WO2000055348A1

WO2000055348A1 - Sunscreen composition

Info

Publication number: WO2000055348A1
Application number: PCT/EP2000/001645
Authority: WO
Inventors: Indu Mani; Govindarajan Raman; Vandana Sharma
Original assignee: Unilever Plc; Unilever Nv; Hindustan Lever Limited
Priority date: 1999-03-17
Filing date: 2000-02-29
Publication date: 2000-09-21
Also published as: AU2916000A

Abstract

A process for preparing protein- and/or peptide-bound melanin, which is soluble in an aqueous solution at pH 2 to 11 and temp 0 °C to 50 °C, comprising the steps of reacting dihydroxyphenylalanine with an oxidant enzyme in the presence of an acidic protein and/or peptide having a pI of 3-6. The soluble protein- and/or peptide-bound melanin of this invention is useful as a sunscreen and as a colouring and/or flavouring for food.

Description

SUNSCREEN COMPOSITION

The present invention relates to a process of producing melanin polymers in soluble form and to compositions including such polymers for providing sunscreen benefits by preventing the damaging effects of ultra-violet light on skin.

BACKGROUND AND PRIOR ART

The treatment of human skin damaged through exposure to ultra-violet (UV) light, i.e. photo-damage, has been subject to much research effort in recent years, particularly with the realisation that skin cancer and other skin disorders can arise where the exposure to sunlight is excessive. The sensitivity of the human skin to UV exposure is dependent on the amount of the pigment melanin present in the skin.

The purpose of a sunscreen is to block the excessive UV radiation from affecting the skin. Sunscreens act by deflecting and scattering the incident light that produces burning and tanning of the skin or by absorbing this light. To prevent darkening of existing melanin in the skin, the skin has to be protected broadly across the UV range of about 320-400 nm. It is known that careful selection of sunscreens can offer this protection to the skin and reduce the darkening and damaging effects of the radiation.

WO 96/25920 (Yale University) , describes the use of melanin polymers in cosmetic compositions for providing a substantive, natural appearing tan and to protect the skin from harmful effects of ultraviolet radiation. Since melanins absorb light throughout the ultraviolet and visible spectra, solubilised melanins are also be effective as glass or plastic tinting agents for eye glasses, contact lenses, car windows, house windows, office buildings etc. Apart from cosmetic benefits, soluble melanins can also be used as colouring agents in coffee, tea, soda, whiskey and other liquors .

WO 92/16189 (Yale University) , disclose compositions and methods for preparing soluble melanins stable at physiological pH and temperature. The method comprises reacting dopachrome and one or more enzymes derived from biological cells or tissues which have a pigmentary system. The melanin produced is soluble in an aqueous solution at a pH of at least 5 to 9 at a temperature of 0 to 100°C.

The commercial value of melanins that are soluble at physiological pH and temperature is very great . Soluble melanins occur in nature as isolated polymers or as bound to proteins. It is known that soluble melanin that is bound to protein has much wider scope as a sunscreen than the unbound melanin and will also be more stable and will not get degraded into toxic monomeric compounds.

It is the object of the present invention to provide a process for preparing solubilised forms of melanin bound to a peptide/protein with a pi in the range of 3 to 6. The melanin formed therein is soluble in an aqueous solution at pH 2 to 11 and temperature 0°C to 50°C. It is another object of the present invention to provide compositions comprising such melanin useful for providing sunscreen benefits by preventing the damaging effects of ultra-violet light on skin.

It is another object of the present invention to provide compositions comprising such melanin useful for colouring and or flavouring foodstuffs such as coffee, tea, soda and other beverages .

According to a first aspect of the present invention there is provided a process for preparing protein- and/or peptide- bound melanin, which is soluble in an aqueous solution at pH 2 to 11 and temperature 0°C to 50°C, comprising the steps of reacting dihydroxyphenylalanine with an oxidant enzyme in the presence of an acidic protein and/or peptide having a pi of 3-6.

The reaction mixture may optionally additionally comprise one or more of the following: Dihydroxyindole, 5,6 dihydroxyindole-2-carboxylic acid, dopachrome, indole-5,6- quinone and/or melanochrome.

Preferably the enzyme is a naturally occurring oxidant enzyme such as a tyrosinase. The enzyme source could be either mushroom or neurospora crassa or other natural sources .

The dihydroxyphenylalanine (DOPA) is preferably DL- or L- DOPA. The proteins used should preferably have a molecular weight of at least 45,000 Da and a pi of 3 to 6. Suitable proteins include Bovine Serum Albumin and Ovalbumin for example. The concentration of protein used may be 1 μM to ImM and is preferably 10-100 μM. The reaction mixture preferably contains DOPA, oxidant enzyme and the acidic protein/peptide in the ratio of 1 : 0.2-0.4 : 0.6-1.2.

Metal ions such as copper, zinc and iron may also be used to alter colour and properties of the melanin formed.

It is thought that the oxidant enzyme converts DOPA to dopaquinone and subsequently to dopacharome . At this stage, in the presence of oxygen and light, dopachrome is converted to a variety of intermediates such as dihydroxyindole, 5,6 dihydroxyindole-2-carboxylic acid, indole-5 , 6-quinone and/or melanochrome . The acidic proteins/peptides present in the reaction mixture assist in the polymerisation of these intermediates resulting in the formation of the soluble, protein-bound melanin of the present invention.

The invention also provides a soluble protein- and/or peptide-bound melanin obtainable by the process according to the first aspect of the present invention.

According to a further aspect of the present invention there is provided a sunscreen composition comprising:

(a) 0.1 to 10% by weight of the composition of the soluble, protein- and/or peptide-bound melanin of the present invention and;

(b) a cosmetically acceptable vehicle. The present invention also provides a synergistic skin lightening composition comprising:

(a) from 0.1 to 10 % by weight of the composition of a skin whitening agent and;

(b) 0.1 to 10% by weight of the composition of soluble, protein- and/or peptide-bound melanin.

The skin lightening caused by the composition of the invention is reversible and without any side effects.

Skin whitening agent may be chosen from niacin, niacinamide or a precursor thereof, extracts of placenta, hydroquinone and derivatives (eg. arbutin) , kojic acid, dicarboxylic acids (azelaic acid, sebacic acid) , represented by the formula

HOOC- (CxHy) -COOH where x=4 to 20 and y=6 to 40, ascorbic acid and derivatives thereof, hydroxy acids (lactic acid, glycolic acid, malic acid, tartaric acid etc) , ferulic acid, retinol and derivatives, polyamino acid sequence with an isoelectric point (pi) between 2 to 5.5, organic sunscreens such as 4- tertiary butyl-4 ' -methoxy dibenzoylmethane, available under the trade name PARSOL 1789 from Givaudan, and/or 2 -ethyl hexyl methoxy cinnamate, available under the trade name PARSOL MCX from Givaudan or other UV A and UV B sunscreens and any other known skin whitening compounds.

The sunscreen and skin lightening composition, according to the invention can include cosmetically compatible carriers, preservatives, emulsifiers, thickeners, perfume, colour, skin benefit materials such as moisturisers, emollients antiageing compounds and other skin benefit agents. The vehicle which forms part of the cosmetic composition is cosmetically acceptable in that it will not harm the skin. The vehicles that can be used in the compositions according to the invention can include powder absorbents, binders and carriers, and liquids such as emollients, propellants, solvents, humectants and thickeners, for example.

Moisturisers and humectants such as polyols, glycerol, cetyl alcohol, carbopol 934, ethoxylated castor oil, paraffin oils, lanolin and its derivatives, etc may also be present.

Silicone compounds such as silicone surfactants like DC3225C (Dow Corning) and/or silicone emollients, silicone oil (DC- 200 Ex-Dow Corning) can be used.

The compositions according to the invention can be prepared for topical application to the skin in the form of conventional products such as lotions, creams, ointments and aerosol products.

The invention will now be illustrated by way of Examples. The Examples are for illustration only and do not in any way restrict the scope of the invention. EXAMPLES

Example 1

Process for the preparation of soluble protein bound melanin

The soluble melanin was prepared by reacting 5μM of DL-DOPA and 0.45 μg of tyrosinase in a 0.125 M sodium acetate buffer pH 5.0, in a final volume of 1 ml . The acidic protein Bovine serum albumin (BSA) with pi 4.6 and a molecular weight

66,000 kD, at different concentrations (Table 1) was tested. The control sample was without the acidic protein. The samples were incubated for 12 hours at 25°C and then centrifuged at 16,000 x g for 10 minutes to pellet the melanin. The absorbance of the supernatant was measured at 500 nm (A 500 nm) to determine the level of soluble or non- pelletable melanin. The pellet was washed and sonicated in lml distilled water and the absorbance measured at 500 nm to determine the level of pelletable melanin. The melanin prepared was also analysed for its properties.

Table 1

The data presented in Table 1 shows that in presence of an acidic protein the level of soluble melanin formed is significantly increased over control. The soluble melanin produced according to the invention is soluble in an aqueous solution at pH 2 to 11 and temperature 0°C to 50°C. It is protein bound, and of a very high molecular weight (100 - 200 Kda) . It can be filtered through a 0.22 μ filter and remains in the soluble form when ultracentrifuged at 1,05,000 x g for 1 hour. The soluble melanin is stable on acidification or alkalisation but gets precipitated on boiling. It can be lyophilised into a crystal form and redissolved in water. It varies in colour from golden brown to black and absorbs throughout the UV and visible wavelengths .

Example 2

Effect of different proteins on the formation of soluble protein bound melanin

The melanin preparation was done as described in example 1 but using proteins with different pi to determine the effect of acidic protein on the formation of soluble melanin. The proteins used are as described in Table 2. Table 2

The data presented in Table 2 shows that the increase in the soluble melanin is due to the presence of an acidic protein and there is no corresponding increase when a basic protein (pi 8.0) was present.

Example 3

Skin lightening composition according to the invention

The invention will now be illustrated by reference to the following example of a cosmetic cream.

Example 4

Sunscreen Composition

This example illustrates a suncare cream incorporating the composition of the invention:

Claims

A process for preparing protein- and/or peptide- bound melanin, which is soluble in an aqueous solution at pH 2 to 11 and temp 0°C to 50°C, comprising the steps of reacting dihydroxyphenylalanine with an oxidant enzyme in the presence of an acidic protein and/or peptide having a pi of 3-6.

A process according to claim 1 wherein the enzyme is a tyrosinase .

3. A process according to claim 2 wherein the source of tyrosinase is neurospora crassa .

4. A process according to any preceding claim wherein the protein and/or peptide has a molecular weight of at least 45,000 Da.

5. A process according to any preceding claim wherein the concentration of protein/peptide in the reaction mixture is lμM to 1 mM.

6. A process according to any preceding claim wherein the ratio of dihydroxyphenylalanine to oxidant enzyme to acidic protein/peptide is 1: 0.2-0.4: 0.6-1.2.

7. A process according to any preceding claim wherein the reaction mixture also comprises copper, zinc or iron ions .

8. A protein- and/or peptide-bound melanin, which is soluble in an aqueous solution at pH 2 to 11 and temp 0°C to 50°C, obtainable by the process of claims 1-7.

9. A sunscreen composition comprising:

(a) 0.1 to 10% by weight of the composition of the soluble protein- and/or peptide-bound melanin of claim 8 and

(b) a cosmetically acceptable vehicle.

10. A colouring and/or flavouring composition for food stuffs comprising the soluble protein-and/or peptide-bound melanin of claim 8.

11. Use of the soluble protein-and/or peptide-bound melanin of claim 8 as a sunscreen.

12. Use of the soluble protein-and/or peptide-bound melanin of claim 8 as a food colouring and/or flavouring.

13. A skin lightening composition comprising:

(a) from 0.1 to 10% by weight of the composition of a skin whitening agent .

(b) 0.1 to 10% by weight of the composition of soluble protein and/or peptide-bound melanin.