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Title:
A COMBINATION OF A NANNOCHLOROPSIS GADITANA EXTRACT AND A PROPOLIS EXTRACT AS ACTIVE COMPONENT IN COSMETIC FORMULATIONS FOR DERMAL USE
Document Type and Number:
WIPO Patent Application WO/2017/216588
Kind Code:
A1
Abstract:
A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal and in particular anti-aging use, wherein an active component of the formulation is constituted of the combination of an extract of Nannochloropsis gaditana in water and 1,3-propanediol and an extract of propolis in water, glycerol and β-hydroxypropyl cyclodextrin at a content of 0.1 to 20% w/w, which is added into the mixture after having been incorporated in a citric acid buffer solution pre-mixture adjusted to pH = 7 with its conjugate base, in order to ensure the stability of the final product, and pH is then checked and adjusted as required by the addition of pH adjusters, which are either a 20% w/w sodium hydroxide (NaOH) solution or a 20%w/w citric acid (C6H8O7) solution for the final adjustment to pH = 7.

Inventors:
KOUTSIANAS NIKOLAOS (GR)
LETSIOU SOFIA (GR)
GARDIKIS KONSTANTINOS (GR)
DRAGANI PANAGIOTA (GR)
Application Number:
PCT/GR2016/000063
Publication Date:
December 21, 2017
Filing Date:
November 21, 2016
Export Citation:
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Assignee:
APIVITA S A (GR)
International Classes:
A61Q19/08; A61K8/92; A61K8/97
Foreign References:
DE4336169A11995-07-13
US20150374759A12015-12-31
US20160128932A12016-05-12
US20150290253A12015-10-15
Other References:
ZILIUS MODESTAS ET AL: "Release of propolis phenolic acids from semisolid formulations and their penetration into the human skin in vitro", vol. 2013, 1 January 2013 (2013-01-01), pages 1 - 8, XP008165194, ISSN: 1741-427X, Retrieved from the Internet DOI: 10.1155/2013/958717
DATABASE WPI Week 200663, Derwent World Patents Index; AN 2006-607191, XP002767707
"International Cosmetic Ingredient Dictionary and Handbook", vol. 2, 2014, PERSONAL CARE COUNCIL, Washington D.C., article "Nannochloropsis Gaditana", pages: 2137, XP002767708
"Sunscreens: Development, Evaluation and Regulatory Aspects", 1997, MARCEL DEKKER INC
ALEXANDRA AMARO-ORTIZ; BETTY YAN; JOHN A. D'ORAZIO: "Ultraviolet radiation, aging and the skin: prevention of damage by topical cAMP manipulation", MOLECULES, vol. 19, no. 5, 2014, pages 6202 - 6219
GREGORIS E; FABRIS S; BERTELLE M; GRASSATO L; STEVANATO R: "Propolis as potential cosmeceutical sunscreen agent for its combined photoprotective and antioxidant properties", INT J PHARM., vol. 405, no. 1-2, 28 February 2011 (2011-02-28), pages 97 - 101
FONSECA YM; MARQUELE-OLIVEIRA F; VICENTINI FT; FURTADO NA; SOUSA JP; LUCISANO-VALIM YM; FONSECA MJ: "Evaluation of the Potential of Brazilian Propolis against UV-Induced Oxidative Stress", EVID BASED COMPLEMENT ALTERNAT MED., 2011
DUDONNÉ S; POUPARD P; COUTIERE P; WOILLEZ M; RICHARD T; MERILLON JM; VITRAC X.: "Phenolic composition and antioxidant properties of poplar bud (Populus nigra) extract: individual antioxidant contribution of phenolics and-transcriptional effect on skin aging", J AGRIC FOOD CHEM., vol. 59, no. 9, 11 May 2011 (2011-05-11), pages 4527 - 36, XP055208075, DOI: doi:10.1021/jf104791t
RAPOSO, M.F.; DE MORAIS, R.M.; BERNARDO DE MORAIS A.M: "Bioactivity and applications of sulphated polysaccharides from marine microalgae", MAR DRUGS, vol. 11, 2013, pages 233 - 352
GOUVEIA L.; RAYMUNDO A.; BATISTA A.P.; SOUSA I.; EMPIS J.: "Chlorella vulgaris and Haematococcus pluvialis biomass as colouring and antioxidant in food emulsions", EUR FOOD RES TECH, vol. 222, 2006, pages 362 - 367, XP019328264, DOI: doi:10.1007/s00217-005-0105-z
MACEDO, M.F.; MILLER, A.Z.; DIONISIO, A.; SAIZ-JIMENEZ, C: "Biodiversity of cyanobacteria and green algae on monuments in the Mediterranean Basin: an overview", MICROBIOLOGY, vol. 155, 2009, pages 3476 - 3490
FUJITANI, N.; SAKARI, S.; YAMAGUSHI, Y.; TAKENAKA, H.: "Inhibitory effects of microalgae on activation of hyaluronidase", J APPL PHYCOL, vol. 13, 2001, pages 489 - 492
LANGTON AK; SHERRATT MJ; GRIFFITHS CE; WATSON RE: "A new wrinkle on old skin: the role of elastic fibres in skin ageing", INT J COSMET SCI., vol. 32, no. 5, October 2010 (2010-10-01), pages 330 - 9, XP007915815, DOI: doi:10.1111/j.1468-2494.2010.00574.x
KYOUNG KIM H; KYOUNG KIM Y; SONG IH; BAEK SH; LEE SR; HYE KIM J; KIM JR: "Down-regulation of a forkhead transcription factor, FOX03a, accelerates cellular senescence in human dermal fibroblasts", J GERONTOL A BIOL SCI MED SCI, vol. 60, no. 1, January 2005 (2005-01-01), pages 4 - 9, XP008145245, DOI: doi:10.1093/gerona/60.1.4
CROUCH SP; KOZLOWSKI R; SLATER KJ; FLETCHER J: "The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity", J IMMUNOL METHODS, vol. 160, no. 1, 15 March 1993 (1993-03-15), pages 81 - 8, XP023653002, DOI: doi:10.1016/0022-1759(93)90011-U
MOSMANN T: "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays", J IMMUNOL METHODS, vol. 65, no. 1-2, 16 December 1983 (1983-12-16), pages 55 - 63, XP023973702, DOI: doi:10.1016/0022-1759(83)90303-4
WINKELMAN J: "The distribution of tetraphenylporphinesulfonate in the tumor-bearing rat", CANCER RES., vol. 22, June 1962 (1962-06-01), pages 589 - 96
VOGEL B; SIEBERT H; HOFMANN U; FRANTZ S: "Determination of collagen content within picrosirius red stained paraffin-embedded tissue sections using fluorescence microscopy", METHODSX, vol. 2, 21 February 2015 (2015-02-21), pages 124 - 34
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Claims:
CLAIMS

1. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal and in particular anti- aging use, where the formulations comprise the sunscreen filters 2-Ethylhexyl 2- Cyano-3,3-Diphenylacrylate at a content of 0% to 10% w/w, 2-Ethylhexyl 2- Hydroxybenzoate at a content of 0% to 5% w/w, 4-tert-Butyl-4'- methoxydibenzoylmethane at a content of 0% to 5% w/w, 2'-(6-(4- Methoxyphenyl)1 ,3,5-triazine-2,4-diyl) bis(5-((2-ethylhexyl)oxy)phenol) Bemotrizinol at a content of 0% to 0% w/w, 2-Ethylhexyl 4-Methoxycinnamate at a content of 0% to 10% w/w, Benzoic acid, 2-[4-(diethylamino)-2-hydroxybenzoyl]-, hexylester at a content of 0% to 10% w/w, 2,4,6-Trianilino-(p-carbo-2'-ethylhexyl-r-oxy)-1 ,3,5- triazine at a content of 0% to 5% w/w, Zinc oxide at a content of 0% to 25% w/w and Titanium oxide at a content of 0% to 25% w/w, anionic emulsifiers or combinations thereof, non-ionic coemulsifiers, demulcents, substances from the group of alcohols, stabilizers from the group of gums or hydrophilic colloids, hygroscopic skin-care agents and water, characterized in that an active component of the formulation is constituted of the combination of an extract of Nannochloropsis gaditana in water and 1 ,3-propanediol and an extract of propolis in water, glycerol and β-hydroxypropyl cyclodextrin, at a content of 0.1 to 20% w/w.

2. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the anionic emulsifiers or the combination thereof included in the formulation are Sodium hydrogen N-(1 -oxooctadecyl)-L-glutamate at a content of 0.1 % to 2% w/w, and/or 2,3-Propanetricarboxylic acid, 2-hydroxy-, ester with 1 ,2,3- propanetriol monooctadecanoate at a content of 0,1 % to 4% w/w, and/or Butanedioic acid, sulfo-, C-C16-18-alkyl esters, disodium salt at a content of 0, 1 % to 2% w/w, and/or Sulfuric acid, mixed cetyl and stearyl esters, sodium salts at a content of 0,1 % to 4% w/w cu 1 -hexadecanol, phosphate, potassium salt at a content of 0, 1 % to 4% w/w.

3. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the non-ionic coemulsifiers included in the formulation are fatty alcohols and in particular one or a mixture of cetearyl alcohol, cetyl alcohol, behenyl alcohol, arachidyl alcohol, and/or sucrose esters (and in particular one or a mixture of Sucrose Polystearate, Sucrose Distearate & Sucrose Stearate), and/or glycoside ethers (and in particular one or a mixture of Cetearyl Glucoside, Coco-Glucoside, Arachidyl Glucoside, C-12-20 AlkyI Glucoside, C 14-22 AlkyI Glucoside) at a content of 0.1-5% w/w.

4. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the demulcents with which the formulation is enriched are mainly natural oils and/or natural glycerides and esters from the groups of those derived from saturated and/or unsaturated, branched and/or unbranched alkanecarboxylic acids having a carbon chain length of 3 to 30 carbon atoms and saturated and/or unsaturated, branched and/or unbranched alcohols having a carbon chain length of 3 to 30 carbon atoms and of those derived from aromatic acids and saturated and/or unsaturated, branched and/or unbranched alcohols having a carbon chain length of 3 to 30 atoms such as heptyl undec-10-enoate, 1 ,2,3- Propanetrio Triheptanoate, Octyl Cocoate, Coco- Capprylate/Caprate at a content of 1 -20% w/w. 5. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the content of the substances from the group of alcohols used for the action against microorganisms ranges from 0.1 % to 2% w/w. 6. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the content of the agents stabilizing the structure of the formulation ranges from 0.01 % to 2% w/w. 7. A combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claim 1 characterised in that the content of the hygroscopic agents and the skin-care agents ranges from 0.001 % to 3% w/w. 8. A method of incorporating the combination of a Nannochloropsis gaditana extract and a propolis extract as an active component in cosmetic formulations for dermal use according to claims 1 to 7, characterized in that the extract combination is added into the mixture after having been incorporated in a citric acid buffer solution pre-mixture adjusted to pH = 7 with its conjugate base, in order to ensure the stability of the final product, and pH is then checked and adjusted as required by the addition of pH adjusters, which are either a 20% w/w sodium hydroxide (NaOH) solution or a 20%w/w citric acid (C6H807) solution for the final adjustment to pH = 7.

Description:
DESCRIPTION

A COMBINATION OF A NANNOCHLOROPSIS GADITANA EXTRACT AND A PROPOLIS EXTRACT AS ACTIVE COMPONENT IN COSMETIC FORMULATIONS FOR DERMAL USE

TECHNICAL FIELD

The present invention relates to the use as well as the method of incorporation of a combination of a Nannochloropsis gaditana extract and a propolis extract as active component in cosmetic formulations. The above-mentioned combination is intended for topical dermal use as a component of a cosmetic or pharmaco-cosmetic product having primarly anti-aging use.

TECHNICAL BACKGROUND

No such product has been reported so far worldwide. In particular, skin is a mirror of what occurs in the human body. Aging process is a complex phenomenon, 20% of which is attributed to intrinsic (genetic) factors and 80% to extrinsic (external) factors. In particular, the acceleration and extent of aging depends mainly on the environment, i.e. ultraviolet radiation (photo-aging), oxidative stress (environmental pollution) and, to a lesser extent, on the daily habits (smoking, poor nutrition) and stress. It is currently recognized that exposure to deleterious external conditions such as ultraviolet radiation or environmental pollution may be responsible for 80% of the visible signs of skin aging, including dryness and wrinkles, and pigmentation disorders, whereas photo-aging is associated with cancer (Amaro-Ortiz, 2014).

The main protection" method against photo-aging is covering the exposed areas of the skin with cosmetic formulations containing substances designated as sunscreen filters (List of ultraviolet radiation filters which are allowed for cosmetic products, Annex VI, REGULATION (EC) no. 1223/2009 OF THE EUROPEAN PARLIAMENT AND COUNCIL, and REGULATION (EU) 2016/621 for the amendment of the annex VI of the Regulation (EC) no. 1223/2009 of the European Council and Parliament for cosmetic products). Despite the extended use of these agents, which have been demonstrated to delay the emergence of solar melanoma, it has been shown that these agents, although delaying DNA damage, cannot provide total protection against UVA and UVB radiation (Amaya Viros, 2014).

In order to eliminate the above-mentioned disadvantage of the sun-screen filters, various strategies have been developed which can act synergistically with sunscreen filters and provide via various mechanisms increased photoprotection in the cosmetic formulations, which are mainly emulsions.

Furthermore, photoprotection may arise by the addition of active substances which contribute in the maintenance and enhancement of the function of structural units of the skin, preventing the emergence of clinical signs of aging.

Furthermore, these strategies can be also applied in cosmetic formulations which are commonly used for daily protection and care during low-sunshine periods, since 80% of the solar radiation can pass through the clouds.

Furthermore, protection against oxidative stress is effected only by incorporating active ingredients that enhance the anti-oxidative defense of the skin. Sunscreen filters modify the penetration of ultraviolet radiation through the skin and are classified as chemical and physical filters. Chemical sunscreen filters are organic molecules absorbing ultraviolet radiation, while physical sunscreen filters are inorganic oxides (titanium dioxide, zinc oxides) and act by reflecting ultraviolet radiation. Sunscreen emulsions may contain either physical or chemical filters, or a combination of chemical and physical filters. The combination of chemical and physical filters present some advantages since: 1) the use of a combination has as a result the incorporation of a lower proportion of filters, thereby reducing the increased risk of irritability from the chemical filters, 2) attractive textures are created, which are difficult to obtain when physical filters are used exclusively, and 3) a synergistic effect (Table, Marcel Dekker, 1997) is obtained which results to the use of a lower concentration of sunscreen filters. Efficiency of Mixed Or anic/Inor anic Sunscreens

However, in the cases where combinations of chemical and physical filters are used, two parameters should be taken into account for complete physicochemical stability and efficiency of the sunscreen products:

A) The almost inevitable use of anionic emulsifiers due to their ability to dissolve the chemical filters and to be incorporated in systems having a high electrolyte content. These emulsifiers present pH higher than 7.

B) the need to maintain pH to ca. 7, to avoid dissolution of the physical filters and formation of water-soluble alkaline complexes of the oxides, which could cause accumulation of ions in the aqueous phase of the emulsion, resulting to a) a variation of their distribution in the emulsion and instability in the efficiency of the product and b) a variation in the surface tension of the interface between the two phases of the emulsion and a destabilization thereof.

The above-described parameters make the addition of further components in the formulation difficult, since this must be performed in a way that will ensure maintenance of pH in the acceptable levels. It is remarkable that the majority of the extracts of physical components present a low pH (4.5 - 5.5) mainly for antimicrobial stability. Thus, their incorporation in the formulations of the above category should be performed in a manner that will ensure the maintenance of the pH of the emulsion at ca. 7.

By the present invention it is possible to provide a combination of a Nannochloropsis gaditana extract and a propolis extract and to incorporate this mixture in formulations containing combinations of sunscreen filters in a manner that will achieve the maintenance of pH at the desired levels and thus the maintenance of the stability of the emulsion throughout its life. In particular, the invented extract consists of a combination of two individual components:

1. an extract of propolis in water, glycerol and b-hydroxypropyl cyclodextrin

2. an extract of Nannochloropsis gaditana in water and 1 ,3-propanediol.

Propolis is a resinous substance produced by bees. The composition of propolis varies depending on the flora of the geographic region of the bees and its collection period. It contains on average 50% resins, 30% waxes, 10% aromatic components, 5% pollen and 5% various other components. The bio-active components of propolis are polyphenols, terpenes, steroids as well as sugars and aminoacids. Its major polyphenols are flavonoids and phenolic acids. Due to its high antioxidant content, propolis provides protection against premature aging by inhibiting the harmful free radicals generated by UV radiation (Fonseca, 201 1 , Dudonne, 20 1 , Gregoris, 201 ). This action finds application both in the preparation of cosmetics and in traditional medicine. In particular, various patents are known which relate to the beneficial effect of propolis on the skin, aiming mainly at its antimicrobial and anti-inflammatory properties (patent US2015/0374759, patent US2016/0128932, patent US2015/0290253). In the present invention, the method described in Patent No. 1007520 is used for the preparation of propolis, since the specific method ensures maximum concentration of polyphenols in the extract and their stability in time. Also, the specific method allows the use of any type of propolis as starting material.

The second component of the invented extract is an extract of the microalgae Nannochloropsis gaditana.

Recently, there has been a great interest in natural products derived from marine biodiversity. Algae and mainly microalgae are a major source of bioactive compounds that can be used as functional components (Raposo, 2013), such as carotenoids, phycobilins, fatty acids, polysaccharides, peptides, vitamins and sterols (Raposo, 2013). However, out of ca. 30000 species of microalgae assumed to be present, only a few thousands strains are kept in collections worldwide, only a few hundreds have been studied for their chemical content, and only five of them have been grown in industrial quantities (Gouveia, 2006, Macedo, 2009). Studies on the marine microalgae demonstrate their use as components in cosmetics (Fujitani, 2001 , Blackburn, 2012). In particular, it has been shown that they present antiallergic, anti-inflammatory properties. Also, microalgae inhibit the action of hyaluronidase, an enzyme that decomposes collagen fibers in the skin, destroys the natural hyaluronic acid produced in the body, and thus it deactivates it from the connection of collagen and elastin molecules (Fujitani, 2001). This prevents the formation of new collagen and elastin, and causes premature aging and loosening. As regards the genus Nannochloropsis spp., to which Nannochloropsis gaditana belongs, only Nannochloropsis oculata, which presents tightening properties, has been employed for cosmetic use (Blackburn, 2012).

On the contrary, Nannochloropsis gaditana, which is employed in our invention in combination with a propolis extract, has not been employed so far as a cosmetic component. Nannochloropsis gaditana is a marine microalga and was described for the first time by D. J. Hibberd (Hibberd, 1981 ). In particular, it is a small unicellular (diameter <5 pm), mixotrophic, non-flaggelated microalgae which belongs to the class of Eustigmatophyceae.

The present invention is directed to a method of incorporating a combination of Nannochloropsis gaditana extract and a propolis extract in cosmetic formulations and its use as an active component which presents substantial anti-aging effect at two levels.

The use of a propolis extract and a spirullina extract in cosmetics as separate components in soap for cosmetic use has been mentioned. Also, the use of a propolis extract and an algae extract as components in make-up cosmetics has been mentioned. However, it is the first time that a combination of Nannochloropsis gaditana extract and a propolis extract is employed for cosmetic use Disclosure of the invention

In order to make the invention fully understood by those skilled in the art, the description of the composition of an exemplary emulsion with sunscreen filters, in which composition a combination of Nannochloropsis gaditana extract and a propolis extract as an active component is used, is given.

The composition of the formulation includes a combination of an extract of

Nannochloropsis gaditana in water and 1 ,3-propanediol and an extract of propolis in water, glycerol and β-hydroxypropyl cyclodextrine, as an active component at a content of 0.1 to 20 %w/w.

Also, the specific composition contains a combination of the following sunscreen filters:

INCI (International Nomenclature Chemical Name or Content

of Cosmetic Ingredients)* Description* (%w/w)

2-Ethylhexyl 2-Cyano-3,3- 0-10%

Octocrylene Diphenylacrylate

2-Ethylhexyl 2- 0-5%

Ethylhexyl Salicylate Hydroxybenzoate

4-tert-Butyl-4'- 0-5%

Butyl Methoxydibenzoylmethane methoxydibenzoylmethane

2,2'-(6-(4-Methoxyphenyl)- 0-10%

1 ,3,5-triazine-2,4-diyl)bis(5-((2-

Bis-Ethylhexyloxyphenol ethylhexyl)oxy)phenol) /

Methoxyphenyl Triazine Bemotrizinol

2-Ethylhexyl 4- 0-10%

Ethylhexyl Methoxycinnamate Methoxycinnamate

Benzoic acid, 2-[4- 0-10%

Diethylamino Hydroxybenzoyl (diethylamino)-2- Hexyl Benzoate hydroxybenzoyl]-, hexylester

2,4,6-Trianilino-(p-carbo-2'- 0-5%

ethylhexyl-1 '-oxy)-1 ,3,5-

Ethylhexyl Triazone triazine

Zinc Oxide Zinc oxide 0-25%

Titanium Dioxide Titanium oxide 0-25%

*Cosmetic Ingredient database-Cosing:

http://ec.europa.eu/qrowth/sectors/cosmetics/cosinq/index en. htm

The composition of the formulation also comprises:

1 . Anionic emulsifier or a combination thereof:

INCI (International Nomenclature Chemical Name or Content

of Cosmetic Ingredients)* Description* (%w/w)

Sodium hydrogen N-(1 - 0.1 -2%

Sodium Stearoyl Glutamate oxooctadecyl)-L-glutamate 1 ,2,3-Propanetricarboxylic 0.1-4%

acid, 2-hydroxy-, ester with

1 ,2,3-propanetriol

Glyceryl Stearate Citrate monooctadecanoate

Butanedioic acid, sulfo-, C- 0.1-2%

C16-18-alkyl esters, disodium

Disodium Cetearyl Sulfosuccinate salt

Sulfuric acid, mixed cetyl and 0.1-4%

Sodium Cetearyl Sulfate stearyl esters, sodium salts

1-hexadecanol, phosphate, 0.1-4%

Potassium Cetyl Phosphate potassium salt

2. Non-ionic coemulsifiers such as fatty alcohols (and in particular one or a mixture of cetearyl alcohol, cetyl alcohol, behenyl Alcohol, Arachidyl alcohol), and/or sucrose esters (and in particular one or a mixture of Sucrose Polystearate, Sucrose Distearate & Sucrose Stearate), and/or glycoside ethers (and in particular one or a mixture of Cetearyl Glucoside, Coco-Glucoside, Arachidyl Glucoside, C-12-20 Alkyl Glucoside, C 14-22 Alkyl Glucoside) at a content 0.1-5 %w/w.

3. The composition of the formulation is enriched with demulcents, such as natural oils including Argania Spinosa oil or Olea europea oil, and/or natural glycerides including those derived from Cocos nucifera fruit and esters from the groups of those derived from saturated and/or unsaturated, branched and/or unbranched alkanecarboxylic acids having a carbon chain length of 3 to 30 carbon atoms and saturated and/or unsaturated, branched and/or unbranched alcohols having a carbon chain length of 3 to 30 carbon atoms and of those derived from aromatic acids and saturated and/or unsaturated, branched and/or unbranched alcohols having a carbon chain length of 3 to 30 atoms such as heptyl undec-10- enoate, 1 ,2,3- Propanetrio Triheptanoate, Octyl Cocoate, Coco- Capprylate/Caprate at a content of 1-20 %w/w.

4. The composition of the formulation further contains compounds from the group of alcohols having an action against microorganisms such as 2- phenoxyethanol, 1 ,2-propanediol, 3-(2-ethylhexyloxy) and octane-1 ,2-diol at a content of 0.1-2 %w/w.

5. As stabilizers of the composition of the formulation, stabilizing agents from the group of gums and hydrophilic colloids such as xanthan gum and cetyl hydroxyethyl cellulose at a content of 0.01-2 %w/w are employed.

6. Finally, the composition of the formulation is supplemented with hygroscopic agents and skin-care agents such as salts of hyaluronic acid, panthenol, tocopherol acetate and plant extracts such as crithmum (Crithmum maritimum), olive (Olea europea), helichrysum (Helichrysum italicum) at a content of 0.001 -3%.

7. The rest of the composition of the emulsion is supplemented with water.

The preparation of the composition of the formulation containing the invented combination of a Nannochloropsis gaditana extract and a propolis extract, as active component thereof, is performed by the following steps.

In the first step, all hydrophilic and non-heat-sensitive components of the composition are mixed in a vessel heated at 80 °C. Thereafter, in the second step, all the lipophilic components of the composition are mixed in a second vessel and heated at 80-85 °C until full homogenization of the mixture. In the third step, the mixture of the lipophilic components is transferred in the vessel containing the hydrophilic components and at the same time by a suitable homogenization equipment high-shear mixing is applied to reduce the surface tension between the two inhomogeneous systems and to generate a stable micelle dispersion of the one within the other. The homogenization procedure is performed at a shear rate of 3000 revolutions/minute for 10-30 minutes at a constant temperature of 80 °C and under regular microscopic inspection of the mixture. The homogenized mixture is then cooled with occasional stirring.

Thereafter, at room temperature, the heat-sensitive hydrophilic components, except the invented combination, are added under mild occasional stirring.

The invented combination is added to the mixture mentioned above, after having been incorporated in a citric acid buffer solution pre-mixture adjusted to pH = 7 with its conjugate base. The addition of the invented combination by means of a buffer solution ensures the stability of the formulation throughout its life time.

Thereafter, the pH is checked and adjusted as necessary by the use of pH adjusters which are either a 20% w/w sodium hydroxide solution (NaOH) or a 20% w/w citric acid solution (C 6 H 8 0 ) to achieve a final pH = 7.

The viscosity of the product obtained should be between 30000 and 80000 cps (BROOKFIELD RV, spindle/speed: SA25/3rpm).

Experimental demonstration of the use of a combination of a Nannochloropsis gaditata extract and a propolis extract.

In order to establish the use of the combination of a Nannochloropsis gaditana extract and a propolis extract as an anti-aging component in a cosmetic formulation, an in vitro study of the individual components (propolis, Nannochloropsis gadiata) was performed, by simulating various aging conditions in order to approach optimally the skin aging process. In this study, human skin fibroblasts were employed.

Initially, the bioactivity of the Nannochloropsis gadiatana under oxidative stress conditions was studied. It is known that oxidative stress constitutes a major form of attack on the skin and is considered to be one of the major factors causing aging. Under oxidative stress, formation of free radicals is activated. Free radicals are highly active molecules, which are generated as by-products of the normal metabolism (endogenous) and environmental stress (exogenous) and are responsible for cellular damages, targeting mainly on the cells including skin cells. In the experimental procedure, hydrogen peroxide (H 2 0 2 ) was employed as free-radical inducing agent. In particular, the experimental procedure includes the following steps: a 48-hour incubation of the Nannochloropsis gaditana extract at 1 % concentration on the fibroblasts, removal of the nutrient medium and washing with a phosphate/salt buffer solution (PBS) so as to avoid intermolecular reactions between the extract and H 2 0 2 , addition of a hydrogen peroxide solution (0.1 , 0.5, 1 mM) for three hours, addition of a nutrient medium and measurement of cell viability based on ATP (Crouch, 1993).

From the specific experimental procedure, it was observed that the

Nannochloropsis gaditana extract (1 %) protects cells against oxidative stress, since a 70% increase in cell viability (ATP increase) was observed with the Nannochloropsis gaditana extract (1 %) under oxidative stress conditions (H 2 0 2 addition) compared to cells without a Nannochloropsis gaditana extract.

Further investigation of this significant result led to the study of the variations that are caused by the influence of the Nannochloropsis gaditana extract on the gene expression of skin fibroblasts under oxidative stress conditions. Therefore, realtime polymerase chain reaction RT-PCR (Giulietti A et al.) was employed. From the transcriptomic study, transcripts of fibrillin (FBN) were overexpressed in fibroblasts with a 1 % Nannochloropsis gaditana extract (p<0.05) of the compared to fibroblasts without extract under oxidative stress conditions. Fibrillin is a glycoprotein necessary for the formation of the elastin fibers, since it constitutes the scaffold for the proper positioning of elastin in order to maintain skin elasticity (Langton, 2010). Furthermore, transcripts of FOXO3 were overexpressed in fibroblasts with 1 % Nannochloropsis gaditana extract in comparison to fibroblasts without the extract. FOX03 is a protein, which is considered to belong to the longevity and anti-aging proteins (Kyoung, 2005).

Thereafter, the bioactivity of the propolis extract under oxidative stress conditions (premature aging) was investigated.

Ultraviolet radiation is an important source of oxidative stress for the skin.

Apart from the generation of free radicals, ultraviolet radiation affects the defense enzymes of the skin against oxidation, leaving it more vulnerable to permanent cell damage (acceleration of the skin aging). When the skin is exposed for a long time to ultraviolet radiation without protection by sunscreen filters, its protection depends exclusively on the endogenous anti-oxidative defense systems.

In the experimental procedure performed, the quantity of collagen and elastin produced by the fibroblasts with or without a propolis extract under photo-oxidative stress conditions (UVA+UVB 48mJ/cm 2 ) was determined. A UV lamp was employed as a free-radical forming agent. Specifically, incubation of the propolis extract on the cells lasted for 4 days under photo-oxidative stress conditions (UVA+UVB 48mJ/cm 2 ). On the fifth day, collagen production was measured by the Sirius Red method (Vogel, 2-015), the elastin production was measured by the TPPS method (Winkelaman, 1962) and the cell vitality was measured by the MTT method (Mosmann, 1983). Initially it was observed that the propolis extract (1 %) protects the cells against photo-oxidative stress since a 40% increase in cell viability was observed with the propolis extract (1 %) under photo-oxidative stress conditions in relation to the cells without a propolis extract. From this study it was concluded that the propolis extract protects the cells against photo-oxidative stress, preserving the ability of the cells to produce collagen and elastin, the essential proteins of the extracellular space responsible for the elasticity and robustness of the skin.

Based on the above-mentioned experimental applications, the following were confirmed:

The combination of a Nannochloropsis gaditana extract and a propolis extract has a protective role against the aging of human skin cells. This is confirmed by 1 ) the protective role of the propolis extract against photo-oxidative stress, by the use of which the ability of skin cells to produce collagen and elastin was enhanced, and 2) the protective role of the Nannochloropsis gaditana extract against oxidative stress, by the use of which the expression of transcripts of fibrillin in skin cells was increased, enhancing skin elasticity since this protein offers the scaffold for the proper conformation of elastin. This is confirmed by the increased expression of the transcripts of FOXO3 under the use of the Nannochloropsis gaditana extract, enhancing its anti-aging use. References

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