Process for producing epigallocatechin gallate monoglucoside

Abstract

Methods for selectively preparing the monoglucosides of epigallocatechin and epigallocatechin gallate, and the use of these products in cosmetics, are provided.

Description

[Technical Field]

[0001] The present invention relates to a method for selectively preparing the monoglucosides of epigallocatechin and epigallocatechin gallate and to their use in cosmetics.

[0002] Phenolic compounds (also called phenols), or polyphenols, constitute one of the most numerous and widely distributed groups of substances in the plant kingdom. Polyphenols are products of plant secondary metabolism. The term "phenolic compounds" encompasses a wide range of substances containing an aromatic ring with one or more hydroxyl substituents. The structures of natural polyphenols range from simple molecules such as phenolic acids to highly polymeric compounds such as condensed tannins. Three groups of phenols important to humans are phenolic acids (C6-C1, C6-C2, and C6-C3), flavonoids (C6-C3-C6), and high molecular weight polyphenols (greater than 30 carbon atoms). Indeed, phenols, particularly polyphenols, exhibit a wide variety of beneficial biological activities in mammals, including antiviral, antibacterial, immunostimulatory, antiallergic, antihypertensive, antiischemic, antiallergic, antithrombotic, hypocholesterolemic, antilipoperoxidant, hepatoprotective, anti-inflammatory, anticarcinogenic, antimutagenic, antitumor, antithrombotic, and vasodilatory properties. They are potent antioxidants in vitro.

[0003] Flavonoids comprise a large group of low molecular weight polyphenolic substances, benzo-γ-pyrone derivatives, diverse in chemical structure; they represent the most common and widely distributed group of plant phenolics. The general structure of flavonoids is that of diphenylpropane (C6-C3-C6), which usually consists of two aromatic rings (cycles A and B) linked through three carbons to form an oxidized heterocycle (cycle C). The basic structure and system used for numbering the carbons of the flavonoid nucleus are shown below: [ka]

[0004] Flavonoids often occur as glycosides, which makes the molecules more water-soluble and less reactive to free radicals. Flavonoids have ideal structural chemistry for free radical scavenging activity. Among flavonoids, epigallocatechin (1) and epigallocatechin gallate (EGCG) (2) are of particular interest for cosmetic applications due to their antioxidant, anti-inflammatory, antibacterial, antiallergic, antiviral, and antipigmentation activities, as well as their ability to provide UV protection, activate the skin barrier, and promote cellular activity.

[0005] The structures of these two flavonoids are shown below: [ka]

[0006] Due to their low aqueous solubility and / or high susceptibility to oxidation, the use of phenols in pharmaceutical or cosmetic preparations requires specific, tailored formulations. These formulations must also satisfy constraints related to the end use, making it often difficult to reach a compromise between acceptability, concentration, and stability. More water-soluble and / or oxidation-resistant forms of phenols, such as glycosides, are not always available in nature, and when they do exist, complex extraction and purification procedures from plant material may be required. Both chemical and biochemical (enzymatic) approaches have been attempted to increase water solubility and / or stability. Because phenolic compounds possess several free hydroxyl groups, attempts at chemical modification of phenolic compounds often result in nonselective reactions, producing a variety of different molecules. Further purification steps are then required to recover the desired product(s).

[0007] WO 2007 / 144368 discloses an enzymatic method for producing O-α-glucosides of phenols using glucansucrase from Leuconostoc species. Among other things, epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside are formed. The enzymatic reaction is typically carried out in a buffered aqueous medium containing dimethyl sulfoxide (DMSO) as a cosolvent. The method described in WO 2007 / 144368 produces epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside as mixtures of mono-, diglucosides, and triglucosides, respectively, with relatively low regioselectivity. Typically, the monoglucosides and diglucosides are obtained in a molar ratio of about 30:70 to 60:40.

[0008] It is therefore an object of the present invention to provide a method for preparing epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside in a more selective manner without compromising yield or scalability. This problem has been solved by the method of the present invention as described below. Surprisingly, it has been found that epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside, respectively, selectively prepared by the method of the present invention, exhibit improved skin penetration and bioaccumulation, and are therefore better suited for use in cosmetic applications, especially skin care applications.

[0009] In a first aspect, the present invention provides a method for preparing epigallocatechin gallate 4'-O-α-monoglucoside. In a second aspect, the present invention provides a method for preparing epigallocatechin 4'-O-α-monoglucoside. In a third aspect, the present invention provides epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside, respectively, prepared by the method of the present invention. In a fourth aspect, the present invention provides a method for providing radiant skin glow and / or removal of blemishes, in which epigallocatechin gallate 4'-O-α-monoglucoside is topically applied to the skin. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the semi-quantitative results of skin penetration. [Figure 2] Figure 2 shows the results of measurements of the biomechanical properties of the skin. [Figure 3] FIG. 3 shows the evaluation results of skin firmness.

[0011] The present invention relates to a method for preparing epigallocatechin gallate 4'-O-α-monoglucoside, which comprises incubating epigallocatechin gallate with sucrose and glucansucrase from Leuconostoc species, the incubation being carried out in a medium containing 2-propanol and water. Surprisingly, it was found that the use of 2-propanol as a cosolvent in the medium significantly improved the selectivity of the enzymatic reaction, allowing the monoglucoside to be selectively obtained with high regioselectivity. Notably, the regioselectivity observed was better than when a diol or triol was used as a cosolvent.

[0012] The method of the present invention can be carried out at a concentration suitable for industrial production (e.g., about 15 to 30 mM), and epigallocatechin gallate 4'-O-α-monoglucoside can be obtained in a yield of more than 30%. Preferred reaction conditions are further indicated below.

[0013] For comparison, under otherwise identical conditions: - When other short-chain alcohols were used as cosolvents, much lower conversions were observed (ethanol: 8%, 1-propanol: 0.71%, 1-butanol: 0.08%, and 2-butanol: 0.25%; vs. 48.5% conversion for 2-propanol). - When 1,3-propanediol alone was used as cosolvent, a conversion of 60% was achieved, with 45% being monoglucosides (of which 58% are 4'-O-α-monoglucosides), 14% being diglucosides, and 1% being triglucosides. - When only 2,3-butanediol was used as cosolvent, a conversion of 57% was achieved, with 45% being monoglucosides (of which 66% are 4'-O-α-monoglucosides), 14% being diglucosides, and 1% being triglucosides. - When only 1,3-butanediol was used as cosolvent, a conversion of 67% was achieved, with 47% being monoglucosides (of which 65% are 4'-O-α-monoglucosides), 19% being diglucosides, and 1% being triglucosides. - When only 2,4-butanediol was used as cosolvent, a conversion of 64% was achieved, with 44% being monoglucosides (of which 62% are 4'-O-α-monoglucosides), 19% being diglucosides, and 1% being triglucosides. - With DMSO as cosolvent, a conversion of 61% was achieved, with 40% monoglucosides (of which 63% are 4'-O-α-monoglucosides), 19% diglucosides, and 1% triglucosides. - If no co-solvent was used, no conversion was observed.

[0014] The present invention further relates to a method for preparing epigallocatechin gallate 4'-O-α-monoglucoside, which comprises incubating epigallocatechin with sucrose and glucansucrase from Leuconostoc species, the incubation being carried out in a medium containing 2-propanol and water. Again, the use of 2-propanol as a cosolvent in the medium was found to improve the selectivity of the enzymatic reaction, especially the regioselectivity of glycosylation, and the enzyme efficacy was significantly better than with other short-chain alcohols.

[0015] The enzymatic reaction is achieved using sucrose, an abundant and inexpensive substance used in the food and feed industries. This reaction involves the transfer of the glucose moiety of sucrose to the hydroxyl group of the catechol ring. Once the first glucosyl residue is attached to the hydroxyl group of the catechol ring, further transfer of the glucose moiety of sucrose to either another hydroxyl group of the catechol ring or the hydroxyl group of the fixed glucose can occur, resulting in diglucosides or even triglucosides. The process of the invention makes it possible to significantly reduce or even completely avoid the formation of diglucosides and triglucosides and to obtain monoglucosides with high regioselectivity.

[0016] In the method of the present invention, a glucansucrase from a Leuconostoc species is used. WO 2007 / 144368 mentions several suitable glucansucrasses, which are incorporated herein by reference. In a preferred embodiment, glucansucrase from Leuconostoc mesenteroides NRRL B-512F (ATCC 10830a) is used.

[0017] The process of the invention is carried out in an aqueous medium, which is preferably buffered at a pH convenient for enzyme activity (as is well known by the skilled artisan). The nature of the buffer used in the medium is not critical. However, it is preferred that the buffer be Ca. 2+ or Mg 2+ Buffers that can chelate divalent cations, such as acetic acid, ...

[0018] In one embodiment, sodium acetate or potassium acetate is used at a concentration ranging preferably from 5 to 100 mM, more preferably from 10 to 50 mM, and even more preferably from 15 to 40 mM. In one embodiment, the pH of the buffer is 5.00 to 5.60, more preferably 5.10 to 5.50, and even more preferably 5.20 to 5.40.

[0019] In the method of the present invention, the incubation is carried out in a medium containing 2-propanol and water. In one embodiment, the medium contains 5% to 20% (v / v), more preferably 8% to 18% (v / v) 2-propanol. These concentration ranges have been found to prevent enzyme denaturation under reaction conditions, thereby providing high yields of the desired product even at relatively low enzyme concentrations.

[0020] Optionally, the medium may further comprise a lower diol and / or triol, in particular a lower diol and / or triol selected from the group consisting of 1,3-propanediol, 1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 2,4-butanediol, 1,2,3-propanetriol (glycerol), and mixtures thereof. Surprisingly, it has been found that the regioselectivity of the reaction is even better when a combination of 2-propanol and 1,3-propanediol is used. The media used in the methods of the present invention should not contain co-solvents or other additives that inhibit enzyme reactivity and / or reduce selectivity.

[0021] In one embodiment, the medium does not contain any dimethyl sulfoxide. In one embodiment, the medium does not contain any ethers of ethylene glycol (glyme).

[0022] Epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside are obtained by incubating the respective substrates with sucrose and glucansucrase, respectively. In one embodiment, epigallocatechin gallate is incubated at an initial concentration of 5-50 mM, more preferably 10-40 mM, and most preferably 15-35 mM to obtain epigallocatechin gallate 4'-O-α-monoglucoside. Alternatively, to obtain epigallocatechin 4'-O-α-monoglucoside, epigallocatechin is incubated at an initial concentration of 5-50 mM, more preferably 10-40 mM, and most preferably 15-35 mM.

[0023] The substrate is incubated with sucrose and glucansucrase under conditions that allow the enzyme to become active and synthesize the desired glucoside to the greatest extent possible. In particular, the concentrations and relative ratios of sucrose, glucansucrase and substrate are selected to allow the enzymatic reaction to proceed and provide the desired product in optimal yield and selectivity. In one embodiment, the reaction mixture comprises sucrose at a concentration of 50 to 400 g / l, more preferably 150 to 350 g / l, and most preferably 250 to 320 g / l. In one embodiment, the temperature of the reaction mixture is maintained at 27-33°C, more preferably 28-32°C, and most preferably 29-31°C.

[0024] Optionally, the reaction mixture may further comprise calcium cations, for example in the form of calcium chloride or any other water-soluble calcium salt, to improve the stability of the enzyme. In one embodiment, the reaction mixture comprises calcium chloride at a concentration of 0.4 to 4.0 g / l, more preferably 0.7 to 3.5 g / l, and most preferably 0.9 to 2.25 g / l.

[0025] In one embodiment, epigallocatechin gallate and epigallocatechin are incubated at initial concentrations of 5 to 50 mM, more preferably 10 to 40 mM, and most preferably 15 to 35 mM, respectively.

[0026] The method of the invention can be carried out using any suitable glucansucrase, such as a dextransucrase or an amylosucrase. In one embodiment, the glucansucrase is a dextransucrase. It was found that dextransucrase can efficiently and selectively glucosylate epigallocatechin gallate and epigallocatechin.

[0027] The concentration of glucansucrase, preferably dextransucrase, should be adjusted to the concentration of the substrate and / or sucrose. In particular, using too little glucansucrase can result in reduced yield, and using too much glucansucrase can result in reduced regioselectivity. In one embodiment, the concentration of glucansucrase in the reaction mixture is 0.4 to 4.0 U / ml, more preferably 0.7 to 3.5 U / ml, and even more preferably 0.9 to 2.25 U / ml.

[0028] Due to the reaction conditions of the present invention, the desired epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside, respectively, are formed with extremely high selectivity. In one embodiment, monoglucosides are formed in a ratio of at least 2:1, more preferably at least 3:1, even more preferably at least 4:1, and most preferably at least 5:1, to the sum of diglucosides and triglucosides. Ideally, only or essentially only monoglucosides are formed. The selective formation of monoglucosides is highly desirable because this product complies with Chinese cosmetic applications.

[0029] In one embodiment, the 4'-O-α-monoglucoside is formed with a regioselectivity of at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 92% on a molar basis compared to other monoglucosides. It has been found that this high regioselectivity can improve bioaccumulation in the skin. Preferably, the monoglucosides are formed with high selectivity and in high stereoselectivity.

[0030] In one embodiment, 4'-O-α-monoglucosides (i.e., epigallocatechin gallate 4'-O-α-monoglucoside or epigallocatechin 4'-O-α-monoglucoside, depending on the substrate) represent at least 60%, more preferably at least 65%, even more preferably at least 70%, and most preferably at least 75% of all glucosides formed. In this context, the term "glucosides" encompasses all glucosides formed, i.e., any monoglucoside, diglucoside, and triglucoside, and glucosylation can occur at any position.

[0031] The present invention further encompasses epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside, respectively, formed by the methods of the present invention. As explained above, these products are formed with significantly greater selectivity than previously observed. Furthermore, they have also been found to exhibit improved skin penetration and bioaccumulation. The products of the present invention are also microbiome-compatible.

[0032] In a further aspect, the present invention provides a method for providing radiant skin glow and / or removal of blemishes, in which epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside are topically applied to the skin. The method is cosmetic, i.e., non-therapeutic.

[0033] Surprisingly, epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside have a much higher bioavailability in the skin than the respective aglycones. For example, epigallocatechin gallate 4'-O-α-monoglucoside was primarily accumulated in the skin at a depth of approximately 65 μm, almost twice the amount of the aglycone, epigallocatechin gallate. A glucoside mixture containing monoglucosides, diglucosides, and triglucosides was found to have a skin penetration depth of approximately 50 μm. Detailed results are presented in the Examples section below.

[0034] In several in tubo, in vitro, ex vivo and clinical studies, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside have further been found to exhibit various activities that make them interesting for cosmetic applications, including antioxidant, antiglycation and skin-lightening activities. For example, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention significantly reduced reactive oxygen species (ROS) by -66% using an in vitro model, demonstrating strong antioxidant activity.

[0035] Furthermore, in tubular studies have shown that epigallocatechin gallate 4'-O-α-monoglucoside of the present invention is a potent anti-glycation active agent, as demonstrated by an IC50 value of 0.118 mM, which is better than the positive reference used in the study (aminoguanidine) and other well-known molecules such as vitamin C. The epigallocatechin gallate 4'-O-α-monoglucoside of the present invention also has excellent whitening activity: in an ex vivo study, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was topically applied to human skin explants and compared with kojic acid as a positive control. Epigallocatechin gallate 4'-O-α-monoglucoside provided a 34% reduction in melanin content, which is similar to kojic acid.

[0036] Clinical evaluation of Asian female volunteers revealed that epigallocatechin gallate 4'-O-α-monoglucoside of the present invention can improve skin uniformity, bring about a lighter skin tone, and reduce pigmented spots, resulting in a more uniform and brighter skin tone.In fact, a significant increase in skin uniformity was observed—up to 3.8 times better than placebo—after twice-daily application for 56 days.In addition, a strong reduction in melanin content in the pigmented spot area was also observed, up to 21.7% compared to placebo.Therefore, compared to the common benchmark of 2% vitamin C, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention induces similar effects and even works better.

[0037] The epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was also found to significantly improve skin firmness by up to 21 times compared to placebo after 56 days. Additionally, a significant increase in dermal density of up to 16.6% was observed. In another clinical study on Caucasian volunteers with pigmented spots on their hands, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was found to significantly reduce skin unevenness, improve skin tone, and reduce pigmented skin.In fact, a significant reduction of up to 24% in skin unevenness was observed, which means that epigallocatechin gallate 4'-O-α-monoglucoside is more effective than vitamin C.

[0038] A detailed description of the biological tests is presented further below in the Examples section.

[0039] Overall, these results indicate that epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside of the present invention are extremely well suited for skin care products providing whitening effects, anti-wrinkle, skin brightening and whitening activity. As a result, the epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside of the present invention offer many benefits, making them of interest for cosmetic applications such as anti-aging, face care, and general skin care products.

[0040] In one embodiment, the epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside of the present invention are each formed by a method according to the present invention.

[0041] Preferably, 4'-O-α-monoglucosides are present in a high proportion compared to other glucosides. In one embodiment, the ratio of epigallocatechin gallate 4'-O-α-monoglucoside to other epigallocatechin gallate glucosides is at least 2:1, more preferably at least 3:1, even more preferably at least 4:1, and most preferably at least 5:1.

[0042] In one embodiment, the ratio of epigallocatechin gallate 4'-O-α-monoglucoside to other epigallocatechin gallate glucosides is at least 2:1, more preferably at least 3:1, even more preferably at least 4:1, and most preferably at least 5:1. As mentioned above, monoglucosides exhibit particularly high bioavailability in the skin.

[0043] In one embodiment, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside each form part of a cosmetic composition. Cosmetic compositions typically comprise a cosmetically acceptable carrier and one or more cosmetic active agents—in this case epigallocatechin gallate 4′-O-α-monoglucoside and / or epigallocatechin 4′-O-α-monoglucoside.

[0044] Thus, in one embodiment, a cosmetic composition comprising epigallocatechin gallate 4'-O-α-monoglucoside and a cosmetically acceptable carrier is applied topically to the skin. In one embodiment, a cosmetic composition comprising epigallocatechin 4'-O-α-monoglucoside and a cosmetically acceptable carrier is applied topically to the skin.

[0045] The cosmetic composition comprises epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside in an amount effective to provide the desired activity. In one embodiment, the cosmetic composition comprises from about 0.0001% to about 0.03%, more preferably from about 0.001% to about 0.02%, and most preferably from about 0.005% to about 0.01% epigallocatechin gallate 4'-O-α-monoglucoside. In one embodiment, the cosmetic composition comprises from about 0.000075% to about 0.0225%, more preferably from about 0.00075% to about 0.015%, and most preferably from about 0.00375% to about 0.0075% epigallocatechin gallate 4'-O-α-monoglucoside.

[0046] The cosmetic compositions used according to the invention may contain, in addition to epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside, one or more further cosmetic active agents. For example, the cosmetic composition may include skin lightening agents, dark spot erasers, skin toning agents, hydrating agents, moisturizing agents, UV protection agents, anti-aging actives, and / or soothing agents. In one embodiment, the cosmetic composition comprises one or more additional cosmetic actives selected from the group consisting of skin lightening agents, dark spot erasers, skin toning agents, hydrating agents, moisturizing agents, UV protection agents, and / or anti-aging actives.

[0047] Examples of skin lightening agents that may be used in the compositions of the present invention include niacinamide, 12-hydroxystearic acid, resorcinol, phenylethyl resorcinol, 4-alkyl substituted resorcinol compounds, glutathione precursors, vitamin B6, vitamin C, vitamin A, caffeine, gallic acid and its derivatives, galardin, adapalene, aloe extract, sage extract, ginger extract, ammonium lactate, arbutin, azelaic acid, butylhydroxyanisole, butylhydroxytoluene, citric acid esters, deoxyarbutin, 1,3-diphenylpropane derivatives, 2,5-dihydroxybenzoic acid and its derivatives, 2-(4-acetoxyphenyl)-1,3-dithiane, 2-( These include 4-hydroxyphenyl)-1,3-dithiane, ellagic acid, glucopyranosyl-1-ascorbic acid, gluconic acid, glycolic acid, 4-hydroxy-5-methyl-3[2H]-furanone, 4-hydroxyanisole and its derivatives, 4-hydroxybenzoic acid derivatives, hydroxycaprylic acid, inositol ascorbate, lactic acid, lemon extract, linoleic acid, magnesium ascorbyl phosphate, 5-octanoylsalicylic acid, salicylic acid, 3,4,5-trihydroxybenzyl derivatives, acetylglucosamine, Pitera extract, Thin White, calcium pantothenate (Melanoblock), Seppi White, soybean extract (Bowman-Birk inhibitor), and mixtures thereof. When 12-hydroxystearic acid is used in the composition, it is used as a skin lightening agent, not as a fatty acid.

[0048] Additionally or alternatively, the cosmetic compositions of the present invention may further comprise plant extracts, such as cranberry, hibiscus, guarana, acerola, ginger, licorice, pomegranate, lotus, rhodiola and / or schizandra extracts. The cosmetic composition may also include one or more additives.

[0049] In one embodiment, the cosmetic composition further comprises a fatty acid. The fatty acids, when present in the composition together with the soap, provide what is called a vanishing effect, i.e., the composition vanishes onto the skin without leaving any significant streaks of the composition when applied to human skin. Preferably, the fatty acids that may be present in the composition are selected from fatty acids having from 10 to 30 carbon atoms, more preferably from 12 to 25, even more preferably from 14 to 20, and even more preferably from 16 to 18. Examples of fatty acids that may be used in the composition include pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, arachidic acid, behenic acid, erucic acid, and mixtures thereof.

[0050] In one embodiment, the cosmetic composition further comprises a soap. Soaps, when present in combination with fatty acids in the composition, provide a quenching effect. The soap is preferably prepared by in-situ neutralization of the fatty acids present in the composition. Thus, the soap preferably has a carbon chain length corresponding to the chain length of the fatty acids in the composition. The soap is formed from the fatty acids by using an alkali metal hydroxide, for example, sodium hydroxide or potassium hydroxide. Of the two, potassium hydroxide is more preferred. Thus, the soap is preferably a potassium soap (potassium salt of a fatty acid). Preferably, the composition comprises from about 0.1 wt% to about 10 wt%, more preferably from about 1 wt% to about 8 wt%, more preferably from about 2 wt% to about 7 wt%, and even more preferably from about 3 wt% to about 6 wt% soap.

[0051] In one embodiment, the cosmetic composition further comprises a surfactant, especially a nonionic surfactant having an HLB value in the range of 9 to 20, preferably 10 to 19, even more preferably 12 to 18, even more preferably 13 to 17, and even more preferably 15 to 17. HLB is calculated using the Griffin method, which gives results on an arbitrary scale of 0 to 20, where Mh is the molecular weight of the hydrophilic portion of the molecule and M is the molecular weight of the whole molecule. Typical values ​​for various surfactants are shown below: Value <10: Lipid soluble (water insoluble) Value >10: Water soluble Values ​​of 4 to 8 indicate antifoaming agents Values ​​of 7 to 11 indicate W / O (water-in-oil) emulsifiers Values ​​of 12 to 16 indicate oil in water emulsifiers Values ​​of 11 to 14 indicate wetting agents Values ​​of 11 to 15 are typical of cleaning agents Values ​​between 16 and 20 indicate a soluble agent or hydrotrope

[0052] Preferably, the nonionic surfactant having an HLB value in the range of 9 to 20 is selected from fatty alcohol ethoxylates, alkylphenol ethoxylates, polyoxyethylene sorbitan alkyl esters and mixtures thereof. Preferably, the nonionic surfactant has at least 9 alkylene oxide groups, preferably at least 9 ethylene oxide groups. Preferably, the composition comprises from about 0.5 wt% to about 5 wt%, more preferably from about 1 wt% to about 4 wt%, and even more preferably from about 2 wt% to about 3 wt% of a nonionic surfactant having an HLB in the range of 9 to 20.

[0053] In one embodiment, the cosmetic composition further comprises a polymer. The polymer acts as a thickener in the composition and improves the sensory properties of the composition. The polymer is preferably selected from the following classes: - acrylate / R-methacrylate copolymers, such as, for example, acrylate / steareth-20 methacrylate copolymer (commercially available as Aculyn® 22) and acrylate / beheneth-25 methacrylate copolymer (commercially available as Aculyn® 28); - acrylate / R-methacrylate crosspolymers, such as acrylate / steareth-20 methacrylate crosspolymer (commercially available as Aculyn™ 88); - acrylate copolymer (commercially available as Aculyn™ 33); - acrylate / R-alkyl acrylate crosspolymers, such as acrylate / C10-C30 alkyl acrylate crosspolymer (commercially available as Pemulen™ TR-2); - Copolymer of ammonium acryloyldimethyltaurate and vinylpyrrolidone (commercially available as Aristoflex® AVC). - copolymer of sodium acryloyldimethyltaurate and vinylpyrrolidone (commercially available as Aristoflex® AVS); and Crosspolymers of acryloyldimethyltaurate with R-alkyl acrylates and methacrylates, such as ammonium acryloyldimethyltaurate / beheneth-25 methacrylate crosspolymer (commercially available as Aristoflex® HMB and Aristoflex® BLV). Preferably, the composition comprises from about 0.1 wt% to about 5 wt%, more preferably from about 0.5 wt% to about 4.5 wt%, even more preferably from about 1 wt% to about 4 wt%, even more preferably from about 1.5 wt% to about 3.5 wt%, and still more preferably from about 2 wt% to about 3 wt% of the polymer.

[0054] In one embodiment, the cosmetic composition further comprises an emollient. Examples of emollients that may be used in the composition are stearyl alcohol, glyceryl monoricinoleate, mink oil, cetyl alcohol, isopropyl isostearate, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecane-2-ol, isocetyl alcohol, eicosanyl alcohol, behenyl alcohol, cetyl palmitate, dibutyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, Included are lanolin, cocoa butter, corn oil, cottonseed oil, olive oil, palm kernel oil, rapeseed oil, safflower seed oil, evening primrose oil, soybean oil, sunflower seed oil, avocado oil, sesame oil, coconut oil, arachis oil, castor oil, acetyl lanolin alcohol, petrolatum, mineral oil, butyl myristate, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and mixtures thereof.

[0055] In one embodiment, the cosmetic composition further comprises a solvent. Examples of solvents that may be used in the composition include ethyl alcohol, isopropanol, acetone, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, and mixtures thereof.

[0056] In one embodiment, the cosmetic composition further comprises a powder. Examples of powders that may be used in the composition include chalk, talc, fuller's earth, kaolin, starch, gum, colloidal silica sodium polyacrylate, tetraalkyl and / or trialkylarylammonium smectite, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethylcellulose, ethylene glycol monostearate, and mixtures thereof.

[0057] In one embodiment, the cosmetic composition further comprises a preservative to protect against the growth of potentially harmful microorganisms. Examples of ingredients that can be used as preservatives in the composition include alkyl esters of para-hydroxybenzoic acid, hydantoin derivatives, propionate salts, and various quaternary ammonium compounds. More preferably, ingredients that can be used as preservatives in the composition include sodium benzoate, iodopropynyl butyl carbamate, methylisothiazolinone, iodopropynyl butyl carbamate, phenoxyethanol, methylparaben, propylparaben, imidazolidinyl urea, sodium dehydroacetate, ethylhexylglycerin, benzyl alcohol, alkanediols, and mixtures thereof. Suitable alkanediols for use as preservatives are C6-C12 alkanes vicinal substituted with hydroxy groups. Illustrative examples include 1,2-octanediol (caprylyl glycol), 2,3-octanediol, 1,2-nonanediol, 1,2-decanediol, 1,2-hexanediol, 3,4-octanediol and mixtures thereof, with caprylyl glycol typically being most preferred. When present in the composition, preservatives are preferably added in an amount of from about 0.001 wt% to about 5 wt%, more preferably from about 0.01 wt% to about 3 wt%, and most preferably from about 0.02 wt% to about 2 wt%.

[0058] The cosmetic compositions may further comprise a range of other optional ingredients including antioxidants, binders, biological additives, buffers, colorants, astringents, fragrances, opacifiers, conditioners, exfoliants, pH adjusters, natural extracts, skin sensitizers, skin soothing agents, and skin healing agents.

[0059] The cosmetic composition is preferably formulated in the form of a water-containing composition such as a lotion, cream, emulsion, gel or mousse, more preferably in the form of a cream or lotion, most preferably in the form of a cream.

[0060] The present invention is further illustrated by the following non-limiting examples.

[0061] Example 1: Synthesis of epigallocatechin gallate 4'-O-α-monoglucoside (EGCG MONOG) In a thermostatically stirred reactor, 2.40 kg (=2.40 L) of demineralized water was introduced. The temperature was adjusted to 30°C ± 1°C. Under gentle stirring, 1.215 kg of sucrose (food grade) was introduced in small increments (approximately six 0.2 kg fractions, each fraction being added once the previous fraction had completely dissolved). Subsequently, 4.0 g of calcium chloride was introduced. 0.47 kg (=0.60 L) of 2-propanol (minimum purity: 98%) was introduced under gentle stirring. 41.0 g of epigallocatechin gallate (EGCG; minimum purity: 95%) was introduced under gentle stirring. Once completely solubilized, 0.162 kg of 0.50 M sodium acetate buffer with a pH of 5.41 was introduced into the reactor. At this point, the weight of the intermediate reaction mixture was 4.292 kg and the volume was 3.942 L.

[0062] 0.840 kg (=0.840 L) of demineralized water was added, the pH was adjusted to 5.30 with 1 ml of 6N H2SO4, and 0.203 kg of enzyme solution (35.0 U / g; 1 U corresponds to the amount of enzyme that releases 1 μmol of fructose from sucrose per minute; assay conditions: 100 g / L sucrose, pH 5.20, 20 mM sodium acetate buffer, 0.02 g / L calcium chloride) was added. The total mass of the reaction mixture was 5.247 kg, and the volume was 4.897 L. The EGCG concentration was 7.8 g / kg (17.0 mmol / kg) or 8.4 g / L (18.3 mM). The dextransucrase concentration was 0.76 U / g or 0.81 U / ml.

[0063] The reaction was carried out for 24 hours. The enzymatic reaction was stopped by adjusting the pH of the reaction mixture to 2.0 using concentrated sulfuric acid. EGCG-related substances were separated from sugars, enzymes, and salts by adsorption using a hydrophobic resin (Diaion HP20, Mitsubishi Chemical Industries, Japan), followed by elution with an ethanol / water mixture (specific gravity at 20°C: 0.862). The ethanol / water mixture used to elute the EGCG-related substances was removed by vacuum evaporation. EGCG-glucosides were separated from EGCG using a liquid-liquid extraction method with ethyl acetate (the concentration of the substances and the relative volume of ethyl acetate were adjusted to maximize the purification yield). Such methods are known to those skilled in the art and are described elsewhere, in particular in WO 2007 / 144368 (pp. 46-47).

[0064] The final composition of the purified preparation was as follows: - Monoglucosides: 41.7317 mmol / kg (equivalent to 87.77% of the total glucosides) - Diglucoside: 5.813 mmol / kg (corresponding to 12.23% of the glucosides) - Triglucosides: Not observed - Residual EGCG: 0.4466mmol / kg

[0065] Epigallocatechin gallate 4'-O monoglucoside was the major constituent, representing 79.10% of the monoglucosides. This purified preparation, containing approximately 69% epigallocatechin gallate 4'-O monoglucoside, was used in the studies described below.

[0066] Example 2: Synthesis of epigallocatechin 4'-O-α-monoglucoside Epigallocatechin 4'-O-α-monoglucoside was prepared under the same reaction conditions as described in Example 1, substituting epigallocatechin for epigallocatechin gallate as the substrate.

[0067] Example 3: Statistical analysis For the in vitro and ex vivo studies described in Examples 4-8, all results are presented as the mean ± standard error of the mean (SEM) of three independent triplicates. The in vivo data in Examples 11-14 are typically expressed in percent variation compared to D0.

[0068] The Shapiro-Wilk test was used to verify whether the raw data followed the Gaussian law. In the case of normally distributed data, means were compared using either an unpaired t-test (≤2 groups) or one-way ANOVA followed by a post-hoc test (≥2 groups). In the case of non-normally distributed data, the Kruskal-Wallis test followed by the Mann-Whitney U test was used for unpaired data.

[0069] For all in vivo studies, the Shapiro-Wilk test was used to verify whether the raw data followed Gauss's law. In cases of normally distributed data, means were compared using either unpaired Student's t-tests or paired Student's t-tests. In cases of non-normally distributed data, the Wilcoxon (paired) or Kruskal-Wallis test, followed by the Mann-Whitney-Yew (unpaired) test, was used for paired or unpaired data, respectively. For analyses of self-assessment questionnaires and daily log results, a chi-square test (a dichotomous analysis comparing the number of related responses) was performed.

[0070] In all cases, results were considered significant: p<0.1 and #, p<0.05 and *, p<0.01 and **, and p<0.001 and ***.

[0071] Example 4: Antioxidant activity (in vitro) Cell culture and treatments A 96-well plate was coated with 50 μg / ml collagen I solution. Normal human epidermal keratinocytes (NHEK) were seeded at 20,000 cells per well in quadruplicate. Cells were incubated for 24 hours at 37°C with 5% CO2 in complete medium (Epilife, Gibco) supplemented with human keratinocyte growth supplement (HKGS) factors (Gibco) and 1% antibiotics (Sigma).

[0072] At the end of the incubation period, cells were treated with the purified preparation from Example 1 (containing 0.1% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside) or with 200 μM resveratrol (used as a positive control for the assay), both in 2',7'-dichlorodihydrofluorescein diacetate, for 24 h at 37 °C with 5% CO2. After 24 h, cells were incubated with 50 μM DCFH-DA probe (culture medium) for 30-40 min at 37 °C with 5% CO2 and then stressed with 5 mM tert-butyl peroxide (TBP). The fluorescence evolution was followed for 1 h.

[0073] result Resveratrol at 200 μM significantly reduced ROS production by 61%, confirming the experiment. 0.1% epigallocatechin gallate 4'-O-α-monoglucoside significantly reduced ROS production by 66%, confirming its antioxidant activity on keratinocytes. [Table 1]

[0074] Example 5: Anti-glycation activity (in vitro) The anti-glycation effect was evaluated using an in tubo assay that measures the spontaneous formation of advanced glycation end products (AGEs).

[0075] treatment The assay was performed in a 96-well microplate. 40 μl of the purified preparation from Example 1 containing 0.1% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside was mixed with 50 μl of BSA and 10 μl of ribose. The extract was diluted in sodium phosphate buffer (0.1 M, pH 7.4). Several dilutions were tested. Each diluted extract was performed in triplicate. A standard range of aminoguanidine was used as an inhibitor reference (positive control). A mixture of sodium phosphate buffer and BSA was used to validate the experiment, and a mixture of sodium phosphate buffer, BSA, and ribose was used as a positive control.

[0076] Two blanks were run on the extract: - Uses phosphate buffer, BSA, and extracts - Uses phosphate buffer, ribose, and extracts The microplates were agitated and incubated for 17 hours at 37° C. Fluorescence readings were taken at λ excitation 340 nm and λ / emission 420 nm. The anti-glycation activity of the extract was calculated as follows:

number

[0077] result The results are summarized in the table below: [Table 2]

[0078] Example 6: Evaluation of skin penetration by Raman spectroscopy (ex vivo) Skin explant culture and treatment Skin explants from Caucasian volunteers were treated with 2% epigallocatechin gallate (i.e., aglycone), a glucoside mixture containing epigallocatechin gallate monoglucosides, diglucosides, and triglucosides, or the purified preparation of Example 1 containing epigallocatechin gallate 4'-O-α-monoglucoside for 8 hours at 37°C with 5% CO2. At the end of the incubation period, the skin explants were frozen at optimal cutting temperature (OCT; VWR) of -80°C and then cut longitudinally at a thickness of 20 μm using a cryotome. For Raman imaging analysis, three tissue sections were selected for each explant and deposited on a CaF2 support. Corrected data maps were processed using software based on the least-squares fitting method running in the Matlab environment. This method involves mathematically modeling reference spectra across a spectral image and determining the contribution and distribution of these spectra within the image. The reference spectra were modeled spectra of the following products: aglycone, glucoside mixture, 4'-O-α-monoglucoside, and untreated skin control.

[0079] result After 8 hours of topical application, the skin penetration of epigallocatechin gallate 4'-O-α-monoglucoside was found to be significantly higher than that of the aglycone (+90%) and the glucoside mixture (+20%). Figure 1 shows a semi-quantitative representation of the results. Thus, the epigallocatechin gallate 4'-O-α-monoglucoside of the present invention has better skin bioavailability than the aglycone and glucoside mixture.

[0080] Example 7: Evaluation of whitening activity using Fontana-Masson staining for melanin quantification (ex vivo) Skin explant culture and treatment Fresh human skin explants were obtained from skin phototype III and VI donors aged 46 ± 25 years. The explants were topically treated with 0.4% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside (purified preparation from Example 1) or 2% (v / v) vitamin C, both in water, for 7 days and incubated at 5% CO2 and 37°C. 2% kojic acid (Sigma-Aldrich) was applied topically and incubated under the same conditions, serving as a positive control. The untreated condition consisted of the vehicle (distilled water). The culture medium was refreshed every other day. After the end of the incubation period, the explants were sampled and placed in formalin for Fontana-Masson staining, then dehydrated and embedded in paraffin.

[0081] Melanin quantification by Fontana-Masson staining and image analysis Formalin-fixed skin explants were dehydrated and embedded in paraffin. 4-µm-thick sections were dewaxed and then stained for melanin using the Fontana-Masson silver method. Images were collected using an Axio Observer inverted fluorescence microscope (Zeiss) in bright-field mode. Melanin was quantified using two open-source optical imaging software programs. Photomicrographs (JPEG format) of Fontana-Masson tissue sections were opened in GIMP (GNU Image Manipulation Program). The dark-brown color signals corresponding to melanin grains in the stained sections were selected, copied, and pasted into a new image and saved as a JPEG file; this JPEG file consisted of only black / brown (melanin grains) on a white background. This image was subsequently opened using the ImageJ program. The image was then inverted and the average intensity was obtained.

[0082] result 2% kojic acid and 2% vitamin C significantly reduced melanin content by 34% and 28%, respectively, compared to the untreated condition, confirming the experiment. 0.4% epigallocatechin gallate 4'-O-α-monoglucoside significantly reduced melanin content by 34% compared to the untreated condition.

[0083] Example 8: Transcriptome analysis To assess the impact of the preparation of the present invention on melanin production, co-culture studies with keratinocytes and melanocytes were performed.

[0084] Cell culture and treatments Cell culture was performed with primary cells isolated from biopsies. Cells were co-cultured at a ratio of 10 keratinocytes to 1 melanocyte. Both cell types were derived from a 7-year-old Caucasian male donor. The experiments were carried out under the following conditions: - NHEK-NHEM co-culture untreated - NHEK-NHEM co-culture + diluted version of the purified preparation from Example 1 containing 0.01% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside (abbreviated as "EGCG MONOG 0.01%") The product EGCG MONOG 0.01% was filtered and diluted in Keratinocyte Growth Medium 2 (supplied by Promocell).

[0085] Cells were seeded in 6-well plates for 24 hours in the presence of Keratinocyte Growth Medium 2. After seeding, treatment was applied in the presence of 0.01% EGCG MONOG for 24 hours. Untreated wells were used as a basal reference to compare the impact of EGCG MONOG and gene expression after 24 hours of incubation. After treatment, RNA was extracted from the cells using TRIzol™ (available from Sigma) and reverse transcribed into cDNA. The cDNA was mixed with various primers representing the melanogenesis pathway (PrimePCR custom plate, pigmentation, BioRad). mRNA expression was measured by semi-quantitative PCR on a Bio-Rad CFX96 Real Time PCR System (available from BioRAD). mRNA expression levels were calculated and normalized to a reference gene (B2M). Values ​​were reported relative to untreated controls and represented activating or inhibitory effects.

[0086] result After 24 hours of treatment with EGCG MONOG 0.01%, RT-qPCR revealed that EGCG MONOG 0.01% significantly reduced the expression of genes involved in the regulation of melanin production (KIT -93%, EDNRB -68%, MC1R -96%, SOX10 -60%, MITF -108%). EGCG MONOG 0.01% also significantly reduced melanin synthesis (TYR -58% and TYRP1 -95%), melanosome biogenesis (AP3B1 -59%, GPR143 -365%, PMEL -212%, DTNBP1 -59%, and LYST -48%), and melanin uptake by keratinocytes (F2RL1 -49%) compared to untreated co-cultures.

[0087] Example 9: Consumption of EGCG MONOG by Bacillus sp. We hypothesized that because the active agent of the present invention is glycosylated, it can be used as a substrate by bacteria. To test this hypothesis, Bacillus sp. was cultured in the presence of 1% EGCG MONOG, and its consumption was monitored by HPLC for 48 hours.

[0088] material and method Bacillus sp was isolated from normal and healthy skin (GAB04-01) and used for this study. The bacterial strain is stored at 80°C in the presence of 20% w / v glycerol.

[0089] In preparation for this study, selected samples were thawed and aliquots (cryotubes of bacteria stored at -80°C) were spread onto tryptic soy agar plates, followed by incubation at 30°C. A single colony was used to inoculate 10 ml of tryptic soy broth (TSB) into stoppered plastic tubes and incubated overnight at 37°C under agitation. The optical density at 600 nm was measured after a 10-fold dilution with TSB medium (Biophotometer D30). An aliquot of the sample was first diluted with fresh culture medium (Difco® Sporulation Medium (DSM) 0.5) to obtain an optical density of 1.216. 1 mL of this suspension was used to inoculate 40 mL of fresh culture medium (DSM 0.5). 24 hours after the start of the culture, the medium was changed by adding 1% of the purified preparation of Example 1 containing 0.1% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside or water (negative control) to the medium (i.e., samples were treated with 39.6 mL of DSM 0.5 and either 0.4 mL of EGCG MONOG or 0.4 mL of water). The optical density of the reaction medium immediately after inoculation was 0.030 ± 0.003.

[0090] The kinetics of EGCG MONOG in the presence of Bacillus sp. was followed using HPLC-UV-MS with the following parameters: - Elution profile: 1.00 ml / min - Column: X-Bridge C18 - Injection: 10μl - Temperature: 40℃ - Spectrophotometer: 270nm

[0091] result After only 24 hours, the amount of EGCG MONOG was reduced by 0.307 mM. At the same time, EGCG aglycone or other EGCG derivatives (such as EGCG diglucoside) were not detected, proving that the reduction of EGCG MONOG was related to bacterial consumption and not its degradation. Detailed results are shown in the table below: [Table 3]

[0092] Example 10: Growth promotion of Lactobacillus acidophilus To further investigate the impact of the active agents of the present invention on bacteria, the growth of Lactobacillus acidophilus in MRS medium supplemented with different concentrations of EGCG MONOG was evaluated for 48 hours.

[0093] material and method L. acidophilus (DSM 20079) was cultured on De Man, Rogosa Sharpe (MRS) agar under anaerobic conditions at 37°C. After amplifying the bacteria in the culture medium, a bacterial suspension was prepared for each bacterial strain using assay medium (50% MRS broth in PBS) and adjusted to an optical density at 600 nm (OD600nm) of 0.2. The bacterial suspension was then transferred to a 96-well plate, some of which contained 0.015% to 1% test compound. Strains cultured in 0.01% milk powder served as negative controls. The optical density of the bacterial suspension was measured using a wavelength of 600 nm and showed a value of 0.1. The bacterial cultures were incubated under agitation (~280 rpm) and anaerobic conditions at 37°C. The OD was read kinetically using a microplate spectrophotometer (EPOCH2, BioTek Instruments) for 48 hours to analyze bacterial growth. All experimental conditions were performed in triplicate.

[0094] result The experiment was validated, as 0.01% milk powder significantly increased bacterial growth. The active agent of the present invention was able to significantly increase the growth of Lactobacillus acidophilus, with a dose-dependent effect of 6%, 9%, and 27% at concentrations of 0.13%, 0.4%, and 1%, respectively, compared to cultures without EGCG MONOG.

[0095] Example 11: Analysis of metabolites released by Lactobacillus acidophilus Since the active agents of the present invention were able to stimulate the growth of L. acidophilus, it was decided to analyze the metabolites released by the bacteria in the culture medium by high-field NMR.

[0096] material and method The supernatants of the cultures described above in Example 10, both the untreated strain cultures and the L. acidophilus cultures treated with 1% EGCG MONOG, were stored frozen at -80°C and then analyzed using high-field NMR. For each of these two conditions, three samples were analyzed to assess the reproducibility of the test (n=3).

[0097] For each sample, 400 μl of supernatant was taken and 100 μl of DO / TSP (3-(trimethylsilyl)propionic acid-2,2,3,3-d4 acid sodium salt, internal reference) was added at a concentration of 1 g / l. Samples were analyzed in high field mode on a Bruker ASCEND 500 spectrometer operating at a proton frequency of 500 MHz using a 5 mm cryosonde probe. 1 The compounds were analyzed by H NMR. NMR spectra were calibrated to TSP-d4 at 0 ppm using Topspin software version 4.3.0 (Bruker Biospin, Germany). Quantification of metabolites was performed by utilizing an internal standard (TSP-d4) added at a known concentration. Compounds were quantified by the relative ratio of the peak integral intensity to the intensity of the internal standard.

[0098] result It was found that the addition of 1% of the active agent of the present invention altered the carbon source of the strain for its metabolites: untreated bacteria primarily used sugars as a carbon source, while treatment with EGCG MONOG resulted in a 17% decrease in sugar consumption, favoring polysorbate consumption, which increased significantly by 51%. In addition, the following culture, L. acidophilus, an active agent of the present invention, released significantly more trigonelline, up to 65%.

[0099] Example 12: Compositions used in clinical studies A clinical trial was conducted using a facial cream containing 0.4% of the purified preparation of Example 1 containing epigallocatechin gallate 4'-O-α-monoglucoside or 2% ascorbic acid (vitamin C) versus a placebo cream to evaluate its effect on volunteers with dull skin, large facial aging spots (greater than 3 mm), and shallow wrinkles. For this study, volunteers applied the cream twice daily for 56 days. For the clinical trials described in Examples 14-17, the following compositions were used:

[0100] INCI formulation Placebo: Aqua / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, Ceteth-20, Steareth-20, Isodecyl Neopentanoate, Phenoxyethanol, 1-2-Hexanediol, Caprylyl Glycol, Dimethicone, Fragrance Active Ingredients: Aqua / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, Ceteth-20, Steareth-20, Isodecyl Neopentanoate, Epigallocatechin Gallate Monoglucoside, Phenoxyethanol, 1-2-Hexanediol, Caprylyl Glycol, Dimethicone, Fragrance Vitamin C: Aqua / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, Ceteth-20, Steareth-20, Isodecyl Neopentanoate, Ascorbic Acid, Phenoxyethanol, 1-2-Hexanediol, Caprylyl Glycol, Dimethicone, Fragrance

[0101] composition [Table 4]

[0102] Example 13: Panels used in clinical studies The clinical studies described in Examples 14-16 were conducted as follows: A single-center study was conducted on 38 Asian volunteers with dull skin and large facial spots (greater than 3 mm) presenting with shallow age spots. The volunteers were divided into two groups: - Group 1: 20 Asian women with a mean age of 54±5 years who will be tested with the active cream compared to the placebo cream. - Group 2: 18 Asian women with a mean age of 53±8 years testing vitamin C cream compared to placebo cream.

[0103] The study was carried out in accordance with Bio EC standard operating procedures and in compliance with the regulations laid down in the Guia para investigaciones con seres humanos (Guidelines for investigations involving humans) and the guidelines of the Scientific Committee for Consumer Safety (SCCS). Volunteers applied a cream containing 0.4% active agent and 2% ascorbic acid (vitamin C) to one half of their face and a placebo cream to the other half every other day for 56 days. During the study, skin color and mottle analysis was performed using Color face®, biomechanical properties were tested using Cutometery®, and dermal thickness was determined using a DUB SkinScanner 75® probe (maximum depth 3.28 mm).

[0104] Example 14: Skin biomechanical properties measurement with Cutometer® method Analysis of the mechanical properties of the skin allows assessing the functional state of the following tissue structures: - elastic structures, such as elastic fibers, curvature of connective bundles, wrinkles of the stratum corneum, etc.; and - Viscous behavior structures, e.g., interstitial fluid, internal adhesion, etc.

[0105] The study was carried out using a Cutometer® MPA 580 (Courage & Khazaka). The measurement principle is based on the suction method: a negative pressure is created in the device, drawing the skin into the cylindrical opening (2 mm diameter) of the probe. Inside the probe, the penetration depth is determined by an optical measurement system. Each suction phase is followed by a relaxation phase.

[0106] In this study, the following programs were used: - Cycle length: 4 seconds - Suction: 2 seconds - Relaxation: 2 seconds - Negative pressure: 450 mbar - Chamber diameter: 2mm - Measurement area: fine lines around the eyes

[0107] The resistance of the skin to negative pressure and its ability to return to its original position are displayed as curves at the end of each measurement, from which parameters can be calculated. During the suction phase, due to the deformation of the skin by the negative pressure, first the elastic resistance and then the viscous component are measured, which together represent the firmness of the skin. During the relaxation phase, the immediate recovery of the skin measures the elasticity of the skin, while the delayed return of the skin to its initial position measures the viscoelastic component.

[0108] Figure 2 shows an example of a curve measured on elastic skin. The following parameters are indicated: [Table 5]

[0109] This study focused on the R0 or Uf parameter, which represents the amplitude of the skin during the suction phase. At equal pressure, the more flexible the skin, the greater the amplitude. Therefore, R0 (or UF) may evaluate viscoelastic dispensability, or in other words, skin firmness. These parameters were measured at D0, D28 and D56.

[0110] result After 28 and 56 days of application, a decrease in the R parameter was observed, indicating an increase in firmness with epigallocatechin gallate 4'-O-α-monoglucoside (-8.7% at D28 and -8.2% at D56 compared to D0). The results for epigallocatechin gallate 4'-O-α-monoglucoside and ascorbic acid, respectively, are shown in Table 2 below. [Table 6]

[0111] [Table 7]

[0112] These measurements confirmed a significant improvement of up to 21-fold in skin firmness after application of a cream containing epigallocatechin gallate 4'-O-α-monoglucoside compared to a placebo cream. As can be seen in Figure 3, the effect of epigallocatechin gallate 4'-O-α-monoglucoside was also significantly better than ascorbic acid after days D28 and D56.

[0113] Example 15: Analysis of Skin Color Using Colorface® method Skin color measurements were performed based on photographs taken with the Colorface® acquisition system. Colorface® is a 2D acquisition system for standardized multimodal photographs of the whole face. The device is equipped with a 24M pixel sensor and also has a specific UV lamp. The acquisition methods were: ultraviolet light, unfiltered photography, cross-polarized photography, parallel-polarized photography, standard 45° photography, and standard 60° photography. Analysis of the photographs for complexion radiance, pigmentation, color (L*, a*, b* parameters) and melanin in the spots was performed using image analysis software. 2D photographs of the face were taken at D0, D28, and D56.

[0114] Uniformity of results After 28 and 56 days of application of the active agent cream, a significant increase in mean pigment uniformity of +1.2% and +1.5%, respectively, was observed with the active agent compared to D0. Placebo showed a significant increase of 0.4% after 28 and 56 days compared to D0. Thus, the effect on pigment uniformity was 3.0-fold greater after 28 days and 3.8-fold greater after 56 days for the product containing epigallocatechin gallate 4'-O-α-monoglucoside compared to placebo. The results for the active agent were significant compared to both D0 and placebo as can be seen in the table below. [Table 8]

[0115] For comparison, the results for ascorbic acid are shown in the table below: [Table 9] Thus, vitamin C was 1.6 and 1.8 times better than placebo on days 28 and 56, respectively.

[0116] Shine Results As can be seen from the results in the table below, a clear increase in skin radiance was observed after 28 and 56 days of application of the active agent cream containing 0.4% epigallocatechin gallate 4'-O-α-monoglucoside: [Table 10] Significant increases of 4.5-fold and 2.6-fold were observed after 28 and 56 days, respectively, compared with placebo.

[0117] For comparison, the results for ascorbic acid are shown in the table below: [Table 11]

[0118] Results in reduced pigment defects As can be seen from the results in the table below, a clear reduction in pigment defects was observed after 28 and 56 days of application of the active agent cream containing 0.4% epigallocatechin gallate 4'-O-α-monoglucoside: [Table 12] Thus, compared to placebo, treatment with the active agent resulted in a significant reduction in pigment defects of -16.1% and -21.7% after 28 and 56 days, respectively.

[0119] For comparison, the results for ascorbic acid are shown in the table below: [Table 13]

[0120] Example 16: Ultrasound measurements using a DUB® Skin Scanner method The DUB Skin Scanner system is a high-frequency, high-resolution ultrasound diagnostic tool for use in dermatology, cosmetics, pharmaceuticals and clinical research. 75 MHz ultrasound is a common method for non-invasive skin analysis. High-frequency ultrasound (75 MHz) can probe the superficial layers of the skin and penetrate up to 3 mm into the skin. Skin ultrasound is based on the direction and measurement of the amplitude of the ultrasound wave population within the skin and the portion reflected by the interface separating two media with different acoustic properties. Ultrasound imaging techniques used for visualization of subcutaneous structures allow for assessment of skin uniformity, including density and skin thickness. Thickness and density were measured three times and averaged for each image. For this study, dermal density analysis was performed on D0, D28, and D56.

[0121] result As can be seen from the results in the table below, a clear increase in dermal density was observed 28 and 56 days after application of the active agent cream containing 0.4% epigallocatechin gallate 4'-O-α-monoglucoside: [Table 14]

[0122] Also, application of a cream containing 2% ascorbic acid resulted in a clear increase in dermal density after 28 and 56 days: [Table 15]

[0123] Example 17: Stain removal investigation using Raman spectroscopy Panel Description A double-blind, placebo-controlled clinical evaluation was performed on 45 female Caucasian volunteers aged 45 to 75 years. The volunteers presented with brown spots on their hands. All subjects who participated in the study provided informed consent at the start of the study. Volunteers applied 0.4% of the purified preparation of Example 1 (containing epigallocatechin gallate 4'-O-α-monoglucoside) or a product containing 2% ascorbic acid, or a placebo, to their hands twice daily (morning and evening) for 56 days. On days D0 and D56, the anti-blemish effect was analyzed by Raman spectroscopy.

[0124] composition The composition described in Example 12 was used.

[0125] Raman spectroscopy The setup included a confocal Raman probe coupled with a dispersive Raman spectrometer. The excitation laser beam was delivered to the remote probe via a 5 μm-core fiber, and the Raman signal was transmitted to the spectrometer via a 100 μm-core fiber. The probe was equipped with a 100x long-working-distance objective lens operating in air.

[0126] The piezoelectric device ensured axial measurements from the surface to a defined depth in the skin, allowing the collection of Z Raman profiles. A color video camera built into the probe allowed visualization of the skin surface. This camera was also useful for controlling the focusing of the laser on the skin.

[0127] The spectrometer is equipped with a Peltier-cooled 1024 x 256-element CCD (Coupled Charge Detector) and a grid with 830 grooves / mm. -1 It allows for a wide spectral range of approximately 6cm -1 The excitation source was a 660 nm laser diode, and the power at the sample was set to 30 mW in accordance with radiation protection standards. Protective glasses were used to protect from laser radiation.

[0128] Excitation at 660 nm was chosen as the best compromise between the generation of parasitic fluorescence and the sensitivity of the CCD camera across the entire spectral range. For skin characterization, the measurement of high wavenumber vibrations is crucial to access information about the moisture content. For data acquisition, the device was controlled by Labspec 5 software.

[0129] The Z-profile was constructed by detailed scanning through the skin. Raman spectra were collected at different focal points in 3-μm increments from Z=0 μm to 30 μm below the skin surface.

[0130] The measurement depths are indicated in the table below along with the corresponding acquisition times, resulting in a complete profile time of 1 minute. [Table 16]

[0131] In total, 519 Raman profiles were recorded: 2 profiles per condition and per area (unstained and brown spotted areas). Each profile contained 14 spectra, resulting in 7266 spectra recorded. These measurements were used to evaluate a spectroscopic marker of skin background fluorescence, which is related to the degree of skin whitening: a decrease in this marker indicates a positive whitening effect, while a stability or increase in this marker means that the product has no skin whitening effect.

[0132] Data analysis Spectral data preprocessing was performed using Matlab 7.2 (The MathWorks Inc., USA). Abnormal profiles (poor S / N (signal / noise) ratio, fluorescence, incomplete profiles with large zero offsets, saturation, etc.) were excluded from the database. Each profile without abnormalities was subjected to background correction, which allowed the Raman signal of the skin to be purified. These background corrections included spectral smoothing with a 9 mm Savitzky-Golay filter, spike correction, baseline correction, and intensity normalization over the entire wavenumber range using a vector function.

[0133] No baseline correction was performed to preserve the background fluorescence of the skin, which allowed for the extraction of skin fluorescence markers related to the degree of skin whitening. After these preprocessing steps, the corrected data was used to determine the spectrum corresponding to the keratinous surface.

[0134] result In a clinical study, spectroscopic markers of background skin fluorescence were measured, which correlate with the degree of skin lightening after hand application of a product containing 0.4% epigallocatechin gallate 4'-O-monoglucoside (purified preparation from Example 1) or 2% ascorbic acid, or a placebo. Skin characteristics were evaluated using Raman spectroscopy after 28 and 56 days of twice-daily application. The results are shown in the following two tables: [Table 17]

[0135] [Table 18]

[0136] As can be seen above, both epigallocatechin gallate 4'-O-α-monoglucoside and placebo resulted in similar reductions in melanin detection after 28 days (-10.2 and -14.3, respectively). However, after 56 days, the reduction was more pronounced with epigallocatechin gallate 4'-O-α-monoglucoside (-20.4), while melanin detection remained roughly the same as with placebo (-11.03). On the other hand, ascorbic acid caused a slight increase in melanin detection after 28 days (+2.25 compared to +D0) that was reversed after 56 days (-17.52).

[0137] Thus, D56 demonstrated that epigallocatechin gallate 4'-O-α-monoglucoside significantly reduced background fluorescence, i.e., melanin detection levels, between unblemished control skin and blemished skin compared to placebo. This significant reduction in color difference between unblemished control skin and blemished skin indicates a reduction in pigmented blemishes. Furthermore, the color difference between the blemishes and the surrounding skin was reduced, indicating improved skin uniformity. Finally, a significant difference between ascorbic acid and epigallocatechin gallate 4'-O-α-monoglucoside in favor of epigallocatechin gallate 4'-O-α-monoglucoside was also identified.

[0138] Example 18: Clinical evaluation of African skin Panel Description A clinical study was conducted on 85 female volunteers aged 19-47 years, divided into three groups: active agent, placebo, and aglycone. The study was conducted in South Africa on female volunteers with dark skin types (phototype IV or higher) and dull skin. All participants provided signed informed consent at the beginning of the study. The study was conducted in accordance with the guidelines of the Declaration of Helsinki. Volunteers received a cream containing 0.4% of the purified preparation from Example 1 containing 0.1% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside (active agent) or 0.01% EGCG aglycone or no active agent (placebo) twice daily on the entire face for 56 days. Skin color changes were monitored by L Chromameter®. * Parametric measurements were used to measure after 14, 28, and 56 days.

[0139] INCI formulation [Table 19]

[0140] Assessment of Skin Color Using the Chromameter® L* Parameter Skin color was assessed by a Chromameter CR400 skin color analyzer, and the parameter L* (lightness) was recorded. The higher the value, the lighter the skin color. Depending on the test type, at least three or at least 15 readings were taken at each test subsite for each time interval, which served as the measurement component of the specific pigmented spot color of the study. Skin color was analyzed at D0, D14, D28, and D56.

[0141] statistical analysis For all in vivo studies, the Shapiro-Wilk test was used to verify whether the raw data followed Gauss's law. In the case of normally distributed data, means were compared using either the unpaired Student's t-test or the paired Student's t-test. In the case of non-normally distributed data, the Wilcoxon (paired) or Kruskal-Wallis test, followed by the Mann-Whitney-Yu (unpaired) test, was used for paired or unpaired data, respectively. Results were considered significant: p<0.1 and #, p<0.05 and * , p<0.01 and ** , and p<0.001 and *** .

[0142] Results: Skin color assessment for areas of uneven skin tone First, we looked at uneven areas of the skin and compared the performance of each product. The 0.4% active agent of the present invention was L * A time-dependent effect was found showing a significant increase of +1.3%, +4.7%, and +6.3% after 14, 28, and 56 days, respectively. These increases are the result of gradual whitening activity. On the other hand, the placebo and aglycone * showed the opposite effect of slight reduction in β-glucan and no whitening activity was demonstrated.

[0143] Results: Overall skin color assessment using 15 half-face measurements Whitening performance was then analyzed using 15 Chromameter® measurements on half the face. The data show that in the presence of the active agent of the present invention, L * The results showed a significant increase in serotonin levels, with significant effects after 14, 28 and 56 days. Interestingly, the aglycone and placebo did not perform as well and had little whitening effect at the time of testing. During clinical evaluation, it was shown that the better bioaccumulation of the active agents of the present invention observed during skin penetration analysis correlated well with better clinical whitening performance.

Claims

1. A method for preparing epigallocatechin gallate 4'-O-α-monoglucoside, comprising incubating sucrose and glucansucrase from a Leuconostoc species, preferably from Leuconostoc mesenteroides NRRL B-512F, with epigallocatechin gallate, wherein the incubation is carried out in a medium containing 2-propanol and water.

2. 2. The method of claim 1, wherein the culture medium comprises 5% to 20% (v / v), more preferably 8% to 18% (v / v) 2-propanol.

3. 3. The method of claim 1 or 2, wherein the medium further comprises a lower diol and / or triol selected from the group consisting of 1,3-propanediol, 1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 2,4-butanediol, 1,2,3-propanetriol (glycerol), and mixtures thereof.

4. The method according to any one of claims 1 to 3, wherein the culture medium is dimethyl sulfoxide-free and ether of ethylene glycol (glyme-free).

5. The method according to any one of claims 1 to 4, wherein epigallocatechin gallate is incubated at an initial concentration of 5 to 50 mM, more preferably 10 to 40 mM, and most preferably 15 to 35 mM.

6. The method according to any one of claims 1 to 5, wherein the glucansucrase is dextransucrase.

7. The method according to any one of claims 1 to 6, wherein the concentration of glucansucrase in the reaction mixture is 0.4 to 4.0 U / ml, more preferably 0.7 to 3.5 U / ml, and even more preferably 0.9 to 2.25 U / ml.

8. 8. The method according to any one of claims 1 to 7, wherein monoglucosides are formed in a ratio of at least 2:1 to the sum of diglucosides and triglucosides, more preferably at least 3:1, even more preferably at least 4:1, and most preferably at least 5:

1.

9. 9. The method according to any one of claims 1 to 8, wherein the 4'-O-α-monoglucoside is formed with a regioselectivity of at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 92% on a molar basis compared to other monoglucosides.

10. 10. The method according to any one of claims 1 to 9, wherein epigallocatechin gallate 4'-O-α-monoglucoside represents at least 60%, more preferably at least 65%, even more preferably at least 70%, and most preferably at least 75% of all glucosides formed.

11. Epigallocatechin gallate 4'-O-α-monoglucoside obtained by the method according to any one of claims 1 to 10.

12. A method for providing radiant skin glow and / or removal of blemishes, wherein epigallocatechin gallate 4'-O-α-monoglucoside is topically applied to the skin.

13. 13. The method of claim 12, wherein epigallocatechin gallate 4'-O-α-monoglucoside is formed by the method of any one of claims 1 to 10.

14. 14. The method of claim 12 or 13, wherein the ratio of epigallocatechin gallate 4'-O-α-monoglucoside to other epigallocatechin gallate glucosides is at least 2:1, more preferably at least 3:1, even more preferably at least 4:1, and most preferably at least 5:

1.

15. 15. The method of any one of claims 12 to 14, wherein a cosmetic composition comprising epigallocatechin gallate 4'-O-α-monoglucoside and a cosmetically acceptable carrier is topically applied to the skin.

16. 16. The method of claim 15, wherein the cosmetic composition comprises from about 0.0001% to about 0.03%, more preferably from about 0.001% to about 0.02%, and most preferably from about 0.005% to about 0.01% epigallocatechin gallate 4'-O-α-monoglucoside.

17. 17. The method of claim 15 or 16, further comprising one or more cosmetic actives selected from the group consisting of skin lightening agents, dark spot erasers, skin toning agents, hydrating agents, moisturizing agents, UV protection actives, and / or anti-aging actives.

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