PREPARATION FOR USE AS ANTIOXIDANT

Abstract

A composition for use as antioxidant, where the composition has at least one polyunsaturated fatty acid component and at least one anthocyanin component. The polyunsaturated fatty acid component is an amino acid salt of the omega-3 fatty acids eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). The anthocyanin component is cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-galactoside, peonidin-3-galactoside, or malvidin-3-glucoside. A method for treating a disease with the composition. A method of providing an antioxidant to a subject with the composition.

Claims

1. A composition for, wherein the composition comprises: at least one polyunsaturated fatty acid component of an amino acid salt of eicosapentaenoic acid (EPA) and an amino acid salt of docosahexaenoic acid (DHA); and at least one anthocyanin selected from the group consisting of cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-galactoside, peonidin-3-galactoside, and malvidin-3-glucoside.

2. The composition according to claim 1, wherein the amino acid salts have at least one organic counter ion selected from the group consisting of lysine, arginine, ornithine and mixtures thereof.

3. The composition according to claim 1, wherein the composition further comprises at least one fruit or fruit extract selected from the group consisting of black currants, bilberries, blackberries, elderberries, sweet cherry, Saskatoon berries, and wild blueberries.

4. The composition according to claim 3, wherein the composition further comprises at least one fruit or fruit extract selected from the group consisting of bilberries and wild blueberries.

5. The composition according to claim 1, wherein the composition further comprises an extract of black currants and bilberries.

6. (canceled)

7. A composition comprising: an amino acid salt of, eicosapentaenoic acid (EPA) and an amino acid salt of docosahexaenoic acid (DHA); and at least one anthocyanin selected from the group consisting of cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-galactoside, peonidin-3-galactoside, and malvidin-3-glucoside.

8. The composition of claim 7, wherein the amino acid salts have at least one organic counter ion selected from the group consisting of lysine, arginine, ornithine and mixtures thereof.

9. The composition according to claim 7, wherein the composition further comprises at least one fruit or fruit extract selected from the group consisting of bilberries and wild blueberries.

10. The composition according to claim 9, wherein the composition further comprises the fruits or fruit extracts of bilberries and wild blueberries in a ratio (weight-%) between 10:1 and 1:1.

11. A method of treating a disease, comprising: administering the composition of claim 1 to a patient in need thereof, wherein the disease is cardiovascular disease, atherosclerosis, hypertension, stroke, diabetes-related cardiovascular disfunction, ischemia/reperfusion injury, hypercholesterolemia, coronary artery disease, or chronic obstructive pulmonary disease (COPD).

12. A method of providing an antioxidant to a subject, comprising: administering the composition of claim 1 to a subject in need thereof.

Description

WORKING EXAMPLES

[0048] Materials:

[0049] The omega-3 lysine salt (AvailOm®) was obtained from Evonik Nutrition & Care GmbH, Darmstadt (Germany) and contains around 32 weight-% of L-lysine and around 65 weight-% of polyunsaturated fatty acids. The major polyunsaturated fatty acids in the composition are the omega-3 fatty acids Eicosapentaenoic acid (C20:5w3c) (EPA) and Docosahexaenoic acid (C22:6w3c) (DHA), summing up to around 58 weight-% of the composition. The composition also contains minor amounts of Docosaenoic acid isomer (incl. erucic acid) (C22:1), Docosapentaenoic acid (C22:5w3c) and of the omega-6 fatty acids Arachidonic acid (C20:4w6) and Docosatetraenoic acid (C22:4w6c). The single ω-3 Fatty Acids (ω-3 FA) and L-Lysin were obtained from Evonik Nutrition & Care GmbH, Darmstadt (Germany), the ω-3 Ethyl Ester (ω-3 EE) were obtained from Solutex GC S. L., Madrid (Spain). oxLDL has been acquired from Thermo Fisher. All the inhibitors, powders and solvents necessary for the preparation of the buffers were purchased by Sigma-Aldrich.

[0050] Healthberry 865® (HB) is a dietary supplement consisting of 17 purified anthocyanins (all glycosides of cyanidin, peonidin, delphinidin, petunidin, and malvidin) isolated from black currant (Ribes nigrum) and bilberries (Vaccinium myrtillus) and was obtained from Evonik Nutrition & Care GmbH, Darmstadt (Germany). The major anthocyanins contained in the berry extract used are cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-galactoside and delphinidin-3-galactoside. The amount of anthocyanin citrate is at least 25 weight-% of the composition. The composition is prepared from black currants and bilberries by a process comprising the steps of alcoholic extraction of black currants and bilberries, purification via chromatography, mixing of the extracts with maltodextrin citrate and water and spray-drying of the mixture. The product composition contains extracts of black currants and bilberries mixed in a weight ratio of around 1:1.

[0051] The single anthocyanins, Delphinidin-3-rutinoside (D3-rut), Cyanidin-3-rutinoside (C3-rut), Delphinidin-3-glucoside (DP3-glu), Cyanidin-3-glucoside (C3-glu), Petunidin-3-glucoside (PT3-glu), Delphinidin-3-galactoside (DP3-gal), Peonidin-3-galactoside (PEO3-gal), Delphinidin-3-arabinoside (DP3-ara), Malvidin-3-galactoside (MAL3-gal), Malvidin-3-glucoside (MAL3-glu), Cyanidin-3-galactoside (C3-gal), Cyanidin-3-arabinopyranoside (C3-arapy) were obtained from Polyphenols AS, Sandnes (Norway).

[0052] Experimental Animals

[0053] All experiments involving animals were conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were approved by review board. Wild-type C57BL/6 mice (weighing— 25 g) (Jackson Laboratories, Bar Harbor, Me., USA) have been used to perform vascular reactivity and molecular studies.

[0054] Vascular Reactivity Studies

[0055] Second-order branches of the mesenteric arterial tree were removed from mice to perform vascular studies. Vessels were placed in a wire or pressure myograph system filled with Krebs solution maintained at pH 7.4 at 37° C. in oxygenated (95% O.sub.2/5% CO.sub.2). First, an analysis of vascular reactivity curves was performed. In particular, vasoconstriction was assessed with 80 mmol/L of KCl or with increasing doses of phenylephrine (from 10.sup.−9 M to 10.sup.−6 M) in control conditions. Endothelium-dependent and-independent relaxations were assessed by measuring the dilatory responses of mesenteric arteries to cumulative concentrations of acetylcholine (from 10-9 M to 10-6 M) or nitroglycerine (from 10-9 M to 10-6 M) respectively, in vessels precontracted with phenylephrine at the dose necessary to obtain a similar level of precontraction in each ring (80% of initial KCl-evoked contraction). Caution was taken to avoid endothelial damage; functional integrity was reflected by the response to acetylcholine (from 10.sup.−9 M to 10.sup.−6 M).

[0056] Vascular responses were then tested administering increasing doses of Healthberry 865®-865 or single anthocyanins. Some experiments were performed in presence of selective inhibitors, such as phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor (LY274002, 10 μM, 1 h), Akt inhibitor (Akt inh, 1 μM, 1 h) or the NOS inhibitor N-w-nitro-1-arginine methyl ester (L-NAME, 300 μM, 30 min) before data for dose-response curves were obtained.

[0057] Evaluation of NO Production by DAF

[0058] Production of NO was assessed as previously described (Carrizzo et al. 2016). AvailOm® (100 μg/mL) or acetylcholine (10-6 M) was administered to the mesenteric artery in the last 30 min of 4-amino-5-methylamino-2,7,-difluorofluorescein diacetate (DAF-FM) incubation, alone and after 20 min exposure to L-NAME (300 umol/L, 30 min). Mesenteric segments were cut in 5-μm thick sections, observed under a fluorescence microscope, subsequently counterstained with haematoxylin and eosin and observed under a light microscope.

[0059] Analysis of Total ROS Production

[0060] Dihydroethidium (DHE, Life Technologies) was used to evaluate production of reactive oxygen species (ROS) in mouse mesenteric arteries, as previously described. Briefly, vessels were incubated with 5 μM of DHE for 20 min and subsequently observed under a fluorescence microscope (Zeiss). Images were acquired by a digital camera system (Olympus Soft Imaging Solutions). A second, estimation of total ROS production in mouse vessels was performed with the membrane-permeable fluorescent probe an analog of 2,7-Dichlorodihydrofluorescein (DCDHF), Dihydrorhodamine 123 (DHR123) (Invitrogen). After treatment, vessels were incubated with Krebs solution containing 5 μM DHR123 for 30 min at 37° C., and then washed two times with PBS prior to fluorescence measurement using a fluorescence microplate reader (TECAN infinite 200 Pro).

[0061] Statistical Analysis

[0062] Data are presented as mean±SEM. Statistical analysis was performed by 2-way ANOVA followed by Bonferroni post hoc test. Repeated measurements were analysed by One-way ANOVA followed Bonferroni post-hoc test. Differences were considered to be statistically significant at p<0.05.

Example 1: AvailOm® Evokes a Direct Vasorelaxant Action on Mice Mesenteric Arteries

[0063] To assess the possible direct vascular action of AvailOm®, vascular reactivity studies on mice vessels were performed, administering increasing doses of AvailOm® (5-300 ug/mL) on pre-constricted mice mesenteric arteries, considering the concept that alteration of vascular response of resistance arteries reflects in an important contribution to the development of cardiovascular complications. The data demonstrate that AvailOm® exerts a direct dose-response vasorelaxant action (FIG. 1A). This effect is due to the stimulation of nitric oxide production, since the inhibition of eNOS enzyme, by L-NAME, completely abolishes this effect (FIG. 1B). Considering one of the major enzymes involved in eNOS activation, its vascular action in presence of phosphoinositide 3-kinase (PI3K) inhibitor was assessed, demonstrating that this mechanism is not involved in its direct vascular action (FIG. 1C). Interestingly, in presence of selective AMPK inhibitor, dorsomorphin, AvailOm® completely loses its capability to evoke endothelial-dependent vasorelaxation (FIG. 1D). Study performed in absence of endothelial layer demonstrate that endothelium represents the main target of the compound (FIG. 1E). Assessment of vascular response to L-Lysine did not evoke any vasorelaxant effect (FIG. 1F). This result was similarly to that observed with ω-3-FA (FIG. 1F). In contrast, assessment of vasorelaxant properties of ω-3-EE was able to induces a dose-dependent vasorelaxation, however, the effect shown for AvailOm® was tendentially stronger (FIG. 1F).

[0064] FIG. 1 shows in A-D) vascular response of phenylephrine-precontracted mice vessels to increasing doses of AvailOm® (5-300 μg/mL) (N=5) B) Vascular response of phenylephrine-precontracted mice mesenteric arteries to increasing doses of AvailOm® in presence of L-NAME, C) Wortmannin, D) Dorsomorphin or (E) in vessels with endothelium (e+) and without endothelium (e−). F) Comparison of vasorelaxant effect of AvailOm®, ω3-FA, ω3-EE or L-Lysine. Statistical analyses were performed using two-way ANOVA followed Bonferroni post-hoc test. *p<0.05;**p<0.01, ***p<0.001.

Example 2: AvailOm® Prevents Vascular Oxidative Stress Damage Induced by ox-LDL

[0065] Subsequently the possible effect of AvailOm® on oxidative stress induced by oxLDL was assessed. As reported in FIG. 2, a pre-treatment with AvailOm® (100 μg/mL) of vessels exposed to ox-LDL leads to a significant protection from oxidative stress as showed by endothelial response to ACh (FIG. 2A). Of note, the evaluation of oxidative stress by dihydroethidium, demonstrates a complete protection from oxLDL-evoked oxidative stress (FIG. 2B). Interestingly, the assessment of the protection from oxLDL-evoked vascular oxidative stress, revealed that EE form of ω-3 is the major component that owns the cardiovascular beneficial properties (FIG. 2C-D-E). The qualitative and quantitative assessment of oxidative stress by dihydroethidium and DHR123, respectively, demonstrate that ω-3-EE reproduce a similar effect of AvailOm® alone, however, AvailOm® having a slightly stronger effect to protect from vascular oxidative stress in vitro (FIG. 3A-B).

[0066] FIG. 2 shows in A) vascular response of phenylephrine-precontracted mice mesenteric arteries to increasing doses of ACh (10-9 M to 10-5 M) after exposure to ox-LDL for 30 minutes and to 1 hour to AvailOm (100 μg/mL). B-D) Vascular response of phenylephrine-precontracted mice mesenteric arteries to increasing doses of ACh (10-9 M to 10-5 M) after exposure to ox-LDL for 30 minutes and to 1 hour to L-Lysine, ω3-FA or ω3-EE (100 μg/mL). Statistical analyses were performed using two-way ANOVA followed Bonferroni post-hoc test. *p<0.05; **p<0.01, ***p<0.001.

[0067] FIG. 3 shows in A) representative high-power micrographs of 10 μm sections of mice mesenteric arteries loaded with a dihydroethdium probe at the concentration of 5 μM. Vessels were pre-treated with the single compound (100 μg/mL) for 1 hour and then stimulated with ox-LDL for 30 minutes prior to the acquisition. B) Measurement of ROS production by DHR123 in vessels treated with single compounds. Statistical analyses were performed using one-way ANOVA followed Bonferroni post-hoc test. *p<0.05; **p<0.01, ***p<0.001.

Example 3: AvailOm® in Combination with Most Powerful Anthocyanins Exerts Most Potent Vasorelaxant Effect

[0068] In a next step, the possible vascular action of AvailOm® in combination with different anthocyanins Cyanidin-3-O-galactoside (C3-gal) or C3-gal plus Delphinidin-3-o-arabinoside (DP3-ara) was assessed, maintaining a ratio ½:½, maintaining the same overall amount of the substance to be tested. The data demonstrate that in presence of both C3-gal and C3-gal with DP3-ara, AvailOm® is able to exert a most powerful vasorelaxant effect, which can be seen in a significant improvement of endothelial dependent vasorelaxation at 50, 100 and 150 μg/mL in comparison to AvailOm® alone (FIG. 4A). This shows that the effect is not just additive, but has a clear synergy between the omega-3 fatty acid salt and the anthocyanins used.

[0069] The assessment of nitric oxide production by DAF-FM revealed both in presence of C3-gal and C3-gal with DP3-ara a significant improvement of NO production in comparison to AvailOm® or C3-gal alone (100 μg/mL) (FIG. 4B). Measurement of antioxidative action of AvailOm® with C3-rut, the most powerful antioxidant anthocyanin revealed that the protective action of AvailOm® from ox-LDL evoked oxidative stress was significantly reduced in comparison to AvailOm® alone.

[0070] FIG. 4 shows vascular response of phenylephrine-precontracted mice vessels to increasing doses of AvailOm® (5-150 μg/mL) or to C3-gal, or to AvailOm® and C3-gal or AvailOm® and C3-gal and DP3-ara with a ratio ½:½ or ⅓ respectively. (N=5). B) Representative high-power micrographs of 10 μm sections of mice mesenteric arteries loaded for 2 h with 4,5-diaminofluorescein (DAF-FM) reveal nitric oxide production after treatment with AvailOm® or single combination. Bar graph shows the mean fluorescence intensity of N=4 section for each compound. C) Representative high-power micrographs of 10 μm sections of mice mesenteric arteries loaded with dihydroethdium probe at the concentration of 5 μM. Vessels were pre-treated with ox-LDL, oxLDL plus AvailOm® or with oxLDL plus Availom® mixed with C3-rut (½:½) and D) measurement of ROS production by DHR123. D) Vascular response of phenylephrine-precontracted mice mesenteric arteries to increasing doses of ACh (10-9 to M 10-5 M) after exposure to ox-LDL for 30 minutes and exposed to AvailOm® or AvailOm® mixed with C3-rut.

Example 4: AvailOm® in Combination with Anthocyanin Mix Exerts a Potent Vasorelaxant Effect

[0071] The possible action of AvailOm® in combination with different anthocyanins' mixtures on ROS production was analyzed. First of all, the measurement of oxLDL evoked ROS production showed that AvailOm® plus MIX6 (C3-glu+DP3-glu+MaI3-glu+MaI3-gal+PEO3-gal), respecting a ratio of 1:6 of each product, was able to significantly reduce the oxidative stress with a major degree respecting to AvailOm® alone or AvailOm® in combination with MIX 1 (C3-glu+C3-gal), MIX 2 (MaI3-glu+MaI3-gal), MIX 3 (C3-glu+DP3-glu+MaI3-glu), MIX 4 (MaI3-gal+PEO3-gal) or MIX 5:C3-glu+DP3-glu+C3-rut+MaI3-glu+MaI3-gal+PEO3-gal (FIG. 5A). Interestingly, the evaluation of endothelial vasorelaxation under oxLDL-evoked oxidative stress revealed that AvailOm in combination with MIX6 exert the major protection from ROS evoked endothelial dysfunction (FIG. 5B-G) demonstrating an unexpected synergistic effect with AvailOm®.

[0072] FIG. 5 shows in A) measurement of ROS production by DHR123 in vessels treated with ox-LDL alone or with PEG-SOD, AvailOm®, or AvailOm® plus MIX 1: C3-glu+C3-gal; MIX 2: MaI3-glu+MaI3-gal; MIX 3: C3-glu+DP3-glu+MaI3-glu; MIX 4: MaI3-gal+PEO3-gal; MIX 5: C3-glu+DP3-glu+C3-rut+MaI3-glu+MaI3-gal+PEO3-gal or MIX6: C3-glu+DP3-glu+MaI3-glu+MaI3-gal+PEO3-gal. Statistical analyses were performed using one-way ANOVA followed Bonferroni post-hoc test. *p<0.05. B-G) Vascular response of phenylephrine-precontracted mice mesenteric arteries to increasing doses of ACh (10-9 to M 10-5 M) after exposure to ox-LDL for 30 minutes and then to AvailOm® for 1 hour alone or AvailOm® in combination with MIX1, MIX2, MIX3, MIX4, MIX5 or MIX6.*P<0.05 vs oxLDL+AvailOm®. #p<0.05 oxLDL+AvailOm®; § p<0.05 vs ox-LDL+AvailOm®.

Example 5: The Antioxidant Vascular Action of Healthberry 865® is Due to the Combination of the Anthocyanins Contained

[0073] Previously few studies have reported an antioxidant activity of Healthberry 865® in human subjects (Karlsen et al. 2007). To investigate the capability of Healthberry 865® and the single anthocyanins contained on the modulation of oxidative stress, several methodological approaches were performed measuring both, total anti reactive oxygen species (ROS) capacity and their specific action on the modulation of the main machinery of ROS production, the activity of NADPH oxidase enzyme. The studies performed on mice mesenteric arteries revealed that Healthberry 865® owns an important anti-oxidative action, as shown by the significant reduction of Angiotensin II-induced ROS formation (FIG. 6). The antioxidant vascular actions of single anthocyanins contained in Healthberry 865®: Delphinidin-3-rutinoside (D3-rut), Cyanidin-3-rutinoside (C3-rut), Delphinidin glucoside (DP3-glu), Cyanidin-3-glucoside (C3-glu), Petunidin-3-glucoside (PT3-glu), Delphinidin galactoside (DP3-gal), Peonidin-3-galactoside (PEO3-gal), Delphinidin-3-arabinoside (DP3-ara), Malvidin-3-galactoside (MAL3-gal), Malvidin-3-glucoside (MAL3-glu), Cyanidin-3-galactoside (C3-gal) and Cyanidin-3-arabinopyranoside (C3-arapy) were analyzed. A deeper analysis, using single anthocyanins revealed that C3-glu, C3-rut, DP3-glu, MAL3-gal, PEO3-gal, MaI-3-glu are able to reproduce the antioxidant action of Healthberry 865®. Accordingly, the biochemical measurement of ROS generation by DHR1,2,3 probe confirm the results obtained with DHE (FIG. 6B).

[0074] Moreover, the analysis of NADPH oxidase (NOX) activity after stimulation with Angiotensin II, a gold-standard inducer of NOX activation was performed. The results showed that C3-glu, C3-rut, DP3-glu, MAL3-gal, PEO3-gal, MAL3-glu are able to reduce NOX activity. However, these single anthocyanins resulted in a smaller reduction than evoked by Healthberry 865® (FIG. 6C). In fact, C3-glu and MAL3-glu resulted to be the most powerful anthocyanins closer to the potent effect of Healthberry 865®.

[0075] To evaluate the role on oxidative stress, the action of further mixtures was analyzed: MIX 1: C3-glu+C3-gal; MIX 2: MaI3-glu+MaI3-gal; MIX 3: C3-glu+DP3-glu+MaI3-glu; MIX 4: MaI3-gal+PEO3-gal; MIX 5: C3-glu+DP3-glu+C3-rut+MaI3-glu+MaI3-gal+PEO3-gal. Interestingly, the measurement of both total ROS production and that of NADPH oxidase activity revealed highest efficacy of MIX 5.

[0076] FIG. 6 shows representative high-power micrographs of 10 μm sections of mice mesenteric arteries loaded with dihydroethdium probe at the concentration of 5 μM. Vessels were pre-treated with single anthocyanins (50 μg/mL) for 1 hours and then stimulated with Angiotensin II for 15 minutes prior to the acquisition. (A) Measurement of ROS production by DHR123 in vessels treated with single anthocyanins and combination of anthocyanins mixed with a ratio 1:1. (B) NADPH oxidase activity in mesenteric arteries exposed to HB or single anthocyanins or combination of anthocyanins mixed with a ratio 1:1. Data are expressed as increase of chemiluminescence per minute.

Example 6: Mixture of Different Fruits for an Optimized Ratio of Anthocyanins with Antioxidant Activities in Combination with AvailOm®

[0077] In order to achieve an optimal ratio of all anthocyanins, which have a strong vasorelaxant effect, literature values for the content of the single anthocyanins in specific fruits were compared. Since it is postulated that the beneficial anthocyanins shall be present in a nearly equimolar ratio, the fruits with the highest amounts of the respective anthocyanins were combined in different ratios to achieve balanced ratios of the anthocyanins cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-galactoside, peonidin-3-galactoside, malvidin-3-glucoside.

[0078] The content of anthocyanins was analyzed in detail for black currant, red currant, black chokeberry bilberry, cowberry, elderberry (Benvenuti et al., 2004; Kăhkŏnen et al., 2003; Wu et al., 2004), strawberry, sweet cherry and sour cherry (Jakobek et al., 2007), wild blueberries and Saskatoon berries (Hosseinian et al., 2007).

[0079] By mixing fruits with high amounts of the desired anthocyanins, the following contents of the specific anthocyanins were achieved:

TABLE-US-00001 TABLE 1 mixture of bilberry and wild blueberry in the ratio of 1:1 Total amount Total amount in mixture (weight-%/total Anthocyanin (mg/100 g) anthocyanin amount) Ratio cyanidin-3-glucoside 1123 33 16 delphinidin-3-glucoside 431 12 6 malvidin-3-galactoside 127 4 2 peonidin-3-galactoside 65 2 1 malvidin-3-glucoside 222 6 3 others 1483 43 21 Sum 3451 100

[0080] After mixing the desired berries in the ratio of 1:1, the specific anthocyanins are present in different amounts in the mixture, differing by a factor of up to 16.

[0081] By mixing fruits with high amounts of the desired anthocyanins in an optimized ratio, the following contents of the specific anthocyanins were achieved:

TABLE-US-00002 TABLE 2 mixture of bilberry and wild blueberry in the ratio of 4:1 Total amount Total amount in mixture (weight-%/total Anthocyanin (mg/100 g) anthocyanin amount) Ratio cyanidin-3-glucoside 223 9 2, 3 delphinidin-3-glucoside 308 13 3, 1 malvidin-3-galactoside 149 6 1, 5 peonidin-3-galactoside 152 6 1, 6 malvidin-3-glucoside 280 11 2, 9 others 1334 55 13, 6  Sum 2446 100

[0082] After mixing the desired berries in the ratio (weight-%) of 4:1, the specific anthocyanins are present in similar amounts in the mixture, differing by a factor of less than 2. This corresponds to the mixing ratio of anthocyanins from the previous experiments.

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