FR2710653A1 - Process for stimulating the colour of pigment elements secreted by certain animal cells and especially by those of the shell of molluscs - Google Patents

Abstract

This process consists in amplifying the melanin synthesis of the melanocytes by treating the said tissues or cells in the presence of compounds of the general formula: in which R<1>, R<2> and R<3> are chosen from hydrogen or methyl, ethyl, propyl, isopropyl or sulphonated radicals, preferably encapsulated within infracellular vectors. The process applies to improving the yield of the production of black pearls, to the conversion of white cultured pearls into black pearls as well as to varying the colour of nacres and of pearls.

Description

The present invention relates to a method for stimulating the color of pigmentary elements secreted by certain animal cells and in particular
by those of the mantle of molluscs.

In what follows, the present invention will be more particularly described.
in its application to pearly bivalve molluscs, without, however,
results in a limitation to its scope.

We know that melanin is a complex protein substance, quite
common in the animal kingdom. It contributes to the development of pigments which
color the coat of mammals, the feathers of birds, the color of the skin
human, the opacity of the clouds protecting cephalopods (octopus or Octopus
vulgaris), cuttlefish (Sepia officinalis), octopus (Eledone aldrovandii), squid (Liligo
vulgaris and Allotheutis subulata) ... and is involved in the coloring of the shells and pearls of lamellibranch molluscs (Unio anodonte, Aviculina meleagrina, PtncCadines), the flesh of molluscs such as grypheus and the shells of certain
arthropods.

Since ancient times, fine pearls have been regarded as
particularly attractive and desirable. Even today they represent
a symbol of much sought-after luxury. Black pearls in particular have a value which is about five times that of white pearls and, currently black pearls represent in jewelry about 15 to 20% of the pearls collected.

The very high price of black pearls, natural or cultured, prompted unscrupulous traders to tint much more common pearls (akoya from Japan). One of the methods used is to soak white pearls in a solution of nitrate or another silver salt, then in dilute ammonia, before exposing them to light or to the action of hydrogen sulphide gas. . The pearl is thus stably blackened, which then has a deep and uniform black color.

Another method, currently used for the akoya pearl, consists in subjecting it to irradiation with gamma radiation emitted by a cobalt source; we obtain a pearl of a bluish gray.

These falsifications can, however, be easily detected in the laboratory: using a magnifying glass, traces of paint can be observed around the piercing hole of an artificially colored pearl. For irradiated pearls, the core, usually white in Tahitian cultured pearls, of natural color, appears blackened through the drilling hole. In addition, an intense color and a metallic luster are indicative of a treatment.

It is the same for the small diameter of the pearls (less than 98 mm) because the real black pearls are usually larger (with the exception of the keshis, coming from the black-lipped pearl oyster).

At present, the most accurate method of differentiating natural colored pearls from artificially colored pearls is that which uses spectrophotometry. Pinctada margaritifera pearls exhibit specific absorption at 700 nm (which is due to their natural black pigments). Similarly, the X-ray fluorescence method shows two fluorescence peaks at 450 and 620 nm for Pinctada margaritifera and a single peak of 620 nm for
Pinctada maxima and Pinctada fucata martensi.

Mother-of-pearl and pearls are inseparable; their nature (apatite, aragonite and conchioline) and their physiological origin (secretion from the external epithelium of the mantle) are common.

Several researchers have already suspected the presence of melanin in certain nacres and pearls. Thus, Abolins-Krogis A. (1963, Arkiv. Zool.

15,475) identifies eumelanin as a stabilizing factor.

R.A. Nicolaux (Melanins-Hermann Paris 1968, p. 67) hypothesizes the role of melamine as a thermal sensor in seashells.

The melanistic origin of the color of black pearls has recently been demonstrated by two observations:
- Chromatographic migration of black pearl pigments is similar to that of cephalopod eumelanins.

- Microscopic observation of these pigments shows that they are made up of small vesicles of appearance and morphology identical to those of melanosomes.

It was also observed that the pigment extracted from black pearls and nacres from French Polynesia migrated in a manner quite similar to sepiamelanin (Eumelanin from Loligo: Calmar); indeed,
- the pigment thus extracted reacted with hydrogen peroxide such as eumelanin
- microscopic observation of nacres digested by EDTA showed granules stuffed with black pigments similar to melanosomes.

The object of the present invention is to provide pigmented cells and in particular those of nacres and pearls with substances which significantly amplify the synthesis of melanin, without thereby being a precursor or an element of the enzymatic reaction. .

The inventor was able to observe that certain compounds chosen from among those having as central nucleus a polycyclic structure isomer of naphthalene consisting of the joining of a cyclopentene and a cycloheptene by one of their side and in particular azulene , were particularly suitable for stimulating melanin synthesis. It was thus possible to modify the melanin syntheses and to double the quantity of melamine synthesized by the melanocytes of mammals.

These compounds have the following general formula:

The natural or semisynthetic substances capable of developing such an activity often have methyl, ethyl, propyl, isopropyl, sulfonated substitutions, and more particularly, chamazulene and guaiazuluene may be mentioned.

Substitutions of the azulene ring are made on preferred carbons.

Hydrogen atoms are often substituted by aliphatic radicals.

R1 R2 R3
Azulene HHH
Chamazulene CH3 C2H5 CH3
Guaiazulene CH3 C3H7 CH3
Chamazulene is a compound found naturally in chamomile (Matricaria chamomilla), wormwood (Artemisia absinthium) and certain other compounds such as Achillea millefolium.

Guaiazulene is obtained by extraction from zygophylaceae, guaiac wood oil or by semisynthesis from Gurjun's balm.

The addition of a 1% solution of guaiazulene to the culture medium (Mac Coy) of the melanocytes makes it possible to double the production of pigments of the Caucasian melanocytes as shown in the following table (results for 106 cells per 48 h)
Witness Mc Coy 4,5 | 0.32 lig / ml
Mc Coy + 1% Gaiazulene 11.2t0.74 igIml
It was possible to confirm that, in a cosmetic preparation, the activity observed on the cell cultures was found at the level of the skin and that the tanning of the subjects who had been treated with such a preparation lasted twice as long as that of the non-controls. treated.

Analysis of the results and the amount of neosynthesized melanin after application of gaiazulene allows us to foresee that this is a fairly ubiquitous system capable of being applied to all cells capable of synthesizing melanin.

The inventor proposed to apply the ability exhibited by these azulene derivatives such as free or encapsulated chamazulene and guaiazulene to the stimulation of melamine synthesis in human subjects to the color modification of the elements. secreted by certain animal cells and in particular by the mantle of molluscs.

The present invention therefore relates to a method of stimulating the coloring of the pigment elements secreted by certain animal cells and in particular by those of the mantle of molluscs, characterized in that it consists in amplifying the melanin synthesis of melanocytes by treatment of said tissues or cells in presence of compounds having as central nucleus a polycyclic structure isomer of naphthalene consisting of the joining of a cyclopentene and a cycloheptene by one of their side (formula 1) and in particular azulene.

According to a preferred embodiment of the invention, these compounds have methyl, ethyl, propyl, isopropyl, sulfonated substitutions, and they are especially chosen from chamazulene and guaiazuluene.

With the aim of improving the activity thereof, the active products are preferably encapsulated within subcellular vectors, that is to say of a size much smaller than that of the cells. Concentrations range from 10-6-1y2 g of substance per gram of carrier material. Several liposome encapsulation techniques can be used reverse phase (REV), ether injection, ultrasonication, solvent evaporation, French press, Bengham method (vortex). All the protocols are described in the following books:
- P. Buri, F. Puisieux et al: "New pharmaceutical forms, technological, biopharmaceutical and medical aspects"
- F. Puisieux, "Liposomes" -Leserman and Machy "Liposomes in cell biology and pharmacology".

All of these techniques are satisfactory, provided that the elimination of the organic solvent is well controlled.

It should however be noted that the technique which gives the most consistent results is that of the synthesis of liposomes and nanocapsules by positive depression described in FR A-8909275.

By way of example, below are some basic formulations of liposomes prepared according to this technique:
Example l:
Egg lecithin 7%
Cholesterol 0.05%
Tocopherol 0.1%
Linoleic acid 0.05%
Guaiazulene 0.75%
Ascorbyl palmitate 0.01%
Water qs 100
Example 2: (Nanocapsule or nanosphere vectors)
paraffin 2%
Waxes 0.75%
Cholesterol 1.5%
Ascorbyl palmitate 0.08%
Sucrose palmitate 0.5%
Guiazulene 0.7%
Curator qs

Water qs 100
Nanocapsular forms based on cholesterol, cholic acid, sucrose palmitate, amino acid palmitates or small peptide sequences are suitable. Nanocapsular forms with siloxane or acrylic acids are less suitable, while nanospheric forms of polymers such as polylactic-polyglycosides, polypeptides, polysaccharides and polymers of polyunsaturated fatty acids are quite suitable.

Nanoparticles with porous structures (microsponges) and nanoparticles with a solid matrix such as nanospheres are also well suited.

The following table represents the results obtained with guaiazulene encapsulated under the conditions of Example 1:
TABLE I Encapsulated active Amount of thetized melanin (in g / 106 cells)
Witness 5.14 + 0.76
Vitamin A 5.37 + 0.93
Vitamin D2 6.05 i 0.67
Substituted azulene 8.19 + 0.96
In the present invention, the activity of melanocytes was studied in culture media by a pigment cell capable of synthesizing melamine chosen from cells of arthropods, molluscs, etc .; it has been studied for a concentration of active ingredients of between 1 gIl and 4 mgn; the best results were obtained with concentrations in the culture media of between 0.01 mgn and 2 mgll.

The experimental conditions were as follows:
The coats of each species studied are taken under sterile conditions and then rinsed in HBSS medium (Gibco). They are then cut into small fragments of less than 1 mm3. The cells are dissociated using a solution of pronase E (Sigma). The enzymatic digestion is carried out at 37 ° C. with stirring for 15 minutes.

The cell suspension thus obtained is filtered through a nylon cloth the mesh of which has a diameter of 50 m in order to keep only well-dissociated cells. These are rinsed twice in HBSS medium and then cultured in a medium described by Lenoir and Mathieu (1986) with the following modifications:
DMEM powder medium (Gibco) 1 gram
NaCl 2.047
KCI 0.372
H20 bi distilled 100 ml
The cells are incubated in the presence of 1% of vectorized gaiazulene at a density of 5.105 cells per ml of culture medium. The latter is renewed every 24 hours.

After 48 hours of incubation at 15 ° C. in the dark, the cells are counted on a Coulter Counter and then centrifuged at 1000 revolutions / minute for 5 minutes. The pigment contained in the cell pellet is homogenized in 1 ml of
NaOH-DMSO and optical density is read at a wavelength of 470 nm using a microplate reader.

The standard range is produced with synthetic melanin (Sigma).

TABLE II
COMPARISON OF MELANIN SYNTHESIS RESULTS
ON DIFFERENT MOLLUSCS
Microscopy Molluscs Control Gaiazulene
vectorized
Lamellibranchs No Pectunculus cells 0.008 0.008 not synthesizing pigment Glycynaris no melanin
Lamellibranch Few cells Venus 21 * 1.7 36 i 1.8 synthesizing little pigment Mercenaria of melanin
Lamellibranch Bcp of Mytilus cells
Important pigment synthesis Galloprovin- 621 + 14 1303 * 19 melanin cialis
Lamellibranchs Bcp of Ostrea cells
Significant pigment synthesis Gryphae 717 + 14 1177 + 15 of melanin
The melanin dosage is expressed in ng / ml for 106 cells after 48 hours of culture.

On cultures of mollusc cells (grypheus), the inventor was able to amplify the synthesis of melamine from 400 ng per 106 cells in the controls to 1300 ng per 106 cells treated in a conditioned marine environment.

The synthesis of melanin is well proportional to the number of pigment cells observed. Stimulation by gaiazulene, and especially vectorized gaiazulene, gives a doubly constant response and proportional to the amount of melanin synthesized naturally.

The following table makes it possible to compare the melanin content in ng per ml after 48 h of culture per million cells after seeding of 4,106 cells at a density of 5,105 cells per ml of culture medium in the presence of 1% of a solution of 1% of vectorized galazulene or vectorized chamazulene (conditions of example l):
TABLE III
Witness Gaiazulene Chamazulene
Mussels Mytilus galloprovincialis 710 1560 1018
a 20 35 10
Clams Venus mercenaria 36 54 34
a 4 3 1
Oyster Gryphae angulata 720 1180 1016
a 35 25 10
Almond Pectunculus glycymeris ns ns ns
a ns ns ns
Chamazulene appears to be a little less active than guaiazulene; everything suggests that azulene exhibits comparable activity.

Furthermore, in order to determine the activity of the product during the synthesis of calcite and aragonite (making up the layers of the shell), measurements were made in the ClEL ab system This system defines the following axes
- L: luminescence; it varies from 0 for black to 100 for white
- The values a and b define the colors; the greater the absolute values of a or b, the greater the saturation of the hue:
a positive = red tint
a negative - green tint
b positive: yellow tint
b negative = blue tint.

The measurements were carried out both on ostra or crassostrea nacre (gryphae) (A) and on that of the stimulated areas (B); the following results are observed:
A = 3 L: 93.55 with a = 1.26 a: 0.05 with a = 0.05
b: 1.37 with a = 0.21
B n = 3 L: 43.60 with a = 1.55
a: 1.21 with a = 0.36 b: 0.67 with a = 0.95
One can see, thanks to the above description, all the interest of the present invention in pearl farming.

It is in fact thus possible to treat bivalve pearl molluscs such as Pinctada Margaritifera in order to increase the coloring of the mother-of-pearl.

It is also possible to improve the yield of the production of black pearls, to orient traditionally white cultured pearls towards black pearls and to vary the color of mother-of-pearl and pearls from pale blue to jet black.

The treatments are preferably carried out discontinuously, in basins or by placing a slow-release implant, as is conventional in pharmaceutical technique.

Continuous treatments are possible but may pose more problems, in particular because of the time (more than a year) necessary for the construction of a pearl.

Claims (8)

1 - Process for stimulating the color of the pigment elements secreted by certain animal cells and in particular by those of the mantle tissue of molluscs, characterized in that it consists in amplifying the melanin synthesis of melanocytes by treatment of said tissues or cells in the presence of compounds of general formula:

in which R1 1, R2 and R3 are chosen from hydrogen or methyl, ethyl, propyl, isopropyl or sulfonated radicals. 2. Method according to claim 1, characterized in that the compounds are encapsulated within infracellular vectors. 3. Method according to claim 1 and claim 2, characterized in that the compound is azulene. 4. Method according to claim 3, characterized in that the compound is chamazulene. 5. Method according to claim 3, characterized in that the compound is guaiazuluene. 6. Application of the method according to any one of claims 1 to 5 to the improvement of the yield of the production of black pearls. 7. Application of the process according to any one of claims 1 to 5 to the transformation of white cultured pearls into black pearls. 8. Application of the method according to any one of claims 1 to 5 to the variation of the color of nacres and pearls.