Classifications

• • 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
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products 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
  • A61K8/96—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
  • 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
• • 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
• • 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
  • A61Q19/08—Anti-ageing preparations
• • 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
  • A61Q5/002—Preparations for repairing the hair, e.g. hair cure
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
• • 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
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

• the invention relates to protein hydrolysates obtained from azuki beans and their use, in particular, in the cosmetic treatment and/or care of the skin and/or hair.
• the protein hydrolysates are useful in protecting the skin and/or hair against adverse effects of the exposome, including adverse effects arising from air pollution and/or sunlight. Further, the protein hydrolysates are useful in protecting hair against chemical and heat stress, for example, chemical and heat stress arising from personal care regimes. Methods of preparing the protein hydrolysates are also disclosed.
• the totality of extrinsic factors to which an individual is exposed over a lifetime is referred to as the exposome.
• the appearance of skin, the appearance of hair, and aging are influenced by the combined action of intrinsic and extrinsic factors.
• Intrinsic factors that negatively impact skin and hair include, for example, chronological aging, a person’s genetic makeup, and other biological changes that occur from within the skin. For decades, external damaging factors on the skin and hair have been ignored. However, there is growing evidence that the exposome can have an adverse effect on skin and hair appearance [Vierkbtter A1 , et al. Airborne particle exposure and extrinsic skin aging. J Invest Dermatol. 2010; and Paarado C et al “Environmental Stressors on Skin Aging. Mechanistic insights”, Front Pharmacol. 2019; 10:759, published 9 Jul. 2019, doi : 10.3389/fphar.2019.00759],
• extrinsic factors associated with the exposome include exposure to ultraviolet (UV) rays emanating from the sun, high energy visible light (violetblue) emitted by devices such as TV/computer/smartphone screens, as well as harmful chemical agents found in airborne pollution such as smog and cigarette smoke.
• UV ultraviolet
• violetblue high energy visible light
• UV radiation is one of the main external factors that has a detrimental effect on the appearance of skin and hair. Extrinsic aging has been regarded to be mainly the result of sun radiation exposure, also known as photoaging.
• Air pollutants can have major effects on the function of the skin. Alterations that disturb the skin barrier function, in either stratum corneum lipid metabolism or protein components of the corneocytes, adversely affect skin appearance. Air pollutants may induce severe interference of normal functions of lipids, DNA and/or proteins of the human skin via oxidative damage [Eleni Drakaki, Clio Dessinioti and Christina V. Antoniou. Air pollution and the skin. Front. Environ. Sci., 2014],
• ROS reactive oxidative species
• the production of ROS can affect the skin’s defenses by quickly depleting the enzymatic (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase) and nonenzymatic (vitamin E, vitamin C, and glutathione) antioxidant capacity [Valacchi G, et al. Cutaneous responses to environmental stressors. Ann N Y Acad Sci. 2012], In hair, oxidative stress can also have significant negative effects.
• Proteins are the principal components of hair shafts (65% to 95%) and protein carbonylation, which is an irreversible oxidative protein modification that can significantly damage hair.
• the protection of hair proteins from carbonylation due to pollutants is desirable for hair and scalp protection against urban stress. [Baraibar, Martin. Urban pollution induces irreversible carbonylation of hair proteins. Conference: IFSCC, 2018], [008]
• skin and, in particular, hair are also subjected to other stressors from daily personal care routines. Diverse causes of extrinsic damage to the hair shaft can be roughly divided into physical and chemical causes.
• the human hair shaft consists of the cortex with a central axial medulla and an external cuticular layer.
• Chemical causes of hair damage include bleaching and hair dyeing, while physical causes include thermal treatments (such as treatments with straightening irons), friction from hair accessories and washing [Lee Y, et al. Comparison of hair shaft damage after chemical treatment in Asian, White European, and African hair. Int J Dermatol. 2014],
• extrinsic factors counteracting exposome factors (i.e. extrinsic factors) is of particular interest because extrinsic factors can be modified more easily than intrinsic factors.
• US20210177733 discloses a composition and method for enhancing the skin’s ability to defend itself against free radical aggression caused by exposome- induced oxidative stress.
• the compositons comprise leaf extract of Aristoteiia chilensis leaf extract of Buddleja globosa; and leaf extract of Ugni molinae.
• ZoryalysOfrom Greentech is a natural active ingredient “derived from several molecules: Shogaol & Gingerol (grows in sunny areas), Magnolol & Honokiol (lives in polluted areas) and Pracaxi (biomimetic lipid) /).
• ZORYALYS® is described as protecting the hair against protein oxidation and being active from the surface to the heart of the hair fiber allowing a full protection of hair against exposome aggressions. This active is described as maintaining hair mechanical properties and improving hair shine for a healthy and beautiful hair, by maintaining keratin integrity.
• Peptides released from extruded adzuki bean protein through simulated gastrointestinal digestion that exhibit anti-flammatory activity are disclosed in Zhensing Shi et al, “Peptides released from extruded adzuki bean protein through simulated gastrointestinal digestion exhibit anti-flammatory activity”, J. Agric. Food Chem. 2021 , 69, 7028-7036.
• the present invention sets out to meet some or all of the above-identified needs and to solve some or all of the above-identified problems.
• the invention relates to a protein hydrolysate of azuki beans comprising at least one compound (i.e. 1 , 2, 3 or 4 compounds) chosen from:
• the protein hydrolysate may additionally comprise the compound:
• the protein hydrolysate of the invention is useful in alleviating or preventing adverse effects of the exposome on the skin and/or hair.
• Adverse effects to the skin and/or hair include those arising from air pollution and/or sunlight.
• Adverse effects to the hair also include those due to chemical treatment such as dyeing and physical treatment such as from styling aids, e.g. hair dryers or straightening irons.
• Adverse effects to hair can manifest themselves in the form of hair that: is damaged; is less manageable; has more split ends; has reduced fiber alignment; has increased frizz; and/or is less hydrated.
• Adverse effects to skin can manifest themselves in the form of a reduction in barrier function; a dull appearance of the skin; a loss of brightness, luminosity and/or glossiness of the skin; a loss in homogeneity in the texture of the skin; an increase in roughness of the skin; the appearance of dark spots; and/or erythema (skin redness).
• the invention relates to a compound chosen from:
• the invention relates to combination of the protein hydrolysate according to the first aspect of the invention and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc. It has been found that this combination results in a synergistic effect with respect to antioxidant activity.
• the invention also extends to the combination of a compound chosen from:
• the invention relates a process for preparing a protein hydrolysate of azuki beans, wherein the hydrolysate is obtained by: a) providing a suspension of ground azuki beans in water; b) treating the suspension with alkali so as to solubilize proteins in the ground azuki beans; c) enzymatically treating the suspension obtained in b) with a serine endoprotease; d) deactivating the serine endoprotease in the suspension obtained in c) to form a crude hydrolysate; e) recovering a supernatant from the the crude hydrolysate by solid/liquid separation; and f) removing components from the supernatant having a molecular weight of greater than 3 KDa.
• the process is useful in preparing protein hydrolysates according to the first aspect of the invention.
• the invention extends to protein hydrolysates obtained by the process of preparing a protein hydrolysate of azuki beans described herein.
• the invention relates to a personal care or cosmetic composition comprising at least one cosmetically acceptable excipient or ingredient and a protein hydrolysate of azuki beans according to the first aspect of the invention, or a compound chosen from:
• the invention relates to the use of a protein hydrolysate of azuki beans according to the first aspect of the invention, or a compound chosen from:
• Glu-Tyr-Thr-Gly-Asp-Ala-Ala-Gly-Arg SEQ ID No.5
• stereoisomers, and cosmetically acceptable salts thereof or a combination of said compound and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc for the treatment and/or care of the skin and/or hair.
• the treatment and/or care of the skin and/or hair is cosmetic and non- therapeutic.
• the invention relates to a method of treatment and/or care of the skin and/or hair comprising administering a protein hydrolysate of azuki beans according to the first aspect of the invention, or a compound chosen from:
• the treatment and/or care includes: the alleviation or prevention of the adverse effects of the exposome on the skin and/or hair; the alleviation or prevention of the adverse effects of air pollution on the skin and/or hair; the alleviation or prevention of the adverse effects of sunlight on the skin and/or hair; the alleviation or prevention of the adverse effects of chemical treatment of the hair; the alleviation or prevention of the adverse effects of heat stress on the hair; and/or the maintenance and/or improvement of the physical barrier function of the skin.
• the invention is based on the finding of surprising properties of a protein hydrolysate of azuki beans, which properties lend the protein hydrolysate to cosmetic, non-therapeutic applications on the skin and/or hair.
• skin is understood to be the layers which comprise it, from the uppermost layer or stratum corneum to the lowermost layer or hypodermis, both inclusive. These layers are composed of different types of cells such as keratinocytes, fibroblasts, melanocytes and/or adipocytes among others.
• the term “skin” includes the scalp.
• skin includes the skin of mammals, for example, the skin of humans, and includes skin comprising hair.
• hair includes the hair of the scalp, the skin, the eyelashes, the eyebrows, moustache region and/or beard region of a subject.
• treatment as used in the context of this specification when accompanied by the qualification “cosmetic”, it means that the treatment is non- therapeutic and has the aim of improving the aesthetic appearance of the skin and/orthe hair and includes improving the properties of the skin and/or hair such as, but not restricted to, the level of hydration, elasticity, firmness, shine, tone or texture, which properties affect the cosmetic appearance of the skin and/or hair.
• care in the context of this specification refers to the maintenance of the properties of the skin and/or hair.
• the properties of the skin and/or hair are subject to improvement and maintenance through cosmetic treatment and/or care both in healthy subjects as well as those who present diseases and/or disorders of the skin and/or hair, such as and not restricted to, ulcers and lesions on the skin, psoriasis, dermatitis, acne or rosacea, alopecia, among others.
• prevention refers to the ability of the active of the invention to prevent, delay or hinder the appearance or development of a symptom or manifestation of an adverse effect on the skin and/or hair.
• alleviation refers to the ability of the active of the invention to reduce or lessen a symptom or manifestation of an adverse effect on the skin and/or hair.
• the hyphen which represents the peptide bond, eliminates the OH in the 1- carboxyl group of the amino acid (represented here in the conventional non-ionized form) when situated to the right of the symbol, and eliminates the H of the 2-amino group of the amino acid when situated to the left of the symbol; both modifications can be applied to the same symbol (see the table below).
• the invention relates to a protein hydrolysate of azuki beans.
• the azuki bean is also known as Vigna angularis, and is a legume that was originally grown in China and high-altitude terrains near Vietnam, and is consumed worldwide because it is a rich source of proteins (percentage> 20%).
• Vigna angularis is also known as adzuki bean (Japanese: /J'SE (7 +), azuki, adzuki), azuki bean, or red mung bean.
• a protein hydrolysate is a mixture of fragments obtained from the hydrolytic degradation of proteins and comprises, e.g., free amino acids and peptides of different molecular weights and compositions.
• the protein hydrolysate of the invention can be obtained from the enzymatic hydrolysis of proteins of azuki beans.
• the protein hydrolysate comprises at least one compound (i.e. 1 , 2, 3 or 4 compounds) chosen from:
• the protein hydrolysate can comprise:
• the protein hydrolysate can comprise:
• the protein hydrolysate can comprise:
• the protein hydrolysate can comprise:
• the protein hydrolysate can comprise:
• the protein hydrolysate in particular the protein hydrolysate having any of the combinations of compounds labelled (i), (ii), (iii) or (iv) given above, may additionally comprise the compound:
• each of the compounds labelled (i) to (v) above has been found to have excellent antioxidant activity. Without wishing to be bound by theory, it is believed that this activity contributes to the advantageous skin/hair treatment and/or care properties exhibited by the protein hydrolysate of the invention. [0045] When present in the protein hydrolysate, each of these compounds labelled (i) to (v) above is present in the protein hydrolysate in an amount of from 0.001 to 0.15 ppm or 0.005 to 0.08 ppm.
• the compound labelled (i) above can be present in the protein hydrolysate in an amount of from 0.001 to 0.1 ppm, 0.005 to 0.05 ppm, or 0.01 to 0.03 ppm.
• the compound labelled (ii) above can be present in the protein hydrolysate in an amount of from 0.001 to 0.1 ppm, 0.005 to 0.05 ppm, or 0.015 to 0.04 ppm.
• the compound labelled (iii) above can be present in the protein hydrolysate in an amount of from 0.001 to 0.1 ppm, 0.005 to 0.05 ppm, or 0.01 to 0.03 ppm.
• the compound labelled (iv) above can be present in the protein hydrolysate in an amount of from 0.005 to 0.15 ppm, 0.01 to 0.1 ppm, or 0.03 to 0.08 ppm.
• the compound labelled (v) above can be present in the protein hydrolysate in an amount of from 0.001 to 0.15 ppm, 0.001 to 0.01 ppm, or 0.003 to 0.008 ppm.
• the protein hydrolysate can have a molecular weight of less than or equal to 3 kDa.
• the protein hydrolysate can be absent of any components having a molecular weight of greater than 3 kDa, i.e. it can contain no components having a molecular weight of greaterthan 3 kDa. This can be achieved by filtering the hydrolysate.
• the protein hydrolysate can be obtained by treating a suspension of ground azuki beans in water with alkali to solubilize proteins in the ground azuki beans, treating the resultant suspension with a serine endoprotease, deactivating the serine endoprotease in the suspension to form a crude hydrolysate, and purifying the crude hydrolysate.
• the enzymatic hydrolysis of the proteins in the ground azuki beans is carried out in a suspension (a suspension of ground azuki beans in water in which proteins in the auki beans have been solubilized), not on any form of isolated protein.
• Purifying can comprise removing any components having a molecular weight of greater than 3kDa from the crude hyrolysate.
• the protein hydrolysate of the invention is the purified product of this process.
• the process may not comprise steps of isolating proteins from the ground azuki beans and subjecting the isolated proteins to enzymatic hydrolysis.
• the process may not comprise subjecting proteins isolated from azuki beans to enzymatic hydrolysis and, as a result the protein hydrolysate will comprise components other than fragments of proteins, such as polyphenols and other organic compounds.
• the protein hydrolysate can comprise polyphenols in an amount of at least 0.05 mg of polyphenols per milliliter of protein hydrolysate, particularly at least 0.15 mg of of polyphenols per milliliter of protein hydrolysate.
• the amount of polyphenols can be in the range of 0.05 to 0.30 mg/mL, more particularly 0.15 to 0.25 mg/mL.
• the amount of polyphenols may be measured by the Folin-Ciocalteu method and can particularly expressed as mg of gallic acid equivalents per mL of protein hydrolysate.
• the Folin-Ciocalteu method is a well known method for a person skilled in the analysis of compositions derived for botanicals and is extensively descrived in the literature (e.g. Lamuela-Raventos, “Folin-Ciocalteu method forthe measurement of total phenolic content and antioxidant capacity” 2018, Measurement of Antioxidant Activity & Capacity: Recent Trends and Applications).
• the protein hydrolysate can comprise polyphenols in an amount of at least 2 mg of polyphenols per g of protein hydrolysate (in dry weight), particularly at least 6 mg/g. In particular the amount of polyphenols can be in the range 2 to 12 mg/g, more particularly 6 to 9 mg/g.
• polyphenols is meant naturally occurring organic compounds characterized by the presence of multiple phenol units. Polyphenols include for example flavonoids, tannic acid, and ellagitannin.
• the protein hydrolysate can have a dry weight of 2 to 5 mg; a protein content of 19 to 35 w/w %; a DPPH activity of 74 to 80 %; and a polyphenols content of 0.05 to 0.30 mg/mL or 2 to 12 mg/g.
• the protein hydrolysate can be prepared according to the following steps: a) to d) to obtain a crude hydrolyslate, followed by purification of the crude hydrolysate.
• Step a) involves providing a suspension of ground azuki beans in water.
• the azuki beans are raw azuki beans.
• the ground azuki beans can be obtained by grinding azuki beans into a flour.
• the flour can be sifted to remove larger fragments.
• the ground azuki beans have a particle size of less than 250 micrometers.
• Step b) involves treating the suspension of ground azuki beans in water with alkali so as to solubilize proteins of the ground azuki beans in the water.
• the purpose of this step is to increase the content of protein solubilized in the suspension.
• this step involves adjusting the pH of the suspension to a pH of from about 6.5 to about 7.5, for example, to a pH of 7.
• the pH of the suspension affects the amount and type of proteins in the suspension. This can be achieved by adding an alkali such as NaOH to the suspension of ground azuki beans in water.
• step b) is carried out at room temperature, i.e., at 22 ⁇ 2°C.
• Step c) involves enzymatically treating the suspension obtained in step b) with a serine endoprotease (also known as a serine endopeptidase).
• the serine endoprotease can be a subtilisin A.
• Subtilisin A (classification EC 3.4.
• peptidase family S8 is also known as Subtilisin Carlsberg, Subtilopeptidase A, Alcalase®, ALK- enzyme, bacillopeptidase A, Bacillus subtilis alkaline proteinase bioprase, bioprase AL, colistinase, genenasel, Esperase®, maxatase, protease XXVII, thermoase, superase, subtilisin DY, subtilopeptidase, SP 266, Savinase®, kazusase, protease VIII, protin A 3L, orientase 10B, or protease S.
• Subtilisin A is commonly obtained from Bacillus licheniformis.
• Step c) is carried out at a temperature of from 20 to 30°C, and can be carried out at a temperature of from 23 to 27°C or at a temperature of about 25°C.
• Step c) can be carried out for a period of about 30 to about 120, about 45 to about 90, or about 60 minutes, for example.
• the enzyme activity per g of protein in the ground azuki beans is from 0.1 to 0.5 AU/g.
• AU represents Anson Units.
• An Anson Unit is a unit of enzyme concentration which is defined as the amount of enzyme that can digest urea-denatured hemoglobin at the same initial rate as one milliequivalent of tyrosine at standard conditions (25C and pH 7.50).
• the percentage of protein in the ground azuki beans is from 15 to 25 % w/w.
• the concentration of ground azuki beans in water is 20 -100 g (ground azuki beans)/Kg (water).
• Step d) involves deactiviating the enzyme to give a crude isolate.
• step c) the pH of the suspension (undergoing enzymatic hydrolysis) is monitored. A drop of pH is associated with the release of peptide fragments and subsequent build up of acidic carboxylic acids. Typically, during step c) the suspension undergoing enzymatic hydrolysis suffers slight acidification, resulting in a drop of 0.2 to 0.3 pH units, for example.
• the enzyme is deactivated by increasing the temperature of the suspension obtained in c) to at least 70°C, or to from 70 to 90 °C, or to from 75 to 85°C.
• the suspension obtained in step c) can be obtained at this temperature for a period of time, preferably for at least about 20 minutes.
• the product of step d) is crude hydrosylate.
• Step d) The crude hydrosylate product of step d) is then subjected to purification to remove any components having a molecular weight of greater than 3kDa. This can be achieved by solid/liquid separation (step e)) and ultrafiltraton (step f)).
• Step e) is a purification step and involves removing solid material from the crude hydrolysate by solid/liquid separation. Suitable separation techniques are known in the art and include, for example, filtration, sedimentation, decantation and/or centrifugation. Filtration can be carried out using filters having a pore size of lower than 1000 pm, or lower than 20 pm, or lower than 10 pm, or lower than 1 pm, or lower than 0.1 pm.
• Step f) is also a purification step and involves ultrafiltration to remove fragments from the supernatant obtained in step e) that have a molecular weight of greater than 3 kDa.
• Ultrafiltration is a technique that is well know in the art and involves the use of a membrane to separate molecules with different molecular weight, in this case with a cut off threshold of 3 kDa.
• the invention extends to a protein hydrosylate obtained by the aboved described process.
• the invention extends to a compound chosen from:
• Each of the compounds labelled (i) to (v) above has been found to have excellent antioxidant activity. Without wishing to be bound by theory, it is believed that this activity contributes to the advantageous skin/hair treatment and/or care properties exhibited by the protein hydrolysate of the invention.
• the invention also extends to a combination of the protein hydrolysate according to the first aspect of the invention and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc.
• the invention extends to the combination of a compound chosen from:
• This filtrate obtained by the fermentation of Raphanus sativus (radish) root by the microorganism Leuconostoc contains antimicrobial peptides and acts as a preservative.
• One example of commercially filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc is Leucidal® (INCI Name: Leuconostoc/Radish Root Ferment Filtrate) from Active Micro Technologies LCC.
• Leucidal® is typically employed as a preservative. It has been found that the antioxidant activity of the protein hydrolysate of the invention is unexpectedly boosted in the presence of this filtrate.
• the invention includes a composition which may be a personal care composition or a cosmetic composition, and which comprises the protein hydrolysate as described herein, together with at least one cosmetically acceptable excipient or adjuvant.
• the inventon extends to a composition which may be a personal care composition or a cosmetic composition and which comprises: a combination of the protein hydrolysate as described herein and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc; together with at least one cosmetically acceptable excipient or adjuvant.
• the inventon extends to a composition which may be a personal care composition or a cosmetic composition, comprising a compound chosen from:
• composition which may be a personal care composition or a cosmetic composition and which comprises: a combination of a compound chosen from:
• compositions can be prepared by conventional means known to persons skilled in the art. [“Harry’s Cosmeticology”, Seventh edition, (1982), Wilkinson J.B., Moore R.J., ed. Longman House, Essex, GB], [0063]
• the compositions contain a cosmetically effective amount of the protein hydrolysate, or compound chosen from:
• Glu-Tyr-Thr-Gly-Asp-Ala-Ala-Gly-Arg SEQ ID No.5
• stereoisomers, and cosmetically acceptable salts thereof which should be administered, as well as their dosage, will depend on numerous factors, including age, state of the patient, the nature or severity of the condition, disorder or disease to be treated and/or cared for, the route and frequency of administration and of the particular nature of the compounds to be used.
• the terms “cosmetically effective amount” is understood to mean a non-toxic but sufficient amount of extract of the invention to provide the desired effect.
• the protein hydrolysate can be present in an amount of at least 0.001 wt %, particularly from about 0.001 to 10 wt %, preferably from about 0.004 to 5 wt%, based on the total weight of the personal care or cosmetic composition.
• compositions can be compositions for topical application which optionally include cosmetically acceptable excipients necessary for formulating the desired administration form.
• Topical compositions are compositions suitable for the topical application to mammalian keratinous tissue such as skin or hair.
• compositions for topical or transdermal application can be produced in any solid, liquid or semisolid formulation, such as and not restricted to, creams, multiple emulsions such as and not restricted to, oil and/or silicone in water emulsions, water-in- oil and/or silicone emulsions, water/oil/water or water/silicone/water type emulsions and oil/water/oil or silicone/water/silicone type emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, liniments, sera, soaps, shampoos, conditioners, serums, polysaccharide films, ointments, mousses, pomades, powders, bars, pencils and sprays or aerosols (sprays), including leave-on and rinse-off formulations.
• creams such as and not restricted to, creams, multiple e
• topical or transdermal application formulations can be incorporated using techniques known by the person skilled in the art into different types of solid accessories for example and not restricted to, bandages, gauzes, t-shirts, socks, tights, underwear, girdles, gloves, diapers, sanitary napkins, dressings, bedspreads, wipes, adhesive patches, non-adhesive patches, occlusive patches, microelectric patches or face masks, or they can be incorporated into different make-up products such as makeup foundation, such as fluid foundations and compact foundations, make-up removal lotions, make-up removal milks, under-eye concealers, eye shadows, lipsticks, lip protectors, lip gloss and powders among others.
• compositions of the invention can be in a form chosen from creams, multiple emulsions, solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydroglycolic solutions, hydrogels, liniments, soaps, shampoos, conditioners, serums, polysaccharide films, ointments, mousses, pomades, powders, bars, pencils, sprays or aerosols.
• compositions of the invention may include agents which increase the percutaneous absorption of the compounds of the invention, for example and not restricted to, dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone (1 -dodecylazacycloheptane-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol or polyethylene glycol, among others.
• agents which increase the percutaneous absorption of the compounds of the invention for example and not restricted to, dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone (1 -dodecylazacycloheptane-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol or polyethylene glycol, among others.
• compositions are for topical application and are hair care compositions such as conditioners, treatments, hair tonics, styling gels, mousses, shampoos, hair sprays, pomades, setting lotions, coloring and permanent waving compositions.
• hair care compositions such as conditioners, treatments, hair tonics, styling gels, mousses, shampoos, hair sprays, pomades, setting lotions, coloring and permanent waving compositions.
• tonics, conditioners, treatments, and styling gels which may be in the form of a gel, a lotion, a tincture, a spray, a mousse, a cleansing composition or a foam and which may be applied according to individual needs, e.g., once daily as a lotion, tincture, mousse or spray; or once or twice weekly as a conditioner or treatment.
• suitable compositions in the context of this invention are lotions, a shampoo, a serum or eyelashes mascara.
• the cosmetic composition is a serum.
• serum is well known by the person skilled in the art and refers to compositions that are clear, gel-based or liquid. Serum has a fluid texture, and is more concentrated in active agents than a standard care product.
• compositions may include other active ingredients such as antiwrinkle agents, botox-like agents and/or anti-aging agents; firming agents, skin elasticity agents and/or restructuring agents; moisturizing agents; anti-photoaging agents, and/or blue-light protector agents; DNA protecting agents, DNA repair agents, and/or stem cell protecting agents; free radical scavengers and/or anti-glycation agents, detoxifying agents, antioxidant and/or anti-pollution agents; anti-perspirant agents; melanin synthesis stimulating or inhibiting agents; whitening or depigmenting agents; propigmenting agents; self-tanning agents; lipolytic agents or agents stimulating lipolysis, adipogenic agents, etc. Additional examples can be found in CTFA International Cosmetic Ingredient Dictionary & Handbook, 12th Edition (2008).
• the anti-wrinkle agent, botox-like agent and/or anti-aging agent can be Oxygestkin® [INCI: Tropaeolum majus Flower/Leaf/Stem Extract] or EclalineTM. [INCI: Hydrolyzed Lupine Protein Octenylsuccinate] from SILAB; Matrixyl® [INCI: Palmitoyl Pentapeptide-4], Matrixyl® 3000® [INCI: Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide], Matrixyl® Synthe’6 [INCI: Glycerin, Water, Hydroxypropyl Cyclodextrin, Palmitoyl Tripeptide-38], Matrixyl® MorphomicsTM [INCI: Pentylene Glycol, Caprylyl Glycol], EssenskinTM [INCI: calcium hydroxymethionine], Renovage [INCI: Teprenone], Dermaxyl® [INCI: Palmito
• the firming agent, skin elasticity agent and/or restructuring agent can be selected from the group consisting of Argassential [INCI: C10-16 Alkyl Glucoside, Dicaprylyl Ether, Glycerin] or Replexium BC [INCI: Dimethyl Isosorbide, Polysorbate 20, Aqua, Acetyl Tetrapeptide-11 , Acetyl Tetrapeptide-9] marketed by BASF; Prolevis [INCI: Hydrolyzed Vegetable Protein] or Poretect [INCI: Caprylic/capric Triglyceride, Sorbitan Trioleate, Apium Graveolens Seed Extract, Linum Usitatissimum Seed Extract] marketed by Sederma/Croda; Actifirm Ultra Advanced botanical ingredient [INCI: Centella Asiatica Extract, Rosmarinus Officinalis Leaf Extract, Dipropylene Glycol, Alcohol, Echinacea Angustifolia Leaf Extract] or Actifcol Advanced botanical ingredient [INCI: Aqua, Glycerin,
• the moisturizing agent can be selected from the group consisting of qua Shuttle [INCI: Sorbitol, Laminaria Digitata Extract, Diatomaceous Earth] marketed by Infinitec; Aqua-OsmolineTM [INCI: Ceratonia Siliqua (Carob) Seed Extract] marketed by Vincience/ISP/Ashland; HydralphatineTM Asia [INCI: Hydrogenated Starch Hydrolysate, Panthenol, Bambusa Vulgaris Shoot Extract, Nelumbo Nucifera Flower Extract, Nymphaea Alba Root Extract] or HydraporineTM [INCI: Betaine, Hydrogenated Lecithin, Honey, Pectin] marketed by Lucas Meyer Cosmetics/Unipex; PatcH2OTM [INCI: Trehalose, Urea, Serine, Glyceryl Polyacrylate, Algin, Sodium Hyaluronate, Pullulan], Aqu’activTM [INCI: Behenyl Alcohol, Glyceryl Oleate,
• the anti-photoaging agent, and/or blue-light protector agent can be selected from the group consisting of Algept Genofix CPD [INCI: Plankton Extract, Aqua, Lecithin] marketed by Greenaltech; BlumilightTM Biofunctional [INCI proposed: Water/Aqua (and) Butylene Glycol (and) Theobroma Cacao (Cocoa) Seed Extract] marketed by Ashland; Lys’Sun [INCI: Hamamelis Virginiana Leaf Extract, Aqua, Pentylene Glycol, Caprylyl Glycol, Xanthan Gum] marketed by BASF; Vitachelox [INCI: Vitis Vinifera Seed Extract, Camellia Sinensis Leaf Extract, Quercus Robur Wood Extract] marketed by Indena; L-VCG [INCI: Ascorbyl Glucoside]marketed by Freshine Bio-technology; Lumicease blue ingredient [INCI: Glycerin, Aqua, Hydrolyzed Pea Protein, Glucose
• the DNA protecting agent, DNA repair agent, and/or stem cell protecting agent can be selected from the group consisting of; GP4G SP [INCI: Aqua, Glycerin, Aretmia Extract], Heliostatine [INCI: Aqua, Glycerin, Pisum Sativum Extract], Orsirtine [INCI: Aqua, Glycerin, Oryza Sativa Extract], Chronogen [INCI: Aqua, Butylene Glycol, Tetrapeptide (INCI proposed)], Survixyl IS [INCI: Water, Butylene Glycol, Pentapeptide- 31] and Chrondricare [INCI: Aqua, Butylene Glycol Pentapeptide-28] marketed by Vincience/ISP/Ashland; Lanacityn® [INCI: Glycerin, Aqua, Alteromonas ferment extract, Chysanthellum indicum extract] or Melinoil [INCI: Isopropyl Palmitate, Lecithin, Aqua,
• the reactive carbonyl species scavenger, free radical scavengers and/or anti-glycation agent, detoxifying agent, antioxidant and/or anti-pollution agent can be selected, for example and not restricted to, from the group formed by carnosine and its derivatives; GHK [INCI: Tripeptide-1 ] and its salts and/or derivatives or Quintescine IS [INCI: Dipeptide-4] marketed by Vincience/ISP/Ashland; Preregen [INCI: Glycine Soja (Soybean) Protein, Oxido Reductases], Edelweiss GC [INCI: Leontopodium Alpinum Extract], Lipogard [INCI: Squalane, Ubiquinone], Nectapure [INCI: Buddleja Davidii Extract, Thymus Vulgaris Extract], Alpaflor Nectapure [INCI: Buddleja Davidii Extract, Thymus Vulgaris Extract, Glycerin, Water] or Dismutin
• compositions of the invention may also include other active ingredients such as vitamins, minerals, proteins, peptides, fatty acids, antioxidants, antiinflammatory agents, darkening agents and/or mixtures thereof.
• the cosmetic composition may include other active ingredients for promoting hair growth, and/or preventing hair loss.
• Suitable, non limiting examples, in the context of this invention are Growth Oleoactif® [INCI: Helianthus Annuus (Sunflower) Seed Oil (and) Polyglyceryl-3 Diisostearate (and) Carthamus Tinctorius (Safflower) Flower Extract (and) Hibiscus Sabdariffa Flower Extract] marketed by Hallstar.
• the present invention is based on the finding that the protein hydrolysate of the invention is effective in alleviating and/or preventing adverse effects of the exposome on skin and/or hair. Adverse effects of the exposome include adverse effects due to air pollution, sunlight and/or greenhouse gases. It has also been found that the protein hydrolysate is effective in alleviating adverse effects of chemical treatment or heat stress on hair. In addition, it has been found that the protein hydrolysate is effective in maintaining and/or improving the physical barrier function of the skin.
• the physical barrier function of the skin is also referred to herein as the physical barrier of the skin, the skin’s permeability barrier or simply the skin barrier. This barrier is provided by the stratum corneum and the tight junctions in the epidermis.
• the invention provides for the use of the protein hydrolysate described herein, or a compound chosen from:
• Glu-Tyr-Thr-Gly-Asp-Ala-Ala-Gly-Arg SEQ ID No.5
• stereoisomers, and cosmetically acceptable salts thereof or a combination of said protein hydrolysate or said compound and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostoc, for the treatment and/or care of the skin and/or hair.
• the treatment and/or care of the skin and/or hair is cosmetic and non-therapeutic.
• the use of the protein hydrolysate, compounds or combinations described herein can be for the alleviation and/or prevention of adverse effects of the exposome on hair; the alleviation and/or prevention of adverse effects of air pollution on hair; the alleviation and/or prevention of adverse effects of sunlight on hair; the alleviation and/or prevention of the adverse effects of chemical stress on hair; the alleviation and/or prevention of adverse effects of heat stress on hair; and/or the alleviation or prevention of the adverse effects to the hair caused by peroxidation of lipids in hair and/or carbonylation of proteins in hair.
• the use of the protein hydrolysate, compounds or combinations described herein can be for the alleviation and/or prevention of adverse effects of air pollution and sunlight on hair.
• the protein hydrolysate can be used for reducing hair damage. Adverse effects in hair can manifest themselves in the form of a hair with more split ends; reduced fiber alignment and/or increase in frizz. Therefore, the hair is less manageable.
• the protein hydrolysate can be used for increasing hair maneagability, particularly by a reduction of split ends and/or an increase in fiber alignment and/or reduction of frizz and/or a reduction of volume.
• Air pollution includes particulate matter, for example.
• Particulate matter is an airborne pollutant that includes solid particles. Patrticlu late matter may include dust, dirt, soot or smoke.
• Particulate matter can come from both anthropogenic and naturally occurring sources, including automobiles (e.g. exhaust emissions from combustion engines of automobiles), power plants, factories, fires, wood-buring stoves, dust from open land or construction, dust from roads (e.g. brake-, tyre- and road-wear particles), pollen (e.g. cedar pollen) and smoke from tobacco (e.g. cigarette smoke).
• the particulate matter can have a size of 10 microns or less (also referred to as PMw and “dust”).
• the particulate matter can arise from automobile combustion engines (e.g. diesel particulate such as those encountered in Diesel particulate matters NIST1650B.
• the particular matter can comprise or pollen (e.g. cedar pollen).
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the hair wherein the treatment and/or care of the hair is the alleviation or prevention of the adverse effects of sunlight on the hair.
• the sunlight can comprise UV-A radiation.
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the hair wherein the treatment and/or care of the hair is the alleviation or prevention of the adverse effects of bleaching or dyeing on hair.
• the use of the protein hydrolysate, compounds or combinations described herein can be for the alleviation and/or prevention of adverse effects of the exposome on skin; the alleviation and/or prevention of adverse effects of air pollution on skin; the alleviation and/or prevention of adverse effects of sunlight on skin; the alleviation and/or prevention of adverse effects caused by greenhouse gases; and/or the maintenance and/or improvement of the physical barrier function of the skin.
• the use of the protein hydrolysate, compounds or combinations described herein can be for the alleviation and/or prevention of adverse effects of air pollution and sunlight on skin.
• Adverse effects to skin can manifest themselves in the form of a reduction in barrier function; a dull appearance of the skin; a loss of brightness, luminosity and/or glossiness of the skin; a loss in homogeneity in the texture of the skin; an increase in roughness of the skin; the appearance of dark spots; and/or erythema (skin redness).
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the skin wherein the treatment and/or care of the skin is the alleviation or prevention of the adverse effects of sunlight on the skin.
• the sunlight can comprise UV-A radiation.
• Adverse effects can manifest themselves in the form of a loss in barrier function in the skin.
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the skin wherein the treatment and/or care of the skin is the maintenance and/or improvement of the physical barrier function of the skin.
• Adverse effects can manifest themselves in the form of skin having a dull appearance; and/or having a less bright appearance; and/or having a less luminous appearance and/or a less glossy appearance.
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non- therapeutic treatment and/or care of the skin wherein the treatment and/or care of the skin is increasing skin brightness, and/or increasing the gloss of the skin and/or increasing skin luminosity.
• the adverse effects may also lead to the appearance of dark spots.
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the skin wherein the treatment and/or care of the skin is reducing dark spots.
• the treatment and/or care can involve reducing melanin in the skin.
• the adverse effects can manifest themselves in the form of a reduction of skin tone and/or skin texture homoneity and/or an increase of skin roughness.
• the use of the protein hydrolysate, compounds or combinations described herein for the cosmetic, non-therapeutic treatment and/or care of the skin wherein the treatment and/or care of the skin is increasing skin tone and/or increasing the homongeneity of skin texture and/or reducing skin roughness.
• the invention relates to a method of treatment and/or care of the skin and/or hair comprising administering a protein hydrolysate of azuki beans according to the first aspect of the invention, or a compound chosen from:
• Glu-Tyr-Thr-Gly-Asp-Ala-Ala-Gly-Arg SEQ ID No.5
• stereoisomers, and cosmetically acceptable salts thereof, or a combination of said compound and a filtrate obtained by the fermentation of Raphanus sativus root by the microorganism Leuconostocto a subject may be a cosmetic, non-therapeutic treatment and/or care.
• the protein hydrolysate, compound or combination described above may be present in a cosmetic composition, for example a cosmetic composition as described herein.
• the method can be for the treatment and/or care of the skin and/or hair as described above in relation to applications (uses) of the protein hydolysate, compounds and/or combinations of the invention.
• the treatment and/or care is the alleviation or prevention of the adverse effects of the exposome on the skin and/or hair; the alleviation or prevention of the adverse effects of air pollution on the skin and/or hair; the alleviation or prevention of the adverse effects of sunlight on the skin and/or hair; the alleviation or prevention of the adverse effects of chemical treatment of the hair; the alleviation or prevention of the adverse effects of heat stress on the hair; and/or the maintenance and/or improvement of the physical barrier function of the skin.
• the treatment and/or care can be preventative in that it serves to protect the skin and/or hair from adverse effects.
• the protein hydrolysate, compounds, and combinations of the invention are applied to the skin and/or hair prior to exposure of the stressor.
• the treatment and/or care can be reparative or restorative, i.e. where alleviation has a reparative or restorative function, (e.g. with respect to aesetheic properties).
• the protein hydrolysate, compounds, and combinations of the invention can be applied to the skin and/or hair during or after exposure of the stressor.
• topical or transdermal application can be carried out by iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive cure, microinjections, by needle-free injections by means of pressure, by microelectric patches, face masks or any combination thereof.
• the frequency of application or administration can vary greatly, depending on the needs of each subject, with a recommendation of an application from once a month to ten times a day, preferably from once a week to four times a day, more preferably from three times a week to twice a day, even more preferably once a day.
• the invention also provides for the use of the protein hydrolysate of the invention in a therapeutic treatment of the skin.
• the invention provides a protein hydrolysate as disclosed herein for use as a medicament.
• the invention provides a protein hydolysate as disclosed herein for use in the prevention or treatment of a disease or disorder associated with the exposome, exposure of the skin to air pollution, sunlight and/or greenhouse gases, and/or associated with an impaired physical barrier function of the skin.
• the invention provides for the use of a compound of formula (I), its stereoisomers and/or its pharmaceutically acceptable salts for the manufacture of a medicament for the treatment or prevention of a disease or disorder.
• EXAMPLE 1 azuki beans [00106] The first step was the processing of the raw beans into a fine powder of brown granulated appearance. The aim of this stage is to reduce particle size as much as possible in orderto facilitate protein solubilization. The azuki beans were ground into a flour and larger fragments were sifted out.
• Preliminary treatment of the ground azuki beans increases the content of water-soluble protein in the hydrolysis media, prior to enzyme addition.
• the flour was suspended in deionized water at a concentration of 21 .8 g of flour/Kg of water (w/w) and transferred into a suitable reactor vessel equipped with pH and temperature probes providing real-time monitoring.
• Initial pH was raised from 6.5-6.8 to 7.0 by addition of a basic solution (i.e. NaOH 1 N) and, when needed, more solvent was added to reach a desired concentration.
• the conditions were maintained for 1 hour at room temperature (25 ⁇ 2°C).
• the resulting suspension is referred to as the “protein suspension” - it is a suspension of ground azuki beans in water containing proteins from the ground azuki beans that have been solubilised.
• Enzyme was added to the protein suspension and the mixture stirred for 1 hour at room temperature (25 ⁇ 2°C). Agitation conditions were adjusted to ensure homogenous and vigorous stirring, while keeping foam formation at minimum levels. Then, alcalase was added to the protein suspension.
• Alcalase is a subtilisin type enzyme. Preferred hydrolysis conditions are defined by the use of subtilisin-type Alcalase AF 2.4L (CAS no. 9014-01 -1 , EC 3.4.21 .62, Novozymes®) enzymes.
• [00112] Hydrolyzed Azuki suspension (“crude hydrolyzate”) is optimally centrifugated in continuous flow (i.e. using Westfalia CSA-1 centrifuge or similar equipment) at 10.000xG, with an optimal flow of 50 l/h. Supernatant was collected, whereas pellet was discarded as waste material. Supernatant turbidity was controled throughout the operation taking a representative sample, and maintained below 0.2 AU/g of protein at 600 nm (relative to an appropriate blank in these conditions).
• the supernatant obtained from step 3.1. was passed through filters of varying size under pump-generated pressure.
• a 1 ,2 pm pore filter was used to discard the bulk of large particles that were not eliminated during centrifugation. This first filtration step is optional and the purpose is to reduce the number of small pore size filters needed. Then, the obtained filtrate was subsequently filtered through 0.45 and 0.22 pm pore-sized filters.
• Example 3.2 The filtrate obtained in Example 3.2. was further ultrafiltrated through ultrafiltration membrane (i.e. Millipore ref. P2PLBCC05, 0.5 m 2 /cassette) to remove fragments with molecular weight higher than 3 KDa.
• the resulting filtrate obtained is also referred to as “purified hydrolysate” or “permeate”.
• the inhibition percentage is calculated from the following formula:
• the dry weight of the different batches was on average from 2 to 5 mg of dry weight I purified hydrolysate.
• the protein content ranged from 19 to 35% (w/w).
• DPPH activity ranged from 74 to 80%.
• Polyphenol content was from 0.15 to 0.30 mg/mL.
• HFDPC Human Follicle Dermal Papilla Cells derived from scalp were cultivated with Follicle Dermal Papilla Cell Growth Medium at 37°C and 5% CO 2 . These cells were stressed with Particle Matters (PM) at 0.1 pg/cm 2 (from European Reference Material; Ref.CZI 00) followed by UV-A irradiation (LED source, emission peak at 365nm, 3 J/cm2) in 1X PBS buffer (Phosphate Buffered Saline, pH 7.4). The cells were treated with the stressors for 13 min. Just after stress exposure, cells were treated with purified hydrolysate of Example 3 at 1 % (v/v) in culture medium. The anti-oxidant N-acetyl- cysteine (NAC) was used as a positive control. Two (2) hours afterthe intracellular levels of protein carbonylation were measured.
• PM Particle Matters
• LED source emission peak at 365nm, 3 J/cm2
• 1X PBS buffer Phos
• control group was considered at maximum efficiency (100%) and the stress group at minimum efficiency (at 0%): s able 1
• HFDPC Human Follicle Dermal Papilla Cells
• the raw images (were collected in a range of intensity of specific signal from 0 to 65535 and analyzed with Imaged software.
• the intensity of carbonylation was determined by integration of the specific fluorescence signal normalized by the evaluated area.
• control group is considered the maximum efficiency (100%) and the stress group the minimum efficiency (at 0%):
• Asian hair shafts were treated with purified hydrolysate according to Example 3 at 1 % (v/v) (active ingredient solubilized in ultra-pure water, leave on (no washing steps)) for 30 minutes with a gently movement at room temperature. After this time, hair shafts were dried at natural evaporation at room temperature and were stressed with Cedar pollen (Ref. Cedar Pollen-Cj*; LG-5280 Euromedex ; 500 ng/cm2), followed by Particulate Matter application (150 pg/cm2, applied as powder; from European Reference Material; Ref. ERM-CZ100) and UV-A irradiation (LED source, emission peak at 365 nm, 84 J/cm 2 ; 6 hours of irradiation).
• Diesel pollen Ref. Cedar Pollen-Cj*
• LG-5280 Euromedex 500 ng/cm2
• Particulate Matter application 150 pg/cm2 applied as powder; from European Reference Material; Ref. ERM-CZ100
• Fluorescent images were collected with an epifluorescent microscope (ThermoFisher, Evos M5000) and analyzed with Imaged software (Schneider, 2012). Image collection for the different conditions was achieved using identical conditions of acquisition (40X objective). The raw images were collected in a range of intensity of specific signal from 0 to 65535 and analyzed with Imaged software. The intensity of carbonylation was obtained by the integration ofthe specific fluorescence signal normalized by the evaluated area. For hair shaft cross-sections, in each image, the quantification of carbonylation level was independently obtained for the different compartments (cuticle or cortex) and for the whole hair. Three (3) images per condition were used to quantify the carbonylation levels; the mean value and standard deviation were obtained. Data treatment and statistical analysis were achieved by using graphPad Prism (La Jolla, California, USA).
• control group was considered the maximum efficiency (100%) and the stress group the minimum efficiency (at 0%): p-Values were obtained by ANOVA variance analysis followed by
• Image collection for the different conditions was achieved using identical conditions of acquisition (40X objective).
• the raw images were collected in a range of intensity of specific signal from 0 to 65535 and analyzed with Imaged software.
• the maximum intensity of diffused fluorophore was obtained and quantified by the integration of the specific fluorescence signal among the axis of penetration (from cuticle to cortex).
• Efficacy % (test group X) [Fluorescence intensity (Stress) - Fluorescence intensity (test group)]/ [Fluorescence intensity (Stress) - Fluorescence intensity (Control)] * 100 [00140]
• the control group is considered at maximum efficiency (100%) and the stress group at minimum efficiency (at 0%): p-Values were obtained by ANOVA variance analysis followed by Dunnett’s post-hoc test for multi-comparisons in comparison with stress. ****p-Value ⁇ 0.001 ; ***p-Value ⁇ 0.001 ; **p-Value ⁇ 0.01 ; *p- Value ⁇ 0.05
• African hair shafts were stressed heating at 95°C during 30 min. After the stress exposure hair shafts were treated with Azuki purified hydrolysate at 1% (v/v) (active ingredient solubilized in ultra-pure water) for 30 minutes.
• the raw images were collected in a range of intensity of specific signal from 0 to 65535 and analyzed with Imaged software.
• the maximum intensity of diffused fluorophore was obtained and quantified by the integration of the specific fluorescence signal among the axis of penetration (from cuticle to cortex).
• Three (3) images per condition were used to quantify the fluorescence intensity levels (R.F.U.); the mean value and standard deviation were obtained.
• Data treatment and statistical analysis were achieved by using graphPad Prism (La Jolla, California, USA)
• Efficacy % (test group X) [Fluorescence intensity (Stress) - Fluorescence intensity (test group)]/ [Fluorescence intensity (Stress) - Fluorescence intensity (Control)] * 100 [00145]
• the control group is considered at maximum efficiency (100%) and the stress group at minimum efficiency (at 0%): p-Values were obtained by ANOVA variance analysis followed by Dunnett’s post-hoc test for multi-comparisons in comparison with stress. *p-Value ⁇ 0.05
• Sample preparation for HPLC Analysis Samples from the protection test were directly used for HPLC analysis whereas samples of the “protection and defense”; and “defense” tests, were dried on cellulose paper for 30 min. 15-30 mg of hair samples were weighted and 1 mL of MeOH is added and sonicated for 15 min. After sonication, in 10 mL glass tubes, the following reagents are added: 500 pL sample (extracted previously in MeOH), 750 pL H3PO4, 250 pL 2-Thiobarbituric Acid Solution at 42 mM in NaOH 1 N and 450 pL H 2 O-MilliQ. Tubes are vortexed and incubated at 90°C for 60 min.
• HDFa Human Dermal Fibroblasts
• HEKa Human Epidermal Keratinocytes
• Resazurin the active ingredient of PrestoBlueTM Reagent, is a nontoxic, cell-permeable compound that is virtually non-fluorescent.
• the cellular reducing environment reduces resazurin to resorufin, a compound that is highly fluorescent.
• Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence of the media surrounding the cells, being directly proportional to the number of viable cells in each well.
• DNA is susceptible to oxidative damage.
• 2’-deoxyguanosine (dG) has the lowest ionization potential and thus is the most susceptible to oxidation.
• One of the positions where oxidation of dG can occur is at the C8 position, giving rise to 8-oxo-dG.
• 8-oxo-dG is a useful biomarker of oxidative damage in DNA. If not repaired, this lesion, and/or the products derived from it, may be misread by a polymerase during replication and lead to mispairing with A. Then, during the next round of replication, this A will correctly pair with T leading to a G-T transversion mutation.
• HEKa Human Epidermal Keratinocytes
• DNA samples were digested to nucleosides using enzymatic hydrolysis. For each sample, 7uL 15mM 8-HQ (8-Hydroxyquinoline), and 50 uL buffer solution (50 mM ammonium acetate, 0.2 mM ZnCI2, pH 5.3) were added. Then 3 uL of Nuclease P1 (0.39 unit/uL) and 4 uL of alkaline phosphatase (1 unit/uL in 10 mM Tris pH 7.4) were added, and the solution was incubated at 37 °C for 60 min. The solution was centrifuged for 5 min at 14,000 rpm before LC-MS/MS analysis.
• HPLC-ESI-MS/MS system was a SHIMADZU HPLC model Nexera XR in line with a SHIMADZU triple quadrupole mass spectrometer, model LCMS-8040, equipped with an electrospray ionization source. Nitrogen was used as the drying and nebulizing gas. Argon was used as the collision gas for CID.
• a InfinityLab Poroshell 120-EC-C18 column (4.6 x 75mm x2.7um) were used at a flow rate of 0.5mL/min, maintained at 36°C.
• the gradient conditions were: 0 - 9min 5%B - 90%B, 9 - 12min 90%B, 12.1 - 17min 5%B.
• Analytes were quantified using tandem electrospray ionization mass spectrometry in positive-ion mode. Product ions were monitored in multiple-reaction monitoring mode.
• Mass transition for 8-oxodG was m/z 284 ,15> 168.05 and for 8-Hydroxyguanosine m/z 300.00 > 168.05.
• a secondary transition (m/z 284.15 > 140.1 and m/z 300.00 >140.05, respectively) was used to confirm the identity of 8-oxodG and 8-Hydroxyguanosine respectively in samples, which are biomarkers of DNA oxidation.
• Injection volume was 25uL.
• p-Values were obtained by ANOVA variance analysis followed by Dunnett’s post-hoc test for multi-comparisons in comparison with stress. ***p-Value ⁇ 0.001 ; **p-Value ⁇ 0.01 ; *p-Value ⁇ 0.05
• Table 11A p-Values were obtained by ANOVA variance analysis followed by Dunnett’s post-hoc test for multi-comparisons in comparison with stress. ****p-Value ⁇ 0.0001 ; ***p- Value ⁇ 0.001 ; **p-Value ⁇ 0.01 ; *p- Value ⁇ 0.05.
• Suitable preservative [1 % (w/w) Dermosoft 700B (INCI: evulinic Acid (and) Sodium Levulinate (and) Glycerin (and) Aqua), 4% (w/w) LeucidalTM SF Max from Active Micro Technologies (AMT) (INCI: Lactobacillus Ferment) or 4% (w/w) LeucidalTM Liquid
• the purified extract according to Example 3 was further fractionated by using semi-preparative high-performance liquid chromatography (HPLC) and antioxidant activity assayed by DPPH for identifying the active fraction or peptides.
• HPLC semi-preparative high-performance liquid chromatography
• Peptides were separated using an eluent system consisting of eluents A (water/trifluoroacetic acid 1000:1) and B (acetonitrile/trifluoroacetic acid 1000:1). Elution was carried out at a flow of 35 ml/min using the following gradient composition: isocratic of 0% B in 3 min, 0 to 30% of B in 10 min, and 30 to 75% of B in 7 min, monitoring absorbance of eluting peaks at 220 nm.
• Real time ionization and peptide fragmentation was achieved by acquiring FT spectra in a resolution of 30.000 FHMW) followed by FT- MS/MS scan and selecting the 10 most intense ions with HCD fragmentation, collision energy of 35% and dynamic exclusion of 0.5 min. All spectra were acquired in positive ion mode.
• the resin was washed with DMF, prior to removal of Fmoc group by treatment with a 20% solution of piperidine in DMF, followed by washings in the same solvent. Subsequent residues were incorporated in DMF employing DIPCDI and Oxyma as coupling reagents. After verification of reaction completion using ninhydrin test, the solvent was filtrated away by suction. After this, the resin was washed with DMF and the Fmoc-deprotection step was repeated as abovementioned or in 2% DBU in DMF. Coupling-deprotection cycles were repeated for introduction of the remaining amino acids, with increasing Fmoc removal treatment times from the 6 th residue. Oxyma (0.5M) was introduced in the cocktail whenever Asp residue was already incorporated into the growing peptide chain.
• Resazurin the active ingredient of PrestoBlueTM Reagent, is a nontoxic, cell- permeable compound that is virtually non-fluorescent.
• the cellular reducing environment reduces resazurin to resorufin, a compound that is highly fluorescent.
• Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence of the media surrounding the cells, being directly proportional to the number of viable cells in each well.
• Eitgh hundret milligrams (800 mg) of Asian hair are weighted in Petri Dishes and Irradiated in a Solar Simulator (Solar simulator SOL500 Honle, 32.4-36 J/cm2) for 2 h.
• a Solar Simulator Solar simulator SOL500 Honle, 32.4-36 J/cm2
• 600 mg of damaged hair is weighted in Flacon Tubes and 6 mL of azuki purified hydrolysate at 22 pg/mL are added and left on an orbital shaker for 30 min. After the incubation time with the azuki purified hydrolysate, the hair is rinsed with water at 30°C for 30 seconds.
• Leave-on serum was prepared including the following ingredients:
• Face cream was prepared including the following ingredients:
• Table 21 Mean split ends variation of active and placebo leave-on serums after 14 days of product application. * p ⁇ 0.05 calculated using paired Wilcoxon test.
• Results demonstrate that after 14 days of application of the active leve-on serum there is a significant decrease in split ends respect to initial time.
• Results demonstrate that after 14 days of application of the active leve-on serum there is a significant increase in fiber alignment respect to initial time.
• Table 23 Average frizz variation of active and placebo leave-on serums after 14 days of product application. ** p ⁇ 0.01 and * p ⁇ 0.05 calculated using paired Wilcoxon test.
• Results demonstrate that after 14 days of application of active leve-on serum there is a significant decrease in frizz respect to initial time.
• Volunteers’ hair was clinically evaluated by an expert at initial time and after 14 days of product application.
• the efficacy of the product against exposome’s real outdoor conditions was assessed by volunteers’ hair volume variation.
• Hair volume was evaluated by an expert who performed a clinical evaluation grading hair volume through a scale at initial time and 14 days of product application.
• Table 24 Mean hair volume variation of active and placebo leave-on serums after 14 days of product application. Is p ⁇ 0.1 calculated using paired Wilcoxon test.
• Results demonstrate that after 14 days of application of the active leave on serum there is a significant decrease in hair volume respect to initial time in volunteers with hair type 3 and 4.
• TEWL trans epidermal water loss
• Table 25 TEWL variation of active and placebo creams after 14 and 28 days of product application. Significance calculated using Wilcoxon paired test of active versus placebo cream at 14 days of application is * p ⁇ 0.05.
• Results demonstrate that after 14 days of application of active cream there is a significant decrease in TEWL respect to placebo cream.
• Skin texture heterogeneity was assessed by the average perimeter of spots in volunteers’ cheek. Images at initial time and 28 days of product application were acquired with Colorface® and perimeter average of spots in Region Of Interest (ROI) was measured with Framescan software
• Table 26 Spot perimeter average variation of active and placebo creams after 28 days of product application. * p ⁇ 0.05 calculated using a t-student test.
• Results demonstrate that after 28 days of application of active cream there is a significant decrease in spot perimeter respect to initial time, leading to a decrease in skin texture heterogeneity.
• Skin tone homogeneity was evaluated by a dermatologist who performed a clinical evaluation grading skin’s tone homogeneity through a scale at initial and 28 days of product application.
• Table 27 Skin tone homogeneity average variation of active and placebo creams after 28 days of product application. * p ⁇ 0.05 calculated using a t-student test.
• Results demonstrate that after 28 days of application of active cream there is a significant increase in skin’s tone homogeneity according to the expert evaluation respect to initial time.
• Skin erythema was assessed by the average variation of a* parameter which is used for measuring color related to erythema.
• Table 28 a* parameter average variation of active and placebo creams after 28 days of product application. * p ⁇ 0.05 calculated using paired t-student test.
• Skin melanin was evaluated by the average variation b* parameter which is used for measuring color related to melanin.
• Results demonstrate that after 28 days of application of active there is a significant decrease in a* and b* parameters respect to placebo cream in Asian volunteers. According to this, skin erythema and skin melanin decreases after 28 day product application in Asian volunteers. This parameters are related with a visual reduction of skin redness and an improving of skin brightnes, respectively.
• Volunteers’ images of cheeks were acquired at initial time and 28 days of product application with Colorface®.
• the efficacy of the product against exposome’s real outdoor conditions in Caucasian volunteers was assessed by a* parameter obtained from image analysis.
• Skin erythema was evaluated at initial time and after 28 days of product application. Skin erythema is assessed by the mean variation of a* parameter which is used for measuring color related to erythema.
• Skin melanin was evaluated at initial time and after 28 days of product application. Skin melanin is assessed by the average variation of b* parameter which is used for measuring color related to melanin. Table 31 . b* parameter average variation of active and placebo creams after 28 days of product application. * p ⁇ 0.05 calculated using paired t-student test. Results demonstrate that after 28 days of product application there is a significant decrease in b* parameter respect to placebo cream in African volunteers leading to a skin with less melanin (i.e. higher brightness).

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