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WO2002013779A1 - Agents eclaircissants pour la peau - Google Patents

Agents eclaircissants pour la peau Download PDF

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Publication number
WO2002013779A1
WO2002013779A1 PCT/GB2001/003516 GB0103516W WO0213779A1 WO 2002013779 A1 WO2002013779 A1 WO 2002013779A1 GB 0103516 W GB0103516 W GB 0103516W WO 0213779 A1 WO0213779 A1 WO 0213779A1
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WIPO (PCT)
Prior art keywords
composition
agent
skin
compound
formula
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PCT/GB2001/003516
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English (en)
Inventor
Peter Samuel James Cheetham
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Zylepsis Ltd
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Zylepsis Ltd
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Priority to AU2001275764A priority Critical patent/AU2001275764A1/en
Publication of WO2002013779A1 publication Critical patent/WO2002013779A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/347—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/676—Ascorbic acid, i.e. vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/678—Tocopherol, i.e. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/08—Preparations for bleaching the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations

Definitions

  • the present invention relates to the use of vinylguaiacol and/or ethylguaiacol as a skin lightening agent in combination with an agent which decreases the cytotoxicity of vinylguaiacol and/or ethylguaiacol on skin cells.
  • a skin lightener should inhibit mammalian tyrosinase effectively, should be permeable through both the cell and melanocyte membranes and should be safe and non-toxic to skin cells at the concentration it is to be used at.
  • Commonly used skin lighteners are hydroquinone, arbutin and kojic acid. Hydroquinone is the most effective but has significant adverse effects so that it is no longer registered for use in the EC. Many other materials have also been used or documented as skin lighteners, including lactic acid, ferulic acid and nicotinamide as well as plant extracts such as Bearberry extract. Other skin lighteners include glycosides and esters of hydroxy salicylic acid, ascorbyl methylsilanol and liquorice extracts. The use of caffeic acid or an ester or amide thereof to depigment the skin is described in US 5164185. Depigmenting compositions containing di- or tri- caffeoylquinic acid are disclosed in US 5445816. Vinylphenol or vinylsyringol (analogues of vinylguaiacol) do not show the effects of the present invention.
  • the naturally occurring compound ferulic acid is known to have skin lightening activity. Ferulic acid is a phenylpropenyl molecule.
  • Known skin lighteners such as vinylguaiacol or ethylguaiacol may exhibit some cytotoxicity against skin cells. Whilst such cytotoxicity can be tolerated, it would be advantageous to ameliorate it to some extent. The present invention addresses this issue.
  • composition comprising a compound of formula I and an agent, wherein the agent decreases the cytotoxicity of the compound of formula I on skin cells:
  • composition is preferably for use as a skin or hair lightener.
  • the compound of formula I can be provided as a salt, for example as a sodium or potassium salt.
  • the compound of formula I acts as a skin lightening agent.
  • skin lightener or “skin lightening agent” includes an agent which inhibits melanogenesis, especially the enzymes (such as tyrosinase) which are associated with the formation of melanin by mammalian cells, such as melanocytes in the skin.
  • the compound of formula I for the purposes of this invention is provided for a number of different cosmetic and/or therapeutic uses. Examples of such uses are to maintain a light or fair complexion, to reduce generalised over pigmentation, to lighten age spots, to lighten scars and to lighten hair.
  • composition of the first aspect of the invention is applied topically to the skin or hair.
  • the compound of formula I is provided at levels which will inhibit melan ⁇ genesis, more specifically which cause the inhibition of enzymes associated with the formation of melanin such as tyrosinase.
  • a level of a skin lightener, which produces 80% inhibition of melanogenesis is defined as a therapeutic or cosmetically useful dose.
  • a compound of formula I can exhibit one or more useful activities selected from antioxidant, antimicrobial, antibrowning, aroma/flavour and acidulant activities. Skilled persons will appreciate that the antibrowning and skin lightening activities of a compound of formula I are linked. Without being bound in any way by scientific theory, they appear to work by inhibiting enzymes associated with coloured material formation. For example, the compound inhibits polyphenol oxidase (tyrosinase and laccase) enzymes which catalyse formation of brown-black pigments (melanoids) formed by oxidative polymerization of plant phenols such as chlorogenic acid in plant tissue. Similarly, the compound inhibits tyrosinase enzyme in the skin to prevent melanin formation from precursor materials present in skin cells, such as tyrosine.
  • polyphenol oxidase tyrosinase and laccase
  • a compound of the formula I can be used in a wide range of applications because of their versatility.
  • a particular advantage of a compound of formula I is that it displays solubility in both oil and water for ease of formulation.
  • a still further advantage of a compound of formula I is that it possesses a pleasant aroma and this may avoid the need for the separate addition of a perfume in various compositions and formulations.
  • Vinylguaiacol and ethylguaiacol have a fresh camphoraceous/herbal/medicated character bringing to mind some pharmaceutical cough preparations. Thus, they convey hygiene, cleanliness and well being.
  • Vinylguaiacol and ethylguaiacol possess both a pleasant aroma and antimicrobial activity, making them especially suitable for use in formulations for application to the skin.
  • a compound of formula I is provided in combination with an agent which decreases the cytotoxicity of the compound of formula I on skin cells preferably increasing the viability of the skin and cells.
  • a compound or composition which exhibits cytotoxicity against skin cells is one which causes damage to cells and may ultimately result in cell death.
  • a composition or a compound (which may be vinylguaiacol and/or ethylguaiacol or a composition comprising vinylguaiacol and/or ethylguaiacol) which produces 50% cytotoxicity against skin cells is defined as a cytotoxic dose.
  • the agent can decrease the cytotoxicity of the compound of formula I on skin cells in at least two ways.
  • a first way is by adding an agent which makes the cells less susceptible to the cytotoxic effects of the compound or which repairs any damage caused to the cells by the compound of formula I.
  • an increased dose of the compound of formula I can be included to give the same cytotoxic effect as a previous lower dose without the agent.
  • an agent can decrease the cytotoxic effect of the compound of formula I on skin cells by increasing the efficacy of the compound as an inhibitor of melanogenesis. If the dose of the compound required to produce 80% inhibition of melanogenesis is reduced to a much lower concentration, this reduces the risk of cytotoxicity to the skin cells.
  • An agent may achieve this result by working synergistically with the compound of formula I, or by providing separate/unrelated skin lightening effects.
  • this invention provides a composition comprising vinylguaiacol and an agent wherein the difference between the inhibition of melanogenesis (preferably 80% inhibition of melanogenesis) and cytotoxicity (preferably 50% cytoxicity) on skin cells is more than 10 fold (or times).
  • the agent will increase the difference between the toxic dose and the therapeutic dose by between 20 and 200 times, preferably between 30 and 150 times more preferably more than 100 times.
  • the compound of formula I and the agent can be provided in the composition in varying concentrations and ratios.
  • the ratio of the compound of formula I and the agent in the composition can be from about 100:1 to about 1:100, preferably from 10:1 to 1:10. More preferably, ratio of the compound of formula I and agent is about 1: 1.
  • the agent which decreases the cytotoxicity of vinylguaiacol on skin cells is an antioxidant.
  • an anti-oxidant is any compound or substance, which inhibits or slows the rate of oxidative reactions.
  • antioxidants include ascorbic acid, tocopherols (including alpha-tocopherol), carotenoids (including beta-carotene), ferulic acid, selenium, butylated hydroxytoluene, butyrated hydroxyanisole, ascorbyl esters (including ascorbyl palmitate) caffeic acid, propyl gallate, tertiarybutylhydroxyquinone (TBHQ) flavanoids, flavanols flavenoids and ubiquinone.
  • An antioxidant for the purposes of this invention may be provided in an isolated or purified form. Alternatively, the antioxidant may be provided by a plant extract. Examples of plant extracts providing antioxidants include but are not limited to palm oil or rosemary extract.
  • the agent exhibits both antioxidant and skin lightening properties.
  • Such an agent will decrease the cytotoxicity of the compound of formula I while contributing either independently or synergistically to the skin lightening effect.
  • agents with both antioxidant and skin lightening properties include ferulic acid and ascorbic acid.
  • the agent which decreases the cytotoxicity of a compound of formula I on skin cells is one or more of tocopherol, ferulic acid or ascorbic acid.
  • the composition comprises two or more of tocopherol, ferulic acid and ascorbic acid in combination with a compound of formula I.
  • the tocopherol can be one or more selected from alpha-, beta-, gamma- or delta- tocopherol.
  • the tocopherol can be the D or the L isomer or a mixture of D and L isomers including a racemic mixture.
  • the tocopherol can be provided by a mixture of two or more of alpha-, beta-, gamma-, or delta- tocopherol where the tocopherol can be the D or L isomer or a mixture of D and L isomers (including a racemic mixture).
  • the composition may comprise vinylguaiacol, ferulic acid and alpha-tocopherol.
  • Each of the components can be provided in varying concentrations and ratios.
  • the relative ratios of any two of the components may preferably vary from 100 to 1 fold, preferably 10 to 1 fold.
  • the components may be provided in approximately a 1:1:1 ratio.
  • the composition may comprise vinylguaiacol, ferulic acid and ascorbic acid.
  • the relative ratios of the components may vary from 100 to 1 fold, preferably 10 to 1 fold.
  • the components may be provided in approximately a 1: 1:1 ratio.
  • the composition may comprise ethylguaiacol, ferulic acid and alpha-tocopherol in varying concentrations and ratios.
  • the relative ratios of the components may preferably vary from 100 to 1 fold, preferably 10 to 1 fold.
  • the components may be provided in approximately a 1:1:1 ratio.
  • no one component is present at a concentration of more than 100 times any other one of these components.
  • the composition may comprise ethylguaiacol, ferulic acid and ascorbic acid.
  • the relative ratios of the components may vary from 100 to 1 fold, preferably 10 to 1 fold.
  • the components may be provided in approximately a 1:1:1 ratio.
  • no one component is present at a concentration of more than 100 times any other one of the components
  • the composition may comprise vinylguaiacol and ethylguaiacol in combination with one or more of tocopherol, ferulic acid and ascorbic acid.
  • composition of the first aspect of the invention may comprise a skin lightener in addition to the compound of formula I and the agent.
  • the additional skin lightener can include one or more of kojic acid, hydroquinone, arbutin or a plant extract containing arbutin such as Bearberry extract, glycyrrhizic acid or liquorice extracts containing it, protocatechuic acid and lactic acid.
  • composition may contain other active ingredients with complementary activities to the compound of formula I and/or the other ingredients, such as antioxidants, anti-irritants, anti-microbials, anti-dandruff and anti-acne ingredients.
  • actives responsible for exfoliation e.g. alpha- and beta-hydroxy acids
  • UV absorbtion e.g. UV absorbtion
  • inhibition of collagenase/elastase i.e. anti ageing
  • stabilisation of labile antioxidants e.g. alpha- and beta-hydroxy acids
  • the agents can be replaced by a derivative of the agent which has comparible functional characteristics.
  • any isomer of each of the agents can be used in the composition.
  • alpha-tocopherol can be replaced by any isomer of tocopherol, for example, alpha, beta, gamma or delta forms of D- or L-tocopherol, as discussed above.
  • Vinylguaiacol and/or ethylguaiacol may be provided in encapsulated form.
  • a compound of formula I and an agent in the manufacture of a composition for use as a lightener, wherein the agent decreases the cytotoxic effect of the compound of formula I on skin cells.
  • the composition is provided as a hair or a skin lightener.
  • the composition of the first or second aspects of the invention is preferably provided as a formulation which is applied externally to the skin surface or to the hair.
  • the composition can be in the form of a cream or lotion (including oil in water and water in oil creams and lotions), spray, powder, gel, shampoo, conditioner, mousse, serum, oil, stick or patch.
  • the formulation can be provided specifically as a skin lightening product, or the skin lightening preparation can be added to a formulation for a different use (i.e. sunscreen lotion, day cream, moisturiser, perfume, body spray, bath or shower product).
  • the composition of the first or second aspects of the invention can be provided as a cosmetic product, a pharmaceutical product or a personal care product.
  • cosmetic products are products intended for increasing the appeal, visually and olfactively, of the human body.
  • personal care products are products intended for cleaning, smoothing or otherwise improving the health and well-being of the outside of the human body.
  • the third aspect of the invention provides the use of an agent to decrease the cytotoxicity of the compound of formula I on skin cells.
  • the agent can decrease the cytotoxicity of the compound on skin cells either by increasing the dose of the compound required to only produce 50% cytotoxicity of skin cells and/or by increasing the efficacy and therefore decreasing the dose of the compound required to produce 80% inhibition of melanogenesis.
  • a fourth aspect of the invention relates to a method for lightening the colour of skin or hair.
  • This method comprises applying to skin or hair a composition comprising a compound of formula I and an agent which decreases the cytotoxic effect of the compound on skin cells.
  • the method may be cosmetic or pharmaceutical.
  • the composition can be applied to the skin or hair daily, every other day, biweekly or weekly.
  • the composition can be applied to the skin or hair as a lightening preparation or can be incorporated into a product with another use such as a moisturiser, sun screen, body spray, perfume, bath or shower product, shampoo or conditioner.
  • the compound of formula I and the agent can supplied as a composition with both components premixed.
  • the components can be supplied separately and mixed prior to application.
  • the components may also be supplied separately and applied sequentially.
  • the compounds of formula I can be provided from a number of sources.
  • the compounds are provided in the form of an extract derived from a plant material which has been subsequently treated with a micro-organism.
  • Vinylguaiacol and/or ethylguaiacol may be prepared by a bioprocess which comprises treating a substrate with one or more microorganisms selected from Rhodotorula, Saccharomyces (eg, S.cerevisiae), Paecilomyces, Candida and Paenibacillus; wherein the substrate is selected from ferulic acid or caffeic acid.
  • bioprocesses are natural, that is, they involve biological, especially enzymatic processes.
  • Vinylguaiacol and ethylguaiacol are readily biodegradable because they occur in nature.
  • bioprocesses are not limited to the specific examples, but include micro-organisms and/or enzymic and/or cell free extracts and/or engineered cells or enzymes therefrom which exhibit a suitable enzymic activity.
  • the micro-organism or enzyme or cell-free extract derived therefrom may produce the desired product efficiently and in high yields. This may be quantified in terms of: the rate of production of the product (gl _1 day _1 ); the concentration of the product that accumulates (gl "1 ); the yield of the product obtained from the substrate (g of product per g of substrate or % M yield); and the absence of side products which is reflected in the purity of the isolated product (% purity).
  • the strains may exhibit tolerance to high concentrations of both the substrate and the product, for example at least lgl "1 , preferably in the range of 1 to 40gl "1 , more preferably in the range of 5 to 40gl "1 .
  • the strains may also exhibit high metabolic selectivity for the production of the required products, for example the products may be produced in at least 75% reaction molar yield and at least 50% recovered molar yield, and they have the ability to produce the products as non- growing cells so that, for example, expensive nutrients do not have to be supplied and expensive sterile fermentation equipment does not have to be used.
  • the criteria for establishing suitability of the micro-organism or enzyme or cell-free extract for use in the methods of the invention are as follows:
  • the micro-organism or enzyme or cell-free extract derived therefrom may produce at least lg of the desired product per litre of reaction fluid and/or at least 50% molar yield of the desired product from the substrate (eg ferulic acid or caffeic acid) at a concentration of >0.5gl "1 .
  • the desired product may have a purity of at least 90% as determined by positive characterisation of the product by ab initio analytical methods such as NMR.
  • micro-organism or enzyme or cell-free extract may be capable of being used repeatedly, in two phase reaction systems, as immobilised cells, as disrupted cells, and is capable of reacting with impure substrates, especially plant extracts, if required.
  • the bioprocess includes a biphasic reaction mixture.
  • the biphasic reaction mixture includes an aqueous phase, such as water, and a water immiscible (eg, organic liquid) phase such as vegetable oil, for example miglyol.
  • the water immiscible phase acts as a product 'sink' in which the desired product formed from the substrate accumulates. This prevents accumulation of the product in the aqueous phase to levels which may inhibit or terminate the enzymatic reaction. This results in increased product yields compared to when the bioprocess is performed using a monophasic reaction mixture.
  • the product should be produced over a reasonably short period of time eg 1 to 3 days or less.
  • the test microorganism is isolated using the soil isolation protocol described hereinafter.
  • one or more of the components of the composition are provided in the form of an extract from a plant material. Suitable extracts from plant materials include, for example, a maize extract containing vinylguaiacol (eg, in an amount of greater than 50%).
  • vinylguaiacol and/or ethylguaiacol may be in encapsulated form.
  • the growth medium can contain specified amounts of either, or both, a vitamin supplement and a trace elements supplement. These were prepared as follows.
  • Vitamin supplement biotin (2 mgl “1 ), folic acid (2 mgl “1 ), pyridoxine (10 mgl “1 ), riboflavin (5 mgl “1 ), thiamine (5 mgl “1 ), nicotinic acid (5 mgl “1 ), pantothenic acid (5 mgl “1 ), vitamin B 12 (0.1 mgl “1 ), 4-aminobenzoic acid (5 mgl “1 ), and thioacetic acid (5 mgl “1 ).
  • Trace elements supplement: concentrated hydrochloric acid (51.3 mil “1 ), MgO (10.75 gl “1 ), CaCOs (2.0 gl “1 ), FeSO4.7H 2 O (4.5 gl “1 ), ZnSO 4 .7H 2 O (1.44 gl “1 ), MnSO 4 .4H 2 O (1.12 gl “1 ), CuSO 4 .5H 2 O (0.25 gl “1 ), CoSO 4 .7H 2 O (0.28 gl “1 ), and
  • yeast malt medium comprising: 4g glucose, 4g yeast extracts, lOg malt extract per litre deionised water.
  • Rhodotorula glutinis (IMI 379894) was cultured at 30 °C by shaking at 200 rpm on a yeast malt medium containing (per litre of deionised water): glucose 4g; yeast extract 4g and malt extract lOg. After 40 hours incubation, ferulic acid was added to a final concentration of 2gl "1 . The incubation was continued for a further 21 hours during which time the progress of the reaction was monitored by h.p.l.c. analysis using the conditions described above.
  • Example 2 Preparation of Vinylguaiacol Saccharomyces cerevisiae, "Bakers yeast", (2g, purchased from J. Sainsburys pic under the trade mark Sainsburys Easy Blend) was activated by suspending dry yeast powder in deionised water (20 ml) for 30 minutes at 37 °C.
  • the cells were harvested by centrifugation (15 minutes at 4000rpm), resuspended in 50ml of 0.9% (w/v) NaCl, and then disrupted by passage once through a cell disrupter (operating pressure 30,000psi).
  • ferulic acid To 50ml of the resultant disrupted cell suspension was added ferulic acid at an initial concentration of lOgl "1 .
  • Also added at the same time was 50ml of Miglyol to form an upper organic layer to the biphasic biotransformation mixture. The progress of the reaction was monitored by analysis as described above. After incubation at 30°C for 64 hours the reaction had progressed to a 92% conversion to vinylguaiacol.
  • Ferulic acid was released from maize fibre as follows. A lOg portion of maize fibre was shaken (200 rpm) at 30°C, overnight, in a conical flask with 100 ml of 1M sodium hydroxide solution. The resulting solution was acidified to pH 5.5 prior to the addition of 45 ml of a culture of Rhodotorula glutinis (IMI 379894) which had been grown on yeast malt medium in a 250 ml shake flask and incubated with shaking (200 rpm) at 30 °C for 40 hours. At this time a concentration of 0.495 gl -1 ferulic acid was detected.
  • IMI 379894 Rhodotorula glutinis
  • the resulting suspension was itself incubated at 30 °C with shaking (200 rpm) and the vinylguaiacol concentration monitored by hplc. After 10 minutes a 7.9% conversion of ferulic acid to vinylguaiacol was observed; after 1 hour there was a 29% conversion; after 20 hours a 93 % conversion.
  • the reaction mixture was extracted twice with 50ml of n-hexane and the combined extracts dried and evaporated to yield 48mg of an oil comprising 84% vinylguaiacol.
  • Ferulic acid was released from maize fibre as follows. A 50g portion of maize fibre was shaken (200 rpm) at 30°C, for 15 hours, in a conical flask with 500 ml of 1M sodium hydroxide solution. The resulting solution containing 940 g ferulic acid was neutralised by the addition of concentrated hydrochloric acid. This was added to 1 litre of a culture of Rhodotorula glutinis (IMI 379894) which had been grown on yeast malt medium in a 5 litre shake flask and incubated with shaking (200 rpm) at 30 °C for 24 hours. The mixture was adjusted to pH 5.5 and 1 litre of n-hexane was added.
  • IMI 379894 Rhodotorula glutinis
  • the resulting two-phase system was mixed gently (80 rpm) at 30°C. After 24 hours the two liquid phases were separated and the aqueous re-extracted with 500 ml of n-hexane.
  • the combined organic solvent phases which contained 540 mg vinylguaiacol (75 % yield) were dried and evaporated to yield an oil (740 mg) which was 65 % vinylguaiacol by assay. This represents a 66% recovery of vinylguaiacol from ferulic acid.
  • Example 5 Production of Ethylguaiacol from ferulic acid using C. versitalis
  • Candida versitalis (NCYC 1433) was grown from a plate culture inoculum for 6 days in yeast malt medium containing lOg/L malt extract, 4g/L yeast extract, 4g/L glucose, and 2% sodium chloride dissolved in deionised water and autoclaved at 120°C. The 50ml culture was incubated at 30 °C and 200 rpm in a 250ml conical flask.
  • this culture was used to provide a 10% inoculum for a 150ml second culture of the yeast malt medium occupying 50% v/v of the flask. This was incubated at 21-22°C for 24 hrs while agitating at 150 rpm. Then ferulic acid was added to a concentration of 2g/L, together with 100 ml of Miglyol, which alternatively could be added after 50 hrs when the concentration of ethylguaiacol in the aqueous phase had reached 0.25-0.3g/L.
  • the concentration of ethylguaiacol in the Miglyol was 3.64g/L, which represents 92 to 94% of the theoretical maximum yield.
  • the ethylguaiacol could be easily recovered from the Miglyol as a pure chemical by solvent extraction into hexane and then rotary evaporation to dryness.
  • Vinylguaiacol was produced from ferulic acid by microbial bioconversion as described in Example 1 or Example 2.
  • Ethylguaiacol was recovered in a yield of 72% from the starting ferulic acid, and with no residual vinylguaiacol remaining in this recovered ethylguaiacol product.
  • Candida versitalis (Zyl 866; NCYC 1433) was grown (from a 10% inoculum) in
  • Table 1 Preparation of compounds of the invention from ferulic acid and selected microorganisms.
  • Example 8 Preparation of vinylguaiacol from ferulic acid using Paenibacillus polymyxa Paenibacillus polymyxa (Zyl 277; IMI CC Deposit No 382464) cells were grown at 30° C, shaking at 200 rpm for 27 hours on a medium comprising per litre deionised water: (NH SO 4 , 5g; K 2 HPO 4 , 2g; NaCl, 0.2g; glucose, lOg; malt extract, 3g; yeast extract, 3g; MgSO4, 0.22g; CaCb, 0.015g; ferulic acid, 0.5g.
  • the cells were harvested by centrifugation (4,000 x g 15 m') washed with 0.9% (w/v) saline solution followed by resuspension in 0.9% (w/v) saline solution as a 20-fold concentration.
  • An aliquot of concentrated cells (5ml) was added to a solution of sodium alginate (15ml 3.5% w/v) and mixed thoroughly, prior to addition dropwise from a 3ml plastic pipette into 1 litre of 0.2M CaCL solution.
  • the beads formed by this procedure were stored at 4°C overnight in CaCk solution to harden before washed in 21 of tap water.
  • the beads interspersed with an inert packing material were packed into a 100ml glass column.
  • a solution of ferulic acid in tap water 500 ml, 6g/l was pumped continuously through the column at a temperature of 24 °C and the pH of this solution was maintained at pH 7.0.
  • the aqueous stream exiting the top of the column was continuously extracted into hexane (500 ml) to remove vinylguaiacol, prior to returning to the column.
  • Example 9 Production of Vinylguaiacol from Ferulic Acid by Paenibacillus polymyxa (ZYL277) in a Two Phase System Paenibacillus polymyxa (Zyl 277; IMI CC Deposit No 382464) was grown in a bioreactor in a medium containing (g/1) (NH4) 2 SO 4 , 5; K>HPO 4 , 2; NaCl, 0.2; yeast extract, 2; malt extract, 2; glucose, 10; ferulic acid, 0.5; lOml/1 of a solution containing 0.1M MgSO4/0.01M CaCk; at 30°C, pH 6.0, oxygen 70% on a stirrer cascade (100-500 rpm).
  • the hexane layer was removed periodically and replaced with 100ml of new hexane to prevent it becoming saturated with vinylguaiacol.
  • Vinylguaiacol concentrations in both phases at the time of changing the hexane phase are shown below along with the cumulative total ferulic acid added to the aqueous phase (g/1).
  • Tyrosine To 171.4 ⁇ l of a solution of tyrosine (1.5mM) in phosphate buffer (lOOmM, pH6.4) was added of an inhibitor solution (lOO ⁇ g ml "1 ) in phsophate buffer (lOOmM, pH6.4) in a quartz cuvette. The reaction mixture was made up to 980 ⁇ l with phosphate buffer (lOOmM, pH6.4) and the reaction initiated by the addition of tyrosinase (Sigma, 220 ⁇ l, 1100 units/ml in phosphate buffer (lOOmM, pH6.4)), The reaction was monitored by the increase in absorbance at 470nm over 10 minutes.
  • Each 96 well assay plate included a solvent control and a blank control. Eight concentrations of kojic acid were used as a positive control.
  • the concentration of cells was determined by counting an aliquot of the stock cell suspension in a haemocytometer. A seeding suspension of 5 X 10 4 cells/ml was prepared in assay medium. One hundred microlitres of the seeding cell suspension was added to the appropriate wells on each 96 well plate. One , hundred microlitres of growth medium was added to the outer wells to maintain humidity. The cells were incubated at 37 +/- 1°C in a humidified atmosphere containing 5 47- 1 % CO 2 in air for 24 hours.
  • test article Based on the information available for the test article, eight decreasing doses were selected and used in the assay.
  • the highest dose of test article was a concentration of 5 mg/ml, based on its solubility in the solvent.
  • the maximum solvent concentration (other than assay medium) was 1 % .
  • test article and positive control was tested by treating six wells per dilution of B16-F1 cells seeded approximately 24 hours earlier. Prior to treatment the medium was removed, and 200 ⁇ l of the pre-warmed (37 +/- 1°C) test article dilutions, positive control dilutions and solvent control, were added to the appropriate wells. All test article concentrations were dosed into a single outer well (200 ⁇ l) of the corresponding plate to serve as a turbidity control. The remaining wells around the edge of the plate, designated as blanks, received 200 ⁇ l of assay medium prior to incubation. Following dosing, the plates were incubated at 37 +/- 1°C for 96 hours +/- 2 hours.
  • the cells remaining in the assay plate were used to determine the cytotoxicity of the test article (cytotoxicity plate).
  • the remaining media was removed and 100, ⁇ l of growth medium containing 25 ⁇ g/ml of neutral red was added to each well.
  • the plates were returned to the incubator for 3 hours +/- 5 minutes, after which time the NR medium was decanted and the cells washed with 150 ⁇ l of PBS.
  • the PBS was removed by gently tapping the plate.
  • One hundred and fifty ⁇ l of NR desorb solution was added to each well. After a minimum of 20 minutes incubation at room temperature, the plates were agitated and the absorbance of the neutral red was measured at 540 nm (OD540) with an Anthos 2010TM microplate reader.
  • the mean of the six OD405 values for each concentration of test article and positive control was calculated.
  • the turbidity control values were subtracted to obtain a correct OD405 value.
  • the mean of the six OD405 values for the corresponding negative or solvent control were calculated by subtracting the blank control value.
  • the corrected OD405 values of the test article or positive control was divided by the corrected OD405 value for the negative or solvent control to obtain a melanin production ratio.
  • the mean of the six OD405 values for each concentration of test article and positive control was calculated.
  • the blank control value was subtracted to obtain a corrected OD405 value.
  • the mean of the six OD540 values for the corresponding negative or solvent control was calculated in the same way.
  • the corrected OD540 values of the test article or positive control was divided by the corrected OD540 value for the negative or solvent control to obtain a cytotoxicity ratio.
  • vinylguaiacol can be used at higher concentrations and therefore have a greater skin lightening effect without affecting cells or skin viability.
  • ⁇ -tocopherol and ascorbic acid have no skin lightening effects themselves and so act as diluents of the activity of the vinylguaiacol. Nevertheless despite the reduction in melanogenesis inhibition activity, the gap between the concentrations of vinylguaiacol that cause 80% inhibition of melanogenesis and 50% cytotoxicity is increased significantly.
  • Ferulic acid is itself a good inhibitor of melanogenesis and so a good improvement in the gap between the inhibition and cytotoxic effects is achieved without a reduction in the dose of active required to achieve 80% inhibition of melanogenesis.
  • compositions of the invention are provided.
  • cosmetic products are products intended for increasing the appeal, visually and olfactively, of the human body.
  • personal care products are products intended for cleaning, smoothing or otherwise improving the health and well-being of the outside of the human body.
  • the compounds of the invention may be used in food and beverage compositions.
  • Phase A to Phase B. Mix at 80°C. Cool to 60°C and add Phase C. Cool to 50 °C and package.
  • phase B Add phase B to phase A; disperse well and hold at 45 °C. Then add phase C slowly. Hold at 45°C, mix well then add phase D. Cool and mix to 30°C and pack.
  • a formulation was produced containing a compound of the invention by formulating the following ingredients:
  • qs indicates that the components are added "to taste” i.e. to a preferred amount.
  • Exemplary micro-organisms suitable for use in accordance with the present invention have been deposited for the purposes of patent procedures under the Budapest Treaty with the IMI Genetic Resource Reference Collection which is an International Depositary authority recognised under the Treaty.
  • the address of the IMI Collection is CABI Bioscience UK Centre Egham, Genetic Resource Collection, Bakeham Lane, Egham, Surrey, England TW20 PTY telephone 01784 470111, fax 01491 829100, e-mail bioscience@cabi.org.

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Abstract

La présente invention concerne l'utilisation de vinylguaïacol et/ou d'éthylguaïacol comme agent éclaircissant pour la peau conjointement avec un agent qui réduit la cytotoxicité du vinylguaïacol et/ou de l'éthylguaïacol sur les cellules de la peau. Ainsi, l'invention concerne une composition éclaircissante pour la peau qui réduit les effets cytotoxiques sur les cellules de la peau, et un agent.
PCT/GB2001/003516 2000-08-14 2001-08-03 Agents eclaircissants pour la peau Ceased WO2002013779A1 (fr)

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AU2001275764A AU2001275764A1 (en) 2000-08-14 2001-08-03 Skin lightening agents

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GBGB0020000.6A GB0020000D0 (en) 2000-08-14 2000-08-14 Skin lightening agents

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005070383A1 (fr) * 2004-01-24 2005-08-04 Unilever Plc Compositions eclaircissantes pour la peau
WO2011129765A1 (fr) * 2010-04-16 2011-10-20 Davos Life Science Pte. Ltd. Interaction synergique d'au moins un constituant de vitamine e et d'inhibiteurs de tyrosinase pour applications dermatologiques
WO2014205813A1 (fr) * 2013-06-28 2014-12-31 L'oreal Composition de prévention ou de réduction de la pigmentation de la peau et d'éclaircissement du teint de la peau, et utilisations associées
EP3056193A1 (fr) * 2015-02-12 2016-08-17 Ems S.A. Composition cosmétique et usage correspondant
CN119745726A (zh) * 2024-11-18 2025-04-04 广州市海菲生物科技有限公司 一种植物止痒洗发水

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047179A1 (fr) * 1999-02-13 2000-08-17 Zylepsis Limited Agents eclaircissants pour la peau

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047179A1 (fr) * 1999-02-13 2000-08-17 Zylepsis Limited Agents eclaircissants pour la peau
WO2000047045A1 (fr) * 1999-02-13 2000-08-17 Zylepsis Limited Composes et compositions de conservation

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005070383A1 (fr) * 2004-01-24 2005-08-04 Unilever Plc Compositions eclaircissantes pour la peau
WO2011129765A1 (fr) * 2010-04-16 2011-10-20 Davos Life Science Pte. Ltd. Interaction synergique d'au moins un constituant de vitamine e et d'inhibiteurs de tyrosinase pour applications dermatologiques
WO2014205813A1 (fr) * 2013-06-28 2014-12-31 L'oreal Composition de prévention ou de réduction de la pigmentation de la peau et d'éclaircissement du teint de la peau, et utilisations associées
EP3056193A1 (fr) * 2015-02-12 2016-08-17 Ems S.A. Composition cosmétique et usage correspondant
CN119745726A (zh) * 2024-11-18 2025-04-04 广州市海菲生物科技有限公司 一种植物止痒洗发水

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