Use of metal complexes which are mimetics of SOD as food agents and as cosmetics

Abstract

This invention relates to the use of the metal complexes which are mimetics of the superoxide dismutase (SOD) enzyme produced from macrocyclic polyazapyridinophane compounds of formula (1) as food agents and also as cosmetics.

Claims

1. A cosmetic formulation comprising a metal complex mimetic of superoxide dismutase of Formula 1 with at least one metal ion ##STR00004##

2. A food agent comprising a metal complex mimetic of superoxide dismutase of Formula 1 with at least one metal ion ##STR00005##

3. The cosmetic formulation of claim 1, wherein the metal ion comprises Cu(II), Fe(II) or Mn(II).

4. The cosmetic formulation of claim 3, wherein the ion is Mn(II).

5. The food agent of claim 2, wherein the food agent is a food additive, dietary supplement, functional food or functional drink.

6. A personal hygiene product comprising a metal complex mimetic of superoxide dismutase of Formula 1 with at least one metal ion ##STR00006##

7. The cosmetic formulation of claim 1, wherein said formulation is formulated as a cream, paste, milk, gel, lotion, ointment, suspension, dispersion, powder, shampoo, foam, spray or solid bar.

8. The food agent of claim 2, wherein the metal ion comprises Cu(II), Fe(II) or Mn(II).

9. The food agent of claim 8, wherein the ion is Mn(II).

10. The personal hygiene product of claim 6, wherein the cosmetic formulation is formulated as a cream, paste, milk, gel, lotion, ointment, suspension, dispersion, powder, shampoo, foam, spray or solid bar.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Graphical representation of the results obtained after incubation of Medaka embryos in stage 8 with the oxidant H.sub.2O.sub.2 and the compound Pytren-4Q+Mn for 72 hours, as detailed in the example.

(2) FIG. 2: Graphical representation of the results obtained after incubating Medaka embryos in stage 8 with the oxidant H.sub.2O.sub.2 and the compound Pytren-4Q+Mn for 14 days, as described in the example.

EXAMPLE

(3) 1. Materials and Methods

(4) 1.1 Medaka Embryos

(5) Individual adults (strain Cab, Carolina Biological Supply, (North Carolina)), were held in tanks with continuous water flow at 27 C., and a photoperiod of 14 hours of light and 10 hours of darkness. The fish were fed twice a day with Artemia nauplii, which were hatched daily from commercial Sanders cysts, and with dry food once per day.

(6) Fertilized eggs were collected daily, deposited in Yamamoto solution (0.13 M NaCl, 2.7 mM KCl, 1.8 mM CaCl2 and 0.24 mM NaHCO.sub.3, pH 7.3) (Yamamoto T., Medaka (killifish): Biology and strains. 1975, Tokyo: Keigaku Pub. Co), and were kept in the incubator at 26 C. The embryo stage was determined according to the description of Iwamatsu (Iwamatsu T., Stages of normal development in the medaka Oryzias latipes. Mech Dev. 2004, Vol. 121, pages 605-618).

(7) 1.2 Survival Analysis Using Medaka Fish Embryos

(8) Medaka embryos that were in stage 8 (early morula stage, Iwamatsu, 2004) were separated into 2 groups of 60 embryos and one group of 30 embryos. A group of 60 embryos were treated for 4 days with Pytren-4Q+Mn dissolved in Yamamoto solution at a concentration of 250 M. The other group of 60 embryos were incubated for 4 days in Yamamoto solution. This group was used as a negative control. The group of 30 embryos, positive control group, were treated for 4 days with a Yamamoto solution of vitamin C, an antioxidant compound, at a concentration of 100 M. After 4 days of incubation, the embryos were washed with Yamamoto solution. The groups of 60 embryos were separated one by one into two groups of 30 embryos. The embryos of all the groups were individually placed into the wells of 96-well plates, each well containing 200 l of Yamamoto solution with H.sub.2O.sub.2, at different concentrations depending on the extension of incubation time at which mortality was observed. When mortality was observed at 72 hours after being placed in H.sub.2O.sub.2 Yamamoto solution, the concentration of H.sub.2O.sub.2 used was 22.5 mM. Thirty embryos of each initial group of 60 embryos and the 30 embryos of the group treated with vitamin C were treated with H.sub.2O.sub.2 Yamamoto solution at a concentration of 22.5 mM. When mortality was observed at 14 days after being placed in H.sub.2O.sub.2 Yamamoto solution, the concentration of H.sub.2O.sub.2 used was 7.5 mM. Thirty embryos of each initial group of 60 embryos were treated with H.sub.2O.sub.2 Yamamoto solution at a concentration of 7.5 mM. This experiment was replicated in its totality 6 times in the test at 72 hours and 3 times in the test of 14 days.

(9) 1.3 Statistical Analysis

(10) To evaluate survival differences when comparing the 3 test groups, the statistical test for one-way analysis of variance (ANOVA) was applied to determine if there were statistically significant differences. Subsequently, Tukey's post-hoc test was applied to establish which of the 3 groups had statistically significant differences. When 2 test groups were compared, the statistical Students t-test was applied. The level of significance was set at p<0.05 in all cases. All these analyses were performed using the GraphPad Prism 5.0a program.

(11) 2. Results

(12) Tests were performed with compound Pytren-4Q+Mn at a non-toxic concentration for the embryos of 250 M and their antioxidant capacity was measured in two different assays. The first test measured mortality after 72 h incubation with 22.5 M H.sub.2O.sub.2 (shown in FIG. 1). The second test measured mortality after 14 days incubation with 7.5 mM H.sub.2O.sub.2 (shown in FIG. 2). Both tests showed a significant reduction in mortality in response to oxidative stress caused by H.sub.2O.sub.2. The test performed at 72 hours showed that both Pytren-4Q+Mn and vitamin C reduced mortality, which was 48.444.40% in the control and 24.967.11% in the embryos treated with Pytren-4Q+Mn, and 25.995.25% in the embryos treated with vitamin C (Table 1). Therefore the compound of the invention Pytren-4Q+Mn exerts an antioxidant activity similar to that of vitamin C at the tested concentrations.

(13) TABLE-US-00001 TABLE 1 Data obtained in the tests of the example of the invention at 72 hours of incubation. Pytren- 72 hours Control 4Q + Mn Vitamin C Replicate 1 62.5 50 40.63 Replicate 2 56.25 43.75 43.75 Replicate 3 50 12.9 15.63 Replicate 4 43.75 15.63 14.3 Replicate 5 46.9 18.75 20.8 Replicate 6 31.25 8.7 20.8 Mean 48.44 24.96 25.99 Standard 10.78 17.41 12.87 deviation Standard 4.40 7.11 5.25 error

(14) The test performed at 14 days showed that Pytren-4Q+Mn reduced mortality, which was 51.273.12% in the control, 41.471.52% in the embryos treated with Pytren-4Q+Mn (Table 2). The main differences reside in the fact that the detection at 14 days although longer, it is more sensitive because small lethal effects can be detected later.

(15) TABLE-US-00002 TABLE 2 Data obtained in the tests of the example of the invention at 14 hours of incubation. 14 days Control Pytren-4Q + Mn Replicate 1 46.9 39.3 Replicate 2 57.3 40.7 Replicate 3 49.6 44.4 Mean 51.27 41.47 Standard 5.40 2.64 deviation Standard 3.12 1.52 error

(16) The data obtained show that the compound Pytren-4Q+Mn has antioxidant properties at a concentration of 250 M, similar to that of vitamin C at 100 M.