Oleacein for treating or preventing diseases resulting from atherosclerotic plaques

Abstract

A description and explanation is provided of the use of oleacein, especially this obtained from Ligustrum vulgare L., in the manufacturing of a preparation for the treatment and prevention of diseases that are consequences of the atherosclerotic plaque degradation. Such diseases include in particular those selected from the following group: ischemic brain stroke, heart attack and ischemic heart disease.

Claims

1. A method for inhibiting MMP-9 production by atherosclerotic plaque cells in a patient, said method comprising administering oleacein to said patient, wherein said oleacein is obtained from Ligustrum vulgare L.

2. The method of claim 1, wherein said inhibiting stabilizes atherosclerotic plaque and causes a change in phenotype of existing macrophages from pro-inflammatory M1 to anti-inflammatory M2.

3. The method of claim 1, wherein said oleacein is an extract.

4. The method of claim 1, wherein said oleacein is an aqueous extracted oleacein.

Description

EXAMPLE 1

Obtaining Oleacein from the Leaves of Wild Privet (Ligustrum vulgare L.)

(1) Wild privet (Ligustrum vulgare L.) is a decorative plant growing in Europe, often used for hedgerows. The isolation of oleacein contained in the leaves of wild privet was performed using a modification of the method by Kiss et al. (Journal of Ethnopharmacology 120 (2008) 220-225) in the Faculty of Pharmacognosis and Molecular Foundations of Phytotherapy of the Warsaw Medical Academy).

(2) For the isolation, we used 400 g of degraded raw material. In the first stage, privet leaves were extracted four times with distilled water at a temperature of 30 C. in an ultrasound bath, for 30 minutes each time. The extraction was performed at a ratio of 1:10 raw material to solvent. Aqueous extracts were filtered through wool and pooled. The resulting aqueous extract was concentrated through lyophilisation to a volume of about 11. In order to obtain a less contaminated extract and to increase the efficiency of the extraction process of the desirable compound, we used diethyl ether instead of ethyl acetate. Earlier analyses had shown that an ethyl acetate extraction contains more chemical compounds, thereby making the isolation of oleacein more difficult. Consequently, we then subjected the condensed aqueous extract to a 5-fold diethyl ether extract at a ratio of 1:1 solvent to extract. The ether extract was concentrated on a rotary evaporator under reduced pressure, at a temperature of 35 C. We obtained about 5 g of ether extract. The next modification was based on the use of an apparatus and reagents in an isocratic system. According to Kiss et al. (2008), purification of the ethyl acetate extract consisted of the use of a column filled with silica gel (0.125-0.25 mm; 5.5 cm10 cm) using a solvent gradient of chloroform-ethyl acetate (100-0%) as well as ethyl acetate-methanol (100-0%). In the modified method, the ether extract was separated using flash chromatography in a column filled with silica gel (PF-30 SIHP/80G PuriFlash) using a mixture of chloroform and ethyl acetate (85:15) in an isocratic system for 60 minutes (20 ml/min). We obtained 9 fractions, from which fractions 3 and 4 were selected for subsequent separation. These fractions were further separated on a column filled with silica gel (PF-30 SIHP/80G PuriFlash) using a mixture of toluene, methyl acetate and methanol (84:11:5) in an isocratic system for 60 minutes (20 ml/min). We obtained 5 fractions, and from fraction 3, loaded onto a column filled with sephadex, we isolated oleacein using a mixture of chloroform and methanol (9:1), like in the original method. Thus we obtained 1.289 g of the compound. Identity of the compound was confirmed using NMR (See FIG. 1) as well as HPLC-DAD-MS/MS (See FIG. 2).

(3) TABLE-US-00001 Isolation stage Original Method Modified Method 1st extraction 4x water; 4x water; ultrasonic bath; 30 min ultrasonic bath; 30 min 2nd extraction 5x ethyl acetate (1:1) 5x diethyl ether (1:1) 1st separation Water; glass column (0.125-0.25 mm; Water (PF-30 SIHP/80G 5.5 cm 10 cm); gradient PuriFlash); flash-type chloroform-ethyl acetate (100-0%) chromatography; isocratic system and ethyl acetate-methanol (100-0%) of chloroform and ethyl acetate (85:15) 2nd Water (PF-30 SIHP/80G separation PuriFlash); flash-type chromatography; isocratic system of chloroform and ethyl acetate (85:15) Final Sephadex; glass column (2 25 cm); Sephadex; glass column (2 45 cm); purification isocratic system of isocratic system of chloroform-methanol (9:1) chloroform-methanol (9:1)

EXAMPLE 2

Stabilization of Atherosclerotic Plaque Isolated from Carotid Artery

(4) Atherosclerotic plaques (n=15) were obtained from patients subjected to endoartherectomy. The plaque obtained was divided into two portions, which were then incubated in buffered physiological saline (control), or buffered physiological saline in the presence of oleacein at the concentrations of 5, 10 and 20 M for 24 hours at a temperature of 37 C. following prior stimulation with a solution of lipopolysaccharide at a concentration of 1 g/ml. The unstimulated control consisted of physiological saline, in which the plaque was incubated for 2 hours. The effect of oleacein on metalloproteinase-9 (MMP-9) production was determined using immunochemical ELISA (R&D System). The amount of MMP-9 (ng/ml) was calculated per mass of atherosclerotic plaque, and then described in terms of percentage in comparison to the stimulated control. IC.sub.50, i.e. concentration of the compound at which reaction is inhibited by 50%, was determined at a level of 9.071.2 M (averagestandard error).

(5) The graph included in FIG. 1 shows the dependence of MMP-9 production [%] on respective oleacein concentrations (5, 10, 20 M).

(6) The experimental results demonstrate the ability of oleacein to inhibit the production of MMP-9 by the cells contained in atherosclerotic plaque. The lowering of MMP-9 production stabilizes atherosclerotic plaque and retards its degradation. In connection with the effect obtained, oleacein may be used in the treatment and prophylaxis of diseases that are consequences of the degradation of atherosclerotic plaque, in particular ischemic brain stroke, heart attack as well as ischemic heart disease.

EXAMPLE 3

Reduction of Atherosclerotic Plaque Inflammation

(7) The effects of olacein on interleukin 10 (IL-10) production were investigated with the use of the ELISA immunochemical method (R&D system). The IL-10 amounts were converted into the atherosclerotic plaque mass and, subsequently, determined in % compared to the stimulated control. The investigations confirmed increased, olacein-induced IL-10 production up to about 267.161.5%.

(8) In diseases like diabetes, atherosclerosis and other pathological conditions in a human body, there is no change in the CD163 receptor expression in macrophage cells due to haemoglobin-haptoglobin complexes, which prevents from a change in the phenotype of these cells. The pro-inflammatory macrophages have a destabilising effect on the atherosclerotic plaque and cause microbleeding.

(9) Oleacein-haemoglobin (OC+Hb) complexes induce the expression of the macrophage scavenger receptor CD163, which leads to a change in the macrophage phenotypes from the pro-inflammatory M1 form to the anti-inflammatory M2 type. This process significantly affects the atherosclerotic plaque stabilisation in human arteries.

(10) The changes in the macrophage receptor CD163 expression, resulting from macrophage cell stimulation by various agents, were assayed with the use of flow BD FACSCalibur flow cytometer. The findings are presented in the FIG. 2. Symbol description: 1Kcontrol, 2Hbhaemoglobin, 3Hphaptoglobin, 4OC 10oleacein in 10 M, 5OC 20oleacein in 20 M, 6OC 10+Hbhaemoglobin-olacein complex in 10 M, 7OC 20+Hbhaemoglobin-olacein complex in 20 M, 8Hb+Hp 1-1haemoglobin-1-1 haptoglobin complex, 9OC 10+Hb+Hp 1-1olacein-haemoglobin-1-1 haptoglobin complex in 10 M, 10OC 20+Hb+Hp 1-1olacein-haemoglobin-1-1 haptoglobin complex in 20 M, 11Hb+Hp 2-2haemoglobin-2-2 haptoglobin complex, 12OC 10+Hb+Hp 2-2olacein-haemoglobin-2-2 haptoglobin complex in 10 M, 13OC 20+Hb+Hp 2-2olacein-haemoglobin-2-2 haptoglobin in 20 M.