardiovascular system: they decrease the clot formation, inhibit inflammatory processes in blood vessels, increase their relaxation as well as improve the blood flow and oxygen transport to heart. Simultaneously they inhibit dangerous free-radical reactions. Polyphenols included in red wine can lower the oxidation of LDL cholesterol and therefore prevent unfavorable atherosclerotic reactions. In turn extracts of hawthorn demonstrate the activity similar to that of phosphodiesterase HI inhibitors or cardiac glycosides, relaxing activity on coronary and peripheral circulation, however without arrhythmogenic activity. Additionally, they demonstrate antioxidant and anti-inflammatory activity. Hawthorn inflorescences, which together with leaves form the pharmaceutical substrate, include such polyphenols as flavonoids, as well as oligomeric procyanidins, mainly dimeric procyanidin B2, trimeric procyanidin Cl and accompanying catechin and epicatechin. The composition of (dry) hawthorn extracts may vary between one another, but most frequently they contain 2,2% flavonoids and up to about 19% procyanidins. The known source of plant polyphenols is also an extract of the bark of mediterranean pine, containing the whole class of flavonoids, flavan-3-ol derivatives. The extract contains catechin, epicatechin, taxifolin as well as procyanidins. Many of above mentioned polyphenols compounds are found also in other plants, such as berry fruits and tea, especially green one.

Plant extracts containing polyphenols compounds may significantly differ one another due to different composition of polyphenols component. Generally, they contain certain basic group of polyphenols compounds, but there are also significant differences between them, significantly influencing the properties of extracts. The activity of such compounds depends on their bioavailability, strictly connected with their chemical structure— for example the antioxidative activity of polyphenols contained in tea is ascribable mainly to catechins, and in red wine - to reservatrol. Therefore the proposed uses of polyphenols derived from different plants are differ, while the use in the field connected with diseases of cardiovascular system are generally based on their antioxidative

properties.

The antioxidative use of the extract of defatted primrose seeds is known from the description of patent application WO 0018416. From the series of patent applications of

Oriza company there is known the use of said extract as: cell apoptosis inducing agent (JP 20010105658), inhibitor of fats absorption (EP 1312374), Helicobacter pylori controlling agent (JP 20030151745), urease inhibitor (JP 20030339266), agent for treatment of ulceration (JP20040118419). In the Polish patent application P-370446 it was proposed to use polyphenols from primrose in treatment and prevention of osteoporosis. In all of the above mentioned applications there is used a product obtained by extraction of defatted primrose seeds with aliphatic alcohols, acetone, water or mixtures of such solvents

Our research demonstrated that the extract of defatted primrose seeds exhibits the activity of ACE inhibitor and simultaneously neutral endopeptidase inhibitor.

The gist of invention lies in use of the extract of defatted primrose seeds to produce the agent for prevention or limitation of myocardium and coronary vessels damage.

The use of the primrose extract to prepare the formulation for prevention of limitation the process of ischemic-reperfusion damage of myocardium is especially preferred.

It is preferred to use of the extract from defatted evening primrose seeds (Oenothera paradoxa) .

The total fraction of polyphenols compounds is preferably not less than 40% counted as catechin.

The total fraction of proantocyanidin in the extract is preferably at least 30%, counted as cyanidin chloride. In the use according to the invention the extract from defatted primrose seeds is applied in the form of pharmaceutical or dietary formulation, containing, beyond the extract, also auxiliary substances which are pharmaceutically acceptable i/lub used as food additives. The formulation may be in the form suitable for oral administration, especially in the form of tablets, pills, dragees, capsules, powder, granulate, suspension. The formulation designed for use according to the invention may contain

additionally over active substances used in cardioprotection of myocardium and coronary vessels.

The examination of the composition of extracts from defatted evening primrose seeds demonstrate, the contain Ia. ellagic acid, gallic acid, catechin, epicatechin gallate and procyanidins, (i.a. proantocyanidin B3), oenothein B and penta-O-galloyl-β-D-glucose (PGG). PGG, beyond the procyanidin with different polymerization degree, is the dominating compound in the extract from defatted evening primrose seeds and plays an important role w in its therapeutic activity.

The dual action of the extract of primrose seeds, as ACE inhibitors and neutral endopeptidase inhibitors, increases the level of natriuretic (sodiuretic) peptides and vasodilator and, simultaneously, inhibits the renine-angiotensin-aldosteron system. The inhibition of bradykinin degradation allows to retain its concentration on such level, which assures the cardioprotective action on myocardium and coronary vessels.

Extract primrose seeds is obtained from defatted or partly defatted seeds. The defatting effect is obtained by extruding the mature seeds on hydraulic presses under the pressure of several hundreds atmospheres. Such process removes approximately 60% of oil and damages the seed hulls, therefore forming the favored conditions for the penetration of the inside of hull by the solvent in extraction process. Said process is performed at the temperature not exceeding 4O ⁰ C. Subsequently, the defatted seeds are subjected to extraction process with aliphatic alcohol, preferably ethanol, preferably in the mixture with water. The obtained extracts are concentrated, filtered and spray-dried. The product is obtained in powder form.

Extract from defatted primrose seeds has been tested for activity against enzymes, angiotensin-converting ACE and neutral endopeptidase NEP. To this end the sub-extracts were prepared from the extract, which were subsequently separated into fractions, and the compounds were isolated from most active fractions. The test were performed on fractions, sub-extracts and compounds. The tests demonstrated the distinct inhibition of vasopeptidase in a concentration-dependent manner. The HPLC analysis showed the similar qualitative composition of active sub-extracts, with the minor quantitative differences in the sum of polyphenols compounds. The most active compounds appeared

to be oenothein B, procyanidin B3, penta-O-galloyl-β-D-glucose (PGG) and in minor extend methyl gallate. In one of the most active fractions procyanidins were found, the monomers of which are catechin and epicatechin gallate. Procyanidins and PGG determine in a great manner the activity of the whole sub-extract, and the other polyphenols compounds present may act synergistically.

The subject of invention and the performed research confirming the activity of extracts from primrose in the use according to the invention have been closer presented in working examples. Example 1. Preparation of crude extract.

The mature seeds of evening primrose are extruded in hydraulic press, at the temperature not exceeding 4O ⁰ C. The defatted seeds are put in extractor and then flooded with 60% ethanol at 25 - 3O ⁰ C, in an amount of 2-2,5 volumes of seeds. The contents of extractor is leaved for 1 without stirring, and then the alcohol extract is drained from the bottom. The extractor is re-filled with 1 volume of water of the temperature 25 - 3O ⁰ C is leaved for 1 hour, and the aqueous extract is drained from the bottom of apparatus. The extraction with water is repeated four times. The aqueous extracts are pooled the further treatment is performed. Extraction residue is removed from the extractor and sent to the utilization. As a result of extraction process it is obtained approximately 1 volume of alcoholic extract and approximately 4 volumes of aqueous extracts. The next process step is to remove the alcohol from extracts and concentration of the solution of polyphenols. Such process is performed in vacuum evaporator. Alcoholic extracts are distilled separately, without mixing with aqueous extracts. Alcoholic extract is concentrated twice, whereas aqueous extracts 3-4 times.

The alcohol distillates obtained in the process are standardized and reused in the extraction of novel portion of seeds. The decoctions obtained during distillation of extracts are leaved to cool and then they are freed from sediments by centrifuging and optionally filtration. Clear polyphenol solutions are spray-dried. As a result of the described process one obtains polyphenolic extract in the form of

powder, containing at least 40% polyphenols, counted as catechin, of the following composition: procyanidins, including procyanidin B3, 1,2,3,4,6-O-penta-O-galloyl-β-D- glucose, catechin, epicatechin, epicatechin gallate, gallic acid, methyl gallate, ellagic acid, caffeic acid, quercetin, quercetin glucuronide, oenothein B. The total amount of polyphenols was above 40%, and a total amount of procyanidins was above 30%. Example 2

Preparation of sub-extracts and fractions.

20 g of polyphenols extract from Oenothera paradoxa in the powder form, obtained according to example 1, was extracted with: (a) water (2x100 mL), (b) 60% ethanol (v/v, 2x100 mL), (c) isopropanol (2x100 mL), (d) 30% isopropanol (v/v, 2x100 mL), for 60 minutes, at room temperature. After drying of isopropanol solution (c) under vacuum w 4O ⁰ C 2,36 g of residue was obtained. Aqueous solution (a) and aqueous residues after evaporation of solvents from organic solutions (b) and (d) were lyophilized. It was obtained 1,93 g of product from solution (a), 2,58 g product from solution (b) and 2,05 g product from solution (d).

20 g of 30% isopropanol solution was dissolved in water (200 mL) and extracted with ethyl acetate (3x200 mL). The combined extracts were dried in vacuum at 45 ⁰ C, resulting 3,2g of residue. The remaining aqueous solution was lyophilized giving 16,2 g of the product. 2,5 g of the above mentioned extract in ethyl acetate (EA) was dissolved in 10 mL of methanol, absorbed on 5 g of polyamide, applied on the column (diameter 5 cm, height 8 cm), packed with polyamide 6 (particle size 0.05-0.16 mm, Carl Roth) and eluded with methanol (50 mL fractions were collected) and with the solution acetone-water (7:3; 50 mL fractions were collected). 100 fractions were collected, which were pooled into main fractions 1-6A on the basis of their polyphenol composition, evaluated by thin-layer chromatography TLC. All fractions were lyophilized.

7.5 g of the aqueous residue (AR) after extraction with ethyl acetate was dissolved in 40 mL of the mixture methanol-water (7:3), applied on the column (of diameter 4 cm and height 40m) packed with Sephadex LH-20 (particle size 0.025-0.100 mm, Pharmacia, Uppsala, Sweden) and eluted with the solution methanol-water (7:3; 50 mL fractions were

collected). 40 fractions were collected, which were pooled into main fractions 1-5B on the basis of their polyphenol composition, evaluated by thin-layer chromatography TLC. All fractions were lyophilized.

Sub-extracts and fractions were stored at the temperature -18 ⁰ C. The obtained fraction were tested for biological activity. The results were presented on the drawing, in which fig. 1 presents testing of the ethyl acetate solution and fractions 1- 6 A, and fig. 2 presents the results of testing the aqueous residue and fractions 1-5 B.

Example 3

Determination of the contents of polyphenols compounds and proantocyanidin in sub-extracts obtained according to example 2.

The total contents of polyphenolic compounds was determined by Folin-Ciocalteu method, counting as catechin. 0.05g of each sub-extract was dissolved in 25 mL of the solution methanol-water (7:3), and then 0.2 mL of each sub-extract was mixed with 0.5 mL of Folin-Ciocalteu reagent and 10 mL of 10% solution Of Na ₂ CO ₃ and made up to 50 mL with water. The mixture was leaved in darkness for 30 min. Absorbency was measured spectrophotometrically at 700 nm.

The total contents of proantocyanidin was determined according to European Pharmacopoeia IV as cyanidin chloride. 0.2 g of each sub-extract was dissolved in 30 mL of the solution methanol-water (7:3) refluxed for 80 min with 15 mL of 25% HCl. After cooling, the extracts were filtered and made up with the solution methanol-water (7:3) up to 250 mL. 50 mL of each solution was evaporated to approximately 3 mL and added to 15 mL of water. Subsequently the solution was extracted with butanol (3x15 mL). Organic layers were transferred to measuring flask and made up with butanol up to 100 mL. Absorbency was measured spectrophotometrically at 545 nm. The total contents of polyphenols and proantocyanidin in tested sub-extracts was presented in Table 1.

Example 4.

Determination of angiotensin convertase (ACE) and neutral endopeptidase (NEP) activity in sub-extracts obtained according to example 2. Determination of angiotensin convertase (ACE) activity Enzymatic reaction

consisted in cleavage the added substrate Hip-L-His-L-Leu (hippuryl-histydyl-leucine) by ACE enzyme with the formation of product (histydyl-leucine), and subsequently forming with o-phthalaldehyde the fluorizing complex His-Leu-o-phthalidyl, which is determined spectrofluorimetrically. 50 μl of tested solution (solution of lyophilized sub-extract/compound in phosphate buffer), 30 μl phosphate buffer (pH=8,3), 150μl of enzyme solution (1 :300 hog sperm) and 20 μl of substrate (Hip-L-His-L-Leu 24 mM). The reaction mixture was incubated for 30 min. at 37 ⁰ C, and the reaction was inhibited by adding 1000 μl of 0,4M NaOH and thereafter 100 μl of 2% methanolic solution of o-phthaldialdehyde and the mixture was leaved in darkness for 10 min. to generate the fluorizing complex. Subsequently 300 μl of 2M HCl was added and the mixture was leaved for 30 min. in darkness. Fluorescence of the generated product was measured spectrofluorimetrically at λ _eXC = 365 nm and λ _em j _s ⁼ 500 nm with the slot width of 3 nm.

Control test without enzyme was performed to eliminate the fluorescence shown by tested solution itself as well as positive control test without added inhibitor. Determination of neutral endopeptidase (NEP) activity.

Tests for NEP activity were performed in two steps. The enzymatic reaction in the first step in enzymatic cleaving of SAAP-AMC (succinyl-alanyl-alanyl-phenylalanyl-7- amido-4-methylcoumarin) substrate and forming phenylalanyl-7-amido-4-methylcournarin (P-AMC). In the second step of the reaction the added enzyme APN (aminopeptidase N) splits off phenylalanine with liberation fluorescence emitting compound.

50 μl of tested solution (solution of lyophilized sub-extract/compound in HEPES buffer), 50μl 8μM of lizinopril (to inhibit the ACE activity), 50 μl of substrate (400 μM), 350μl HEPES buffer, pH=7,4 and 150 μl of enzyme solution (1:1000 hog sperm)was added to Eppendorf tubes. The reaction mixture was incubated for 1 hour at 37 ⁰ C, and thereafter the reaction was inhibited by adding 50μl of phosphoramidone (50 μM). Subsequently 20 μl solution APN (1:235) was added, the mixture was incubated for 1 hour at 56°C, the reaction was inhibited with 800 μl of acetone. Fluorescence of the formed product was measured spectrofluorimetrically at λ _eXC = 367 nm and λ _em i _s ~ 440 nm with the slot width of 3 nm.

Control test without enzyme was performed to eliminate the fluorescence shown by tested solution itself and control test to eliminate the influence of inhibitor for APN as well as positive control test without added inhibitor.

Statistical analysis.

The determination for each concentration was performed three times in two duplicates, and the results were presented as mean value ± standard deviation (SD). The values IC _5Oi that is the sub-extract concentration inhibiting the enzyme activity by 50%, was read from the curve of the dependence concentration-inhibition percent.

The results of activity tests were presented in Table 1.

Table 1.

Total amount of polyphenols and procyanidins w tested extracts and their ability to inhibit the activity of vasopeptidase w in concentration 25μg/ml, IC ₅₀ values given in brackets.

n.in.- no inhibition (-)- not tested

Example 5

Isolation of active compounds

Ethyl acetate fraction 5A (225 mg) was loaded on the column (diameter 2,5 cm, height 35 cm) packed with Toyopearl HW-40, (Tosoh, Tokyo, Japan) and elated with the solution acetone- water(7:3) (10 mL 50 mL fractions were collected). Fractions 7-9 (5A)

were re-separated once again on the same column, eluting with solution acetone-water (4:6) (10 mL each). 53 g of 1,2,3,4,6-O-penta-O-galloyl-β-D-glucose was obtained (compound 1).

Aqueous fraction 2B (70 mg) was loaded on the column (diameter 2,5 cm, height 20 cm) packed with Toyopearl HW-40, (Tosoh) and eluted with the solution methanol- water (7:3) (10 mL fractions were collected). 10 mg of methyl gallate (compound 2) was obtained.

Aqueous fraction 4B (150 mg) was loaded on the column (diameter 2,5 cm, height 20 cm) packed with Toyopearl HW-40, (Tosoh) and eluted with the solution methanol- water (7:3) (10 mL fractions were collected). 31 mg of (+)-catechin (4α-→8)-(+)-catechin (Procyanidin B3, compound 3).

1,2,3,4,6-O-penta-O-galloyl-β-D-glucose (compound 1): off-white powder; UV, Kn _3x 281 nm; ESI-MS (positive ions) m/z 963.1 [M + Na] ⁺ ; ¹ H NMR (MD ₃ OD) Glucose fragment: δ 6.46 (IH, d, J=8Hz, H-I), 6.20 (IH, t, H-3), 5.85 (IH, m, H-4), 4.78 (IH ₃ m, H-5), 4.51 (IH, 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-I), 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 (signals of carbonyl group), 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-I), 110.63, 110.48, 110.42, 110.40, 110.35 (C-2, C-6).

Methyl gallate (compound 2): light brown powder; UV, λmax 273 nm; ESI-MS (positive ions) m/z 207.0 [M + Na] ⁺ ; ¹ H NMR (MD3OD): δ 6.96, (2H, s, H-2, H-6), 3.75 (3H, s, -OCH ₃ ). ¹³ C NMR (MD3OD): 169.2 (-COOH), 144.8 (C-3, C-5), 138.5 (C-4), 120.7 (C-I), 110.0 (C-2, C-6), 52.7 (-OCH ₃ ). (+)-catechin (4a→8)-(+)-catechin (Procyanidin B3) (compound 3): light brown powder; UV, 7^ _13x 236 nm, 279 nm; ESI-MS (positive ions) m/z 601.1 [M + Na] ⁺ ; thiolysis shows the presence of(+)-catechin and thioether of (+)-catechin; ¹³ C NMR (MD30D): 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-l'u, C-l't), 120.09 (C-6'u), 119.43 (C-6't), 116.13, 115.94, 115.39, 1 15.31 (C-2'u, C-2't, C-5'u, C-5't), 110.09 (C-8t), 100.87 (C-IOu, C-IOt), 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 analysis of active compounds was performed with the use of following methods. Thin-layer chromatography (TLC).

Thin-layer chromatography TLC was performed on silica gel 60 F ₂₅₄ (layer thickness 0.25 mm) and on polyamide 11 F ₂₅₄ (Merck KgaA), Chromatograms were developed in horizontal chambers (Chromdes, Lublin, Poland).

Polyphenol detection: TLC tests were performed on polyamide, with using 60% CH ₃ COOH as eluent or on silica gel using CHCl ₃ -EtOAc-HCOOH (5:4: 1) as eluent. Spots corresponding to compounds were visualized with UV light UV _254J66 and by sprinkling of chromatograms with 5% methanolic solution of FeCl ₃ (navy-blue spots) or with 1% solution of vanillin in H ₂ SO ₄ (pink spots).

High performance liquid chromatography (HPLC). Polyphenol composition of particular sub-extracts and fractions was evaluated by

HPLC with diode detector. Separation was performed on the column Luna C- 18, 25x4.6 mm, 5 μm (Phenomenex, Torrance, CA). The mobile phase was: (A) 2,5% CH ₃ COOH and (B) CH ₃ CN + 2,5% CH ₃ COOH (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 registered for wavelength 200 to 400 run, chromatograms were registered for wavelength 280 and 350 nm.

Samples were dissolved in the mixture of MeOH + 2,5% CH ₃ COOH (1:1) to concentrations 10; 5 or 2.5 mg mL ^"1 .

Thiolysis. The thiolysis of procyanidins was performed in order to establish the qualitative composition of oligomers and polymers. Assays were performed in the following manner: samples were dissolved in methanol (50 μl) and 50 μl of 3.3% hydrochloric acid w methanol and 100 μl of 5% benzyl mercaptan in methanol were added. The mixture was incubated at 40 ⁰ C for 30 min. The monomelic products of procyanidin decomposition was analyzed by HPLC.

The isolated compounds were tested for angiotensin convertase (ACE) and neutral endopeptidase (NEP) activity with using the methods as in example 4, however with preparing the solution of compound in phosphate buffer in the determination of angiotensin convertase (ACE) activity, and in HEPES buffer in the determination of neutral endopeptidase activity. The results are presented in table 2.

Table 2.

Inhibition of vasopeptidase activity with polyphenols at the concentration of 100 μg/ml, IC ₅₀ values (μM) given in brackets

n.in.- no inhibition (-)- not tested