Method for preparing epigallocatechin gallate monoglucoside

Abstract

Methods for the selective preparation of epigallocatechins and mono-glycosides of epigallocatechin gallate, and the use of these products in cosmetics, are provided.

Description

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

[0002] Phenolic compounds (also called phenolics) or polyphenols, constitute one of the most numerous and widespread groups of substances in the plant kingdom. Polyphenols are products of the secondary metabolism of plants. The term "phenolic compound" includes a considerable range of substances having an aromatic ring with one or more hydroxyl substituents. The structure of natural polyphenols varies from simple molecules, such as phenolic acids, to highly polymerized compounds, such as condensed tannins.

[0003] For humans, three important groups are phenolic acids (C6-C1, C6-C2, and C6-C3), flavonoids (C6-C3-C6), and high molecular weight polyphenols (more than 30 carbon atoms). In fact, phenolic substances (especially polyphenols) show a variety of beneficial biological activities in mammals, including antiviral, antibacterial, immunostimulatory, antiallergic, antihypertensive, antiischemic, antiarrhythmic, antithrombotic, cholesterol-lowering, antilipid peroxidation, hepatoprotective, anti-inflammatory, anticancer and antimutagenic, antitumor, antithrombotic, and vasodilatory effects. They are potent antioxidants in vitro.

[0004] Flavonoids consist of a large group of low molecular weight polyphenolic substances, benzo-γ-pyrone derivatives, with a wide variety of chemical structures; they represent the most common and widely distributed group of plant phenolics. The common structure of flavonoids is that of diphenylpropane (C6-C3-C6), which consists of two aromatic rings (rings A and B) connected by three carbons that usually form an oxygen-containing heterocycle (ring C). The basic structure of the flavonoid core and the system used for carbon numbering are shown below:

[0005]

[0006] Flavonoids often exist as glycosides, and glycosylation makes the molecules more water soluble and less reactive toward free radicals. Flavonoids have an ideal structural chemistry for free radical scavenging activity.

[0007] Among flavonoids, epigallocatechin-3-gallate (EGCG) (1) and epigallocatechin-3-gallate (EGCG) (2) have attracted particular attention in cosmetic applications due to their antioxidant, anti-inflammatory, antimicrobial, antiallergic, antiviral, and anti-pigmentation activities, as well as their ability to provide UV protection, activate the skin barrier, and promote cellular activity.

[0008] The structures of these two flavonoids are shown below:

[0009]

[0010] Due to the low water solubility and / or high sensitivity to oxidation of phenolic substances, their use in pharmaceutical or cosmetic preparations requires tailored and specific formulations. Since these formulations must also meet restrictions associated with their end use, it is often difficult to compromise between acceptability, concentration and stability. More water-soluble and / or more antioxidant forms of phenolic substances (such as glycosides) are not always available in nature, and when they exist, complex procedures of extraction and purification from plant materials may be required. Chemical and biochemical (enzymatic) methods have been attempted to increase water solubility and / or stability. Since phenolic compounds have several free hydroxyl groups, attempts to chemically modify phenolic compounds can result in non-selective reactions, generating a variety of different molecules. Additional purification steps are then required to recover the desired (multiple) products.

[0011] WO 2007 / 144368 discloses an enzymatic process for preparing O-α-glucosides of phenolic substances using glucansucrase from Leuconostoc species. Epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside are formed. The enzymatic reaction is usually carried out in a buffered aqueous medium containing dimethyl sulfoxide (DMSO) as a cosolvent.

[0012] The method described in WO 2007 / 144368 provides epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside as a mixture of monoglucoside, diglucoside and triglucoside, respectively, and has relatively low regioselectivity. Typically, monoglucoside and diglucoside are obtained in a molar ratio of about 30:70 to 60:40.

[0013] The problem underlying the present invention was therefore to provide a process for preparing epigallocatechin O-α-glucoside and epigallocatechin gallate O-α-glucoside in a more selective manner, without compromising on yield or scalability.

[0014] This problem has been solved by the method of the invention as described below.

[0015] Surprisingly, it has been found that epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside selectively prepared by the process of the present invention exhibit improved skin permeability and bioaccumulation, respectively. Therefore, they are more suitable for use in cosmetic applications, especially skin care applications.

[0016] In a first aspect, the present invention provides a method for preparing epigallocatechin gallate 4'-O-α-monoglucoside.

[0017] In a second aspect, the present invention provides a method for preparing epigallocatechin 4'-O-α-monoglucoside.

[0018] 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.

[0019] In a fourth aspect, the present invention provides a method for providing a radiant skin complexion and / or age spot removal, wherein epigallocatechin gallate 4'-O-alpha-monoglucoside is topically applied to the skin.

[0020] The present invention relates to a method for preparing epigallocatechin gallate 4'-O-α-monoglucoside, comprising incubating sucrose and dextransucrase from Leuconostoc species with epigallocatechin gallate. The incubation is carried out in a medium comprising 2-propanol and water.

[0021] Surprisingly, it has been found that by using 2-propanol as a cosolvent in the medium, the selectivity of the enzymatic reaction can be significantly improved, selectively providing monoglucosides with high regioselectivity. In particular, the observed regioselectivity is better than if diols or triols are used as cosolvents.

[0022] The method of the present invention can be carried out at a concentration suitable for industrial preparation (eg, about 15 to 30 mM) and provides epigallocatechin gallate 4'-O-α-monoglucoside at a yield of more than 30%. Preferred reaction conditions are further indicated below.

[0023] For comparison, other things being equal:

[0024] - If other short-chain alcohols were used as co-solvents, much lower conversions were observed (ethanol: 8%, 1-propanol: 0.71%, 1-butanol: 0.08%, and 2-butanol: 0.25%; whereas for 2-propanol, 48.5% conversion).

[0025] If only 1,3-propanediol was used as cosolvent, a conversion of 60% was achieved, 45% to monoglucoside (of which 58% to 4'-O-α-monoglucoside), 14% to diglucoside and 1% to triglucoside.

[0026] If only 2,3-butanediol was used as cosolvent, a conversion of 57% was achieved, 45% to monoglucoside (of which 66% to 4'-O-α-monoglucoside), 14% to diglucoside and 1% to triglucoside.

[0027] If only 1,3-butanediol was used as cosolvent, a conversion of 67% was achieved, 47% to monoglucoside (of which 65% to 4'-O-α-monoglucoside), 19% to diglucoside and 1% to triglucoside.

[0028] If only 2,4-butanediol was used as cosolvent, a conversion of 64% was achieved, 44% to monoglucoside (of which 62% to 4'-O-α-monoglucoside), 19% to diglucoside and 1% to triglucoside.

[0029] - In the case of DMSO as co-solvent, a conversion of 61% was achieved, 40% to monoglucoside (of which 63% to 4'-O-α-monoglucoside), 19% to diglucoside and 2% to triglucoside.

[0030] - If no co-solvent was used, no conversion was observed.

[0031] The present invention further relates to a method for preparing epigallocatechin 4'-O-alpha-monoglucoside, comprising incubating epigallocatechin with sucrose and dextransucrase from Leuconostoc species. The incubation is carried out in a medium comprising 2-propanol and water.

[0032] Furthermore, it has been found that the use of 2-propanol as a cosolvent in the medium improves the selectivity of the enzymatic reaction, in particular the regioselectivity of glycosylation. In addition, the enzyme efficiency is significantly better than with other short-chain alcohols.

[0033] The enzymatic reaction is carried out using sucrose, an abundant and relatively cheap substance used in the field of food and feed. The reaction consists in transferring the glucose moiety of the 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 transfers of the glucose moiety of the sucrose may occur, to another hydroxyl group of the catechol ring or to the hydroxyl group of the fixed glucose, providing diglucosides or even triglucosides.

[0034] The process of the invention allows the formation of di- and tri-glucosides to be significantly reduced or even completely avoided and allows mono-glucosides to be obtained with high regioselectivity.

[0035] In the method of the invention a dextransucrase from Leuconostoc sp. is used. Several suitable dextransucrases are mentioned in WO 2007 / 144368, which is incorporated herein by reference.

[0036] In a preferred embodiment, dextransucrase from Leuconostoc mesenteroides NRRL B-512F (ATCC 10830a) is used.

[0037] The method of the present invention is carried out in an aqueous medium. The aqueous medium is preferably buffered at a pH that is favorable for enzyme activity (well known to the skilled person).

[0038] The nature of the buffer used in the medium is not very important. However, preferably, it should be avoided to be able to chelate divalent cations (such as Ca 2+ or Mg 2+ ) buffer. Suitable buffers include, but are not limited to, acetate buffers (e.g., sodium acetate or potassium acetate) or sodium propionate buffers.

[0039] In one embodiment, sodium acetate or potassium acetate buffer is used, preferably in a concentration ranging from 5 to 100 mM, more preferably from 10 to 50 mM and even more preferably from 15 to 40 mM.

[0040] In one embodiment, the pH of the buffer is from 5.00 to 5.60, more preferably from 5.10 to 5.50, and even more preferably from 5.20 to 5.40.

[0041] In the method of the present invention, the incubation is carried out in a medium comprising 2-propanol and water.

[0042] In one embodiment, the medium comprises 5% to 20% (v / v) 2-propanol, more preferably 8% to 18% (v / v). These concentration ranges have been found to prevent enzyme denaturation under the reaction conditions, thereby providing the desired product in high yield even at relatively low enzyme concentrations.

[0043] Optionally, the medium may also contain lower diols and / or lower triols, in particular lower diols and / or lower triols selected from the group consisting of 1,3-propylene glycol, 1,2-propylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 2,4-butylene glycol, 1,2,3-propanetriol (glycerol) and mixtures thereof. Surprisingly, it has been found that the regioselectivity of the reaction is even better if a combination of 2-propanol and 1,3-propylene glycol is used.

[0044] The medium used in the process of the invention should not contain any co-solvents or other additives which inhibit enzyme reactivity and / or reduce selectivity.

[0045] In one embodiment, the medium does not contain any dimethyl sulfoxide.

[0046] In one embodiment, the medium does not contain any ethers of ethylene glycol (glymes).

[0047] Epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside were obtained by incubating the corresponding substrates with sucrose and dextran sucrase, respectively.

[0048] In one embodiment, 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 to obtain epigallocatechin gallate 4'-O-α-monoglucoside.

[0049] Alternatively, to obtain epigallocatechin 4'-O-α-monoglucoside, epigallocatechin is incubated at an initial concentration of 5 to 50 mM, more preferably 10 to 40 mM and most preferably 15 to 35 mM.

[0050] The substrate is incubated with sucrose and glucansucrase under conditions that allow the enzyme to be active and synthesize the maximum possible amount of the desired glucoside.

[0051] In particular, the concentrations and relative ratios of sucrose, dextran sucrase and substrate are selected so that the enzymatic reaction can proceed to provide the desired product with optimal yield and selectivity.

[0052] 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.

[0053] In one embodiment, the temperature of the reaction mixture is maintained at 27 to 33 °C, more preferably at 28 to 32 °C, and most preferably at 29 to 31 °C.

[0054] Optionally, the reaction mixture may also contain calcium cations, for example in the form of calcium chloride or in the form of any other water-soluble calcium salt, to improve the stability of the enzyme.

[0055] In one embodiment, the reaction mixture comprises calcium chloride in 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.

[0056] In one embodiment, epigallocatechin gallate and epigallocatechin are incubated at an initial concentration of 5 to 50 mM, more preferably 10 to 40 mM and most preferably 15 to 35 mM, respectively.

[0057] The method of the invention can be performed with any suitable dextransucrase, for example with a dextransucrase or an amylosucrase.

[0058] In one embodiment, the dextransucrase is a dextransucrase.

[0059] Dextransucrase has been found to be able to efficiently and selectively glycosylate epigallocatechin gallate and epigallocatechin.

[0060] The concentration of the dextransucrase (preferably dextransucrase) should be adjusted according to the concentration of the substrate and / or sucrose. In particular, using too little dextransucrase may result in reduced yield, while using too much dextransucrase may result in reduced regioselectivity.

[0061] In one embodiment, the concentration of the dextransucrase 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.

[0062] Benefiting from the reaction conditions of the present invention, the desired epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside are formed with very high selectivity, respectively.

[0063] In one embodiment, the monoglucoside is 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 relative to the sum of the diglucoside and the triglucoside. Ideally, only the monoglucoside is formed or substantially only the monoglucoside is formed. The selective formation of the monoglucoside is highly desirable because the product meets the Chinese cosmetic application standards.

[0064] In one embodiment, 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% by mole compared to other monoglucosides. It has been found that due to this high regioselectivity, bioaccumulation in the skin can be improved.

[0065] Preferably, the monoglucosides are formed with high selectivity and with high stereoselectivity.

[0066] In one embodiment, 4'-O-α-monoglucoside (i.e., epigallocatechin gallate 4'-O-α-monoglucoside or epigallocatechin 4'-O-α-monoglucoside, depending on the substrate) accounts for at least 60%, more preferably at least 65%, even more preferably at least 70%, and most preferably at least 75% of the total glucosides formed. In this context, the term "glucoside" includes all glucosides formed, i.e., any mono-, di- and tri-glucosides, and wherein the glycosylation occurs at any position.

[0067] The present invention also includes epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside, respectively, formed by the method of the present invention.

[0068] As explained above, these products are formed with significantly higher selectivity than previously observed. In addition, they have been found to exhibit improved skin permeability and bioaccumulation. The products of the present invention are also microbiome friendly.

[0069] In a further aspect, the present invention also provides a method for providing a radiant skin complexion and / or age spot removal, wherein epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside is topically applied to the skin.

[0070] The method is cosmetic, ie, non-therapeutic.

[0071] Surprisingly, it was found that epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside have much higher bioavailability in the skin than the corresponding aglycones.

[0072] For example, epigallocatechin gallate 4'-O-α-monoglucoside was found to accumulate in the skin primarily at a depth of about 65 μm, in an amount almost twice the amount of the aglycone (epigallocatechin gallate). A glucoside mixture comprising monoglucoside, diglucoside, and triglucoside was found to have a skin penetration depth of about 50 μm. Detailed results are provided in the Examples section below.

[0073] In several in vitro, in vitro, ex vivo and clinical studies, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside were additionally found to exhibit a variety of activities making them of interest in cosmetic applications, including antioxidant, anti-glycation and lightening activities.

[0074] For example, using an in vitro model, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was found to significantly reduce reactive oxygen species (ROS) by 66%, demonstrating strong antioxidant activity.

[0075] Furthermore, in vitro tests showed that epigallocatechin gallate 4'-O-α-monoglucoside of the present invention is a potent anti-glycation active substance, as demonstrated by an IC50 value of 0.118 mM, which is better than the positive reference (aminoguanidine) and other well-known molecules (such as vitamin C) used in the test.

[0076] Epigallocatechin gallate 4'-O-α-monoglucoside of the present invention also has excellent whitening activity: in an in vitro test, 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. It was found that epigallocatechin gallate 4'-O-α-monoglucoside provided a -34% reduction in melanin content, which is similar to the reduction provided by kojic acid.

[0077] Clinical evaluation of Asian female volunteers showed that epigallocatechin gallate 4'-O-α-monoglucoside of the present invention can improve skin uniformity and lead to a brighter skin tone and a reduction in pigment spots, resulting in a more uniform bright skin tone. In fact, a significant improvement in skin uniformity was observed - up to 3.8 times better than placebo after 56 days of twice daily application. In addition, a strong reduction in melanin content in the pigment spot area of ​​up to -21.7% was observed compared to placebo. Therefore, compared to vitamin C at 2% as a commonly used benchmark, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention induces similar effects, and even better effects.

[0078] Epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was also found to significantly improve skin firmness after 56 days, up to 21 times better than placebo. In addition, a significant increase in dermal density of up to 16.6% was observed.

[0079] In another clinical study on Caucasian volunteers with pigmented spots on their hands, it was demonstrated that epigallocatechin gallate 4'-O-α-monoglucoside of the present invention was able to significantly reduce skin heterogeneity, while improving skin tone and reducing skin pigmentation. In fact, a significant reduction in skin heterogeneity of up to -24% was observed, meaning that epigallocatechin gallate 4'-O-α-monoglucoside is more effective than vitamin C.

[0080] A detailed description of the biological assays is provided further in the Examples section below.

[0081] In conclusion, these results demonstrate that epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside of the present invention are very suitable for use in skin care products providing whitening effects, anti-age spot, skin tone and lightening activities.

[0082] Therefore, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside of the present invention provide many benefits, making them attractive for cosmetic applications such as anti-aging, facial care, and general skin care products.

[0083] In one embodiment, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside are formed separately by the process according to the present invention.

[0084] Preferably, 4'-O-α-monoglucoside is present in a high proportion compared to the other glucosides.

[0085] In one embodiment, the ratio of epigallocatechin gallate 4'-O-alpha-monoglucoside to other glucosides of epigallocatechin gallate 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.

[0086] In one embodiment, the ratio of epigallocatechin 4'-O-α-monoglucoside to other glucosides of epigallocatechin 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.

[0087] As mentioned above, monoglucosides exhibit particularly high bioavailability in the skin.

[0088] In one embodiment, epigallocatechin gallate 4'-O-α-monoglucoside and epigallocatechin 4'-O-α-monoglucoside separately form part of the cosmetic composition.

[0089] Cosmetic compositions typically comprise a cosmetically acceptable carrier and one or more cosmetic actives - in this example, epigallocatechin gallate 4'-O-α-monoglucoside and / or epigallocatechin 4'-O-α-monoglucoside.

[0090] Thus, in one embodiment, a cosmetic composition comprising epigallocatechin gallate 4'-O-alpha-monoglucoside and a cosmetically acceptable carrier is topically applied to the skin.

[0091] In one embodiment, a cosmetic composition comprising epigallocatechin 4'-O-alpha-monoglucoside and a cosmetically acceptable carrier is topically applied to the skin.

[0092] 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.

[0093] In one embodiment, the cosmetic composition comprises from about 0.0001% to about 0.03% of epigallocatechin gallate 4'-O-alpha-monoglucoside, more preferably from about 0.001% to about 0.02% and most preferably from about 0.005% to about 0.01%.

[0094] In one embodiment, the cosmetic composition comprises from about 0.000075% to about 0.0225% of epigallocatechin 4'-O-alpha-monoglucoside, more preferably from about 0.00075% to about 0.015% and most preferably from about 0.00375% to about 0.0075%.

[0095] The cosmetic compositions used according to the invention may comprise, besides epigallocatechin gallate 4′-O-α-monoglucoside and / or epigallocatechin 4′-O-α-monoglucoside, one or more further cosmetic active substances.

[0096] For example, the cosmetic composition may include skin lighteners, dark spot reducers, skin tone agents, moisturizers, humectants, UV protectants, anti-aging actives, and / or soothing agents.

[0097] In one embodiment, the cosmetic composition comprises one or more additional cosmetic actives selected from the group consisting of brighteners, dark spot reducers, skin tone conditioners, moisturizers, moisturizers, UV protection actives and / or anti-aging actives.

[0098] Examples of whitening agents that can be used in the composition of the present invention include niacinamide, 12-hydroxystearic acid, resorcinol, phenethyl 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, butylated hydroxyanisole, butylated hydroxytoluene, citrate, deoxyarbutin, 1,3-diphenylpropane derivatives, 2,5-dihydroxybenzoic acid and its derivatives, 2-(4-acetoxyphenyl)-1,3-dithiane, 2-(4-hydroxyphenyl)-1,3-dithiane, ellagic acid, glucopyranosyl-1-ascorbate, gluconic acid, glycolic acid, 4-hydroxy-5-methyl-3[2H]-furanone, 4-hydroxyanisole and its derivatives, 4-hydroxybenzoic acid derivatives, hydroxyoctanoic acid, inositol ascorbate, lactic acid, lemon extract, linoleic acid, magnesium ascorbyl phosphate, 5-octanoyl salicylic acid, salicylic acid, 3,4,5-trihydroxybenzyl derivatives, acetylglucosamine, pitera extract, symwhite, calcium pantothenate (Melano-block), seppiwhite, soybean extract (bowman birk inhibitor) and mixtures thereof. When 12-hydroxystearic acid is used in the composition, it acts as a skin lightening agent, not as a fatty acid.

[0099] Additionally or alternatively, the cosmetic composition of the present invention may also comprise plant extracts, such as cranberry, hibiscus, guarana, acerola, ginger, licorice, pomegrentate, lotus, rhodiola rosea and / or schisandra chinensis extracts.

[0100] The cosmetic composition may further comprise one or more additives.

[0101] In one embodiment, the cosmetic composition further comprises a fatty acid.

[0102] Fatty acids, when present in the composition with the soap, provide the so-called vanishing effect, ie, when the composition is applied to human skin, it disappears on the skin without leaving noticeable streaks of the composition.

[0103] Preferably, the fatty acid that may be present in the composition is selected from fatty acids having 10 to 30, more preferably 12 to 25, even more preferably 14 to 20, further more preferably 16 to 18 carbon atoms. 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.

[0104] In one embodiment, the cosmetic composition further comprises soap.

[0105] Soap, when present in the composition in combination with fatty acids, provides a vanishing effect.

[0106] Soap is preferably prepared by in situ neutralization of fatty acids present in the composition. Therefore, preferably the soap has a carbon chain length corresponding to the chain length of the fatty acids in the composition. Soap is formed from fatty acids by using an alkali metal hydroxide (e.g. sodium hydroxide or potassium hydroxide). Of the two, potassium hydroxide is more preferred. Therefore, soap is preferably a potassium soap (a potassium salt of a fatty acid).

[0107] Preferably, the composition comprises from about 0.1% to about 10%, more preferably from about 1% to about 8%, more preferably from about 2% to about 7%, even more preferably from about 3% to about 6% soap by weight.

[0108] In one embodiment, the cosmetic composition further comprises a surfactant, in particular a nonionic surfactant having an HLB value in the range of 9 to 20, preferably 10 to 19, more preferably 12 to 18, even more preferably 13 to 17 and still more preferably 15 to 17. HLB is calculated using the Griffin method, where HLB=20xMh / M, where Mh is the molecular weight of the hydrophilic part of the molecule, and M is the molecular weight of the entire molecule, giving a result in any range of 0 to 20. Typical values ​​for various surfactants are given below:

[0109] Value <10: fat soluble (insoluble in water)

[0110] Value >10: Water soluble

[0111] Values ​​from 4 to 8 indicate defoamers

[0112] A value of 7 to 11 indicates a W / O (water-in-oil) emulsion

[0113] A value of 12 to 16 indicates an oil-in-water emulsion

[0114] Values ​​from 11 to 14 indicate a wetting agent

[0115] 12 to 15 is the usual value for detergents

[0116] A value of 16 to 20 indicates a solubiliser or hydrotrope.

[0117] 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 is a nonionic surfactant having at least 9 oxyalkylene groups, preferably at least 9 oxyethylene groups.

[0118] Preferably, the composition comprises from about 0.5 wt % to about 5 wt %, more preferably from about 1 wt % to about 4 wt %, even more preferably from about 2 wt % to about 3 wt % of a nonionic surfactant having an HLB value in the range of 9 to 20.

[0119] 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 categories:

[0120] - Acrylates / R-methacrylate copolymers, such as acrylates / steareth-20 methacrylate copolymer (available as Aculyn TM 22) and acrylates / beheneth-25 methacrylate copolymer (available as Aculyn TM 28 commercially available);

[0121] - Acrylates / R-methacrylate crosspolymer, such as acrylates / steareth-20 methacrylate crosspolymer (available as Aculyn TM 88 commercially available);

[0122] -Acrylates copolymer (available as Aculyn TM 33 commercially available);

[0123] - Acrylates / R-alkyl acrylate crosspolymer, such as acrylates / C10-C30 alkyl acrylate crosspolymer (available as Pemulen TM TR-2 commercially available);

[0124] -Copolymer of ammonium acryloyldimethyltaurate and vinylpyrrolidone (can be used as AVC is commercially available);

[0125] -Sodium acryloyldimethyltaurate and vinylpyrrolidone copolymer (can be used as AVS commercially available); and

[0126] - Acryloyldimethyltaurate crosspolymer with R-alkyl acrylate and methacrylate, such as ammoniumacryloyldimethyltaurate / beheneth-25methacrylate crosspolymer (can be used as HMB and BLV commercially available).

[0127] 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 %, further more preferably from about 1.5 wt % to about 3.5 wt %, still more preferably from about 2 wt % to about 3 wt % of the polymer.

[0128] In one embodiment, the cosmetic composition further comprises an emollient.

[0129] Examples of emollients that can be used in the composition include 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, eicosanol, behenyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, stearyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecane-2-ol, isocetyl alcohol, eicosanol, behenyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, stearyl alcohol, Butyl myristate, polyethylene glycol, triethylene glycol, 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 seed oil, coconut oil, peanut oil, castor oil, acetylated lanolin alcohol, petrolatum, mineral oil, butyl myristate, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate and mixtures thereof.

[0130] In one embodiment, the cosmetic composition further comprises a solvent.

[0131] Examples of solvents that can be used in the composition include ethanol, isopropyl alcohol, acetone, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, and mixtures thereof.

[0132] In one embodiment, the cosmetic composition further comprises a powder.

[0133] Examples of powders that can be used in the composition include chalk, talc, Fuller's earth, kaolin, starches, gums, colloidal silica sodium polyacrylate, tetraalkyl and / or trialkylaryl ammonium montmorillonites, chemically modified magnesium aluminum silicates, organically modified montmorillonite clays, hydrated aluminum silicates, fumed silica, carboxyvinyl polymers, sodium carboxymethylcellulose, ethylene glycol monostearate, and mixtures thereof.

[0134] In one embodiment, the cosmetic composition further comprises a preservative to prevent the growth of potentially harmful microorganisms.

[0135] Examples of ingredients that can be used as preservatives in the composition include alkyl esters of p-hydroxybenzoic acid, hydantoin derivatives, propionates, and various quaternary ammonium compounds. More preferably, ingredients that can be used as preservatives in the composition are sodium benzoate, iodopropynyl butyl carbamate, methylisothiazolinone, iodopropynyl butyl carbamate, phenoxyethanol, methylparaben, propylparaben, imidazolidinyl urea, sodium dehydroacetate, ethylhexylglycerol, benzyl alcohol, alkanediols, and mixtures thereof. Alkanediols that are suitable for use as preservatives are C6-C12 alkanes substituted by ortho-positions of hydroxyl groups. Illustrative examples include 1,2-octanediol (octanediol), 2,3-octanediol, 1,2-nonanediol, 1,2-decanediol, 1,2-hexanediol, 3,4-octanediol, mixtures thereof, and the like, with octanediol generally being most preferred.

[0136] When present in the composition, the preservative is preferably added in an amount of about 0.001 wt % to about 5 wt %, more preferably about 0.01 wt % to about 3 wt %, and most preferably about 0.02 wt % to about 2 wt %.

[0137] The cosmetic composition may also contain a range of other optional ingredients including antioxidants, binders, biological additives, buffers, colorants, astringents, fragrances, opacifiers, conditioning agents, exfoliating agents, pH adjusters, natural extracts, skin sensates, skin soothing agents and skin healing agents.

[0138] The cosmetic composition is preferably formulated in the form of a composition comprising water, such as a lotion, cream, emulsion, gel or mousse. More preferably, the composition is formulated in the form of a cream or lotion and most preferably in the form of a cream.

[0139] The present invention is further illustrated by means of the following non-limiting examples:

[0140] Example 1: Synthesis of Epigallocatechin Gallate 4'-O-α-Monoglucoside (EGCG MONOG)

[0141] In a constant temperature and stirring reactor, 2.40kg (= 2.40l) of desalted water is introduced. The temperature is adjusted to 30 ± 1 ° C. Under gentle stirring, 1.215kg of sucrose (food grade) is introduced in batches (about 6 0.2kg parts, each part is added after the previous part is completely dissolved). Subsequently, 4.0g of calcium chloride is introduced. Under gentle stirring, 0.47kg (= 0.60l) of 2-propanol (minimum purity: 98%) is introduced. Under gentle stirring, 41.0g of epigallocatechin gallate (EGCG; minimum purity: 95%) is introduced. After complete dissolution, 0.162kg of 0.50M sodium acetate buffer at pH 5.41 is introduced into the reactor. At this time, the weight of the intermediate reaction mixture is 4.292kg, and its volume is 3.942l.

[0142] 0.840 kg (= 0.840 l) of desalted water was added, and the pH was adjusted to 5.30 with 1 ml of 6N H2SO4. The pH and temperature were nominal values ​​of 5.3 ± 0.1 and 30 ± 1 ° C, 0.203 kg of enzyme solution (35.0 U / g; 1 U is equivalent to the amount of enzyme that releases 1 μ mol of fructose from sucrose per minute; under the conditions of the assay: sucrose 100 g / l, pH 5.20 in the case of 20 mM sodium acetate buffer, calcium chloride 0.02 g / l). Overall, the 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 dextran sucrase concentration was 0.76 U / g or 0.81 U / ml.

[0143] The reaction was carried out over a period of 24 hours. The enzymatic reaction was stopped by adjusting the pH of the reaction mixture to a value of 2.0 using concentrated sulfuric acid. EGCG-related substances were separated from sugars, enzymes and salts by adsorption using a hydrophobic resin (Diaion HP20 from Mitsubishi Chemical Industries, Japan), and then eluted with an ethanol / water mixture (specific gravity at 20°C: 0.862). The ethanol / water mixture used to elute EGCG-related substances was removed by vacuum evaporation. EGCG-glucoside was separated from EGCG using liquid-liquid extraction with ethyl acetate (adjusting the concentration of the substance and the relative volume of ethyl acetate to maximize the purification yield). Such methods are known to technicians and are described elsewhere, particularly in WO2007 / 144368 (pages 46-47).

[0144] The final composition of the purified preparation was as follows:

[0145] -Monoglucosides: 41.7317mmol / kg (equivalent to 87.77% of glucosides)

[0146] -Diglucoside: 5.813mmol / kg (equivalent to 12.23% of glucoside)

[0147] -Triglucoside: Not observed

[0148] - Residual EGCG: 0.4466mmol / kg

[0149] Epigallocatechin gallate 4'-O-monoglucoside was the main component, accounting for 79.10% of the monoglucosides.

[0150] This purified preparation, containing approximately 69% epigallocatechin gallate 4'-O monoglucoside, was used in the following tests.

[0151] Example 2: Synthesis of Epigallocatechin 4'-O-α-monoglucoside

[0152] Epigallocatechin 4'-O-α-monoglucoside was prepared under the same reaction conditions as described in Example 1, using epigallocatechin instead of epigallocatechin gallate as the substrate.

[0153] Example 3: Statistical analysis

[0154] For the in vitro and ex vivo studies described in Examples 4 to 8, all results are presented as mean ± standard error of the mean (SEM) of three independent triplicate determinations. In vivo data for Examples 11 to 14 are generally presented as percent change compared to D0.

[0155] The Shapiro-Wilk test was used to verify whether the raw data followed the Gaussian law. In the case of normally distributed data, the means were compared using 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 was used for unpaired data followed by the Mann-Whitney U test.

[0156] For all in vivo studies, the Shapiro-Wilk test was used to verify whether the raw data followed the Gaussian law. In the case of normally distributed data, unpaired or paired Student's t-tests were used to compare the mean values. In the case of non-normally distributed data, for paired or unpaired data, Wilcoxon (paired) or Kruskal-Wallis tests were used, respectively, followed by Mann-Whitney U (unpaired) tests. For the analysis of the self-assessment questionnaire and daily diary results, chi-square tests (dichotomous analysis, which consists in comparing the number of relevant answers) were performed.

[0157] In all cases, the results were considered significant at p < 0.1 (indicated by #), p < 0.05 (indicated by *), p < 0.01 (indicated by **), and p < 0.001 (indicated by ***).

[0158] Example 4: Antioxidant activity (in vitro)

[0159] Cell culture and treatment

[0160] 96-well plates were coated with collagen I solution at 50 μg / ml. Normal human epidermal keratinocytes (NHEK) were seeded at 20'000 cells per well in quadruplicate. Cells were incubated in complete medium (Epilife, Gibco) supplemented with human keratinocyte growth supplement (HKGS) factor (Gibco) and 1% antibiotics (Sigma) at 37°C with 5% CO2 for 24 hours.

[0161] At the end of the incubation time, cells were treated with a purified preparation containing epigallocatechin gallate 4'-O-α-monoglucoside at 0.1% (v / v) from Example 1, or with resveratrol at 200 μM (used as a positive control for the assay), both in 2',7'-dichlorodihydrofluorescein diacetate, at 37°C with 5% CO2 for 24 hours. After 24 hours, cells were incubated with DCFH-DA probe at 50 μM (culture medium) at 37°C with 5% CO2 for 30 to 40 minutes and stressed with tert-butyl peroxide (TBP) at 5 mM. Changes in fluorescence were followed for 1 hour.

[0162] result

[0163] Resveratrol at 200 μM significantly reduced ROS production by 61% and validated the experiment.

[0164] Epigallocatechin gallate 4'-O-α-monoglucoside at 0.1% significantly reduced ROS generation by 66%, demonstrating antioxidant activity on keratinocytes.

[0165]

[0166] Example 5: Anti-glycation activity (in vitro)

[0167] The anti-glycation effect was evaluated using an in vitro assay measuring the spontaneous formation of advanced glycation end products (AGEs).

[0168] deal with

[0169] The assay was performed in a 96-well microplate. 40 μl of the purified preparation containing 0.1% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside from Example 1 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 run in triplicate.

[0170] A standard range of aminoguanidine was used as an inhibitor reference (positive control). A mixture of sodium phosphate buffer and BSA was used for validation experiments, and a mixture of sodium phosphate buffer, BSA and ribose was used as a positive control.

[0171] Two blanks were run on the extracts:

[0172] - with phosphate buffered saline, BSA and extracts

[0173] - with phosphate buffer, ribose and extract

[0174] The microplate was stirred and incubated at 37°C for 17 h. Excitation 340nm and Fluorescence readings were taken at emission 420 nm.

[0175] The anti-glycation activity of the extract was calculated as follows:

[0176]

[0177] Fl: fluorescence of the tested extract; blank extract (ribose or BSA); positive control; negative control.

[0178] Based on the above measurements and calculations, IC 50 , which is the concentration of extract that inhibits AGE formation by half.

[0179] result

[0180] The results are summarized in the following table:

[0181]

[0182] IC of positive control (aminoguanidine) 50 Determined to be 34.7 μg / ml (0.326 mM).

[0183] Example 6: Evaluation of Skin Permeability by Raman Spectroscopy (Ex Vitro)

[0184] Skin explant culture and processing

[0185] Skin explants from Caucasian volunteers were treated with epigallocatechin gallate (i.e., aglycone), a mixture of glucosides comprising epigallocatechin gallate monoglucoside, diglucoside and triglucoside, or a purified preparation containing 2% epigallocatechin gallate 4'-O-α-monoglucoside from Example 1 at 37°C with 5% CO2 for 8 hours. At the end of the incubation period, the skin explants were frozen at an optimal cutting temperature (OCT; VWR) of -80°C and then cut longitudinally using a cryotome with a thickness of 20 μm. For each explant, 3 tissue sections were selected and placed on a CaF2 support for Raman imaging analysis. The processing of the correction data map was performed using software based on the least squares fitting method running in a Matlab environment. The method involves mathematical modeling of the reference spectra in the entire spectral image to determine the contribution and distribution of these spectra within the image. Reference spectra were modeled spectra of the following products: aglycone, glucoside mixture, 4′-O-α-monoglucoside, and untreated skin control.

[0186] result

[0187] It was found that 8 hours after topical application, the skin permeability of epigallocatechin gallate 4'-O-α-monoglucoside was significantly higher than that of the aglycone alone (+90%) and that of the glucoside mixture (+20%).

[0188] Figure 1 Semiquantitative graphical representations of the results are shown.

[0189] Therefore, epigallocatechin gallate 4'-O-α-monoglucoside of the present invention has better skin bioavailability than the aglycone and glucoside mixture.

[0190] Example 7: Evaluation of whitening activity using Fontana-Masson staining for melanin quantification (in vitro)

[0191] Skin explant culture and processing

[0192] Fresh human skin explants were obtained from donors of skin phototypes III and VI at the age of 46 ± 25 years. Skin explants were topically treated with 0.4% (v / v) epigallocatechin gallate 4'-O-α-monoglucoside (purified preparation from Example 1) or with 2% (v / v) vitamin C (both in water) for 7 days and incubated at 37°C, 5% CO2. 2% kojic acid (Sigma-Aldrich) was applied topically and incubated under the same conditions, serving as a positive control. Untreated conditions were treated with vehicle (distilled water). The culture medium was renewed every other day. After the end of the culture, the skin explants were sampled and placed in formalin, then dehydrated and embedded in paraffin for Fontana-Masson staining.

[0193] Fontana-Masson staining and melanin quantification by image analysis

[0194] Skin explants fixed in formalin were dehydrated and embedded in paraffin. Sections of 4 μm thickness were dewaxed and then stained for melanin by the Fontana-Masson silver method.

[0195] Images were collected using an Axio Observer inverted fluorescence microscope (Zeiss) in bright-field mode.

[0196] Melanin was quantified using two open source optical imaging software programs. A micrograph (jpeg format) of a Fontana-Masson tissue section was opened in the GIMP-GNU image processing program. The brown-black signal corresponding to the melanin granules on the stained section was selected, copied and pasted into a new image, and saved as a jpeg file; the jpeg file consists of only black / brown (melanin granules) on a white background. The image was then opened using the ImageJ program. The image was then inverted and the average intensity was obtained.

[0197] result

[0198] Kojic acid at 2% and vitamin C at 2% significantly reduced melanin content by 34% and 28%, respectively, compared to untreated conditions and validated the experiments.

[0199] Epigallocatechin gallate 4'-O-α-monoglucoside at 0.4% significantly reduced melanin content by 34% compared to untreated conditions.

[0200] Example 8: Transcriptome Analysis

[0201] In order to evaluate the effect of the formulations of the invention on melanogenesis, co-culture studies with keratinocytes and melanocytes were performed.

[0202] Cell culture and treatment

[0203] Cell culture was achieved 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.

[0204] The experiments were conducted under the following conditions:

[0205] - Untreated NHEK-NHEM co-cultures

[0206] - NHEK-NHEM co-culture + a diluted version of the purified preparation from Example 1 containing epigallocatechin gallate 4'-O-alpha-monoglucoside at 0.01% (v / v) (abbreviated as "EGCG MONOG 0.01%").

[0207] The product EGCG MONOG 0.01% was filtered and diluted in Keratinocyte Growth Medium 2 (supplied by Promocell).

[0208] Cells were seeded in 6-well plates in the presence of Keratinocyte Growth Medium 2 for 24 h. After seeding, treatments in the presence of EGCG MONOG 0.01% were applied for 24 h. Untreated wells were used as a basal reference to compare the effects of EGCG MONOG and gene expression after 24 h of incubation.

[0209] After treatment, TRIzol TM (Can be obtained from Sigma) extract RNA from cells and reverse transcribe into cDNA.CDNA is mixed with different target primers (PrimePCR custom plates, pigmentation, Biorad) representing melanin generation pathway.The expression of mRNA is measured by semi-quantitative PCR on Bio-Rad CFX96 real-time PCR system (can be obtained from BioRAD).The expression level of mRNA is calculated and normalized with reference gene (B2M).

[0210] Values ​​are reported relative to untreated controls to indicate activation or inhibition effects.

[0211] result

[0212] After 24h treatment with EGCG MONOG 0.01%, RT-qPCR showed that EGCG MONOG 0.01% was able to significantly reduce the expression of genes involved in the regulation of melanogenesis (KIT-93%, EDNRB-68%, MC1R-96%, SOX10 -60%, MITF-108%). Compared with untreated co-cultures, EGCG MONOG 0.01% was also able to significantly reduce 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%).

[0213] Example 9: Consumption of EGCGMONOG by Bacillus sp.

[0214] Since the active substance according to the invention is glycosylated, it was assumed that it could be used as a substrate by bacteria. To test this hypothesis, Bacillus sp. was cultivated in the presence of 1% EGCG MONOG and its consumption was followed by HPLC during 48 hours.

[0215] Materials and methods

[0216] Bacillus was isolated from normal and healthy skin (GAB04-01) and used in this study. Bacterial strains were stored at -80°C in the presence of 20% w / v glycerol.

[0217] In preparation for the study, selected samples were thawed and aliquots (bacteria cryovials stored at -80°C) were spread on tryptic soy agar plates and then incubated at 30°C. A single colony was used to inoculate 10 ml of tryptic soy broth (TSB) in a stoppered plastic tube and incubated overnight at 37°C with stirring. The optical density at 600 nm was measured after a 10-fold dilution with TSB medium (Biophotometer D30). First, fresh medium ( An aliquot of the sample was diluted with DSM (0.5) to obtain an optical density of 1.216.

[0218] 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 culture medium was changed by adding 1% of the purified preparation containing epigallocatechin gallate 4'-O-α-monoglucoside at 0.1% (v / v) from Example 1 or water (negative control) to the culture medium (i.e., samples treated with 39.6 ml of DSM 0.5 and 0.4 ml of EGCG MONOG or 0.4 ml of water). The optical density of the reaction culture medium after inoculation was 0.030 ± 0.003.

[0219] The fate of EGCG MONOG in the presence of Bacillus was followed using HPLC-UV-MS with the following parameters:

[0220] -Elution curve: 1.00ml / min

[0221] - Column: X-Bridge C18

[0222] -Injection volume: 10μl

[0223] - Temperature: 40℃

[0224] -Spectrophotometer: 270nm

[0225] result

[0226] After only 24 hours, the amount of EGCG MONOG was reduced by 0.307 mM. At the same time, no EGCG aglycone or any other EGCG derivatives (such as EGCG diglycosides) were detected, proving that the reduction of EGCG MONOG was related to its consumption by bacteria rather than its degradation. The detailed results are shown in the table below:

[0227]

[0228] Example 10: Growth promotion of Lactobacillus acidophilus

[0229] In order to further investigate the effect of the active substances of the present invention on bacteria, the growth of Lactobacillus acidophilus in MRS medium supplemented with different concentrations of EGCG MONOG for 48 hours was evaluated.

[0230] Materials and methods

[0231] Lactobacillus acidophilus (DSM 20079) is cultivated under anaerobic conditions at 37 ° C on DeMan, RogosaSharpe (MRS) agar. After bacterial amplification in the culture medium, for each bacterial strain, a bacterial suspension is prepared using a determination medium (50% MRS broth in PBS), and the optical density (OD600 nm) at 600 nm is adjusted to 0.2. The bacterial suspension is then transferred to a 96-well plate, some of which contain 0.015% to 1% of the test compound. The bacterial strain cultivated with the milk powder at 0.01% serves as a positive control. The optical density of the bacterial suspension is measured using a wavelength of 600nm, and a value of 0.1 is displayed.

[0232] Bacterial cultures were incubated under agitation (-280 rpm) and anaerobic conditions at 37° C. Bacterial growth was analyzed by kinetically reading OD600 nm for 48 hours using a microplate spectrophotometer (EPOCH2, Bio Tek Instruments).

[0233] All experimental conditions were performed in triplicate.

[0234] result

[0235] 0.01% milk powder significantly increased bacterial growth and validated the experiment.

[0236] Compared to cultures without EGCG MONOG, the active substance of the invention at concentrations of 0.13%, 0.4% and 1% was able to significantly increase the growth of Lactobacillus acidophilus by 6%, 9% and 27%, respectively, with a dose-dependent effect.

[0237] Example 11: Analysis of metabolites released by Lactobacillus acidophilus

[0238] Since the active substances according to the invention are able to stimulate the growth of Lactobacillus acidophilus, it was decided to analyse the metabolites released by the bacteria in the culture medium by means of high-field NMR.

[0239] Materials and methods

[0240] The supernatants of the cultures previously described in Example 10 (both untreated strain cultures and cultures of Lactobacillus acidophilus treated with 1% EGCGMONOG) were stored and frozen at -80°C and then analyzed using high-field NMR. For each of the two conditions, three samples were analyzed to assess test reproducibility (n=3).

[0241] For each sample, 400 μl of the supernatant was taken and 100 μl of D2O / TSP (3-(trimethylsilyl)propionic acid-2,2,3,3-d4 sodium salt, internal reference) was added at a concentration of 1 g / l. The samples were analyzed by high field spectrometry on a Bruker ASCEND 500 spectrometer. 1 H NMR was analyzed, the spectrometer was run at a proton frequency of 500 MHz, using a 5 mm cryosonde probe. 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 using an internal standard (TSP-d4) added at a known concentration. Compounds were quantified by the relative ratio of the compound peak integrated intensity to the internal standard intensity.

[0242] result

[0243] It was found that the addition of 1% of the active substance of the invention altered the carbon source used by the strain for its metabolism. Untreated bacteria used mainly sugar as carbon source, while treatment with EGCG MONOG resulted in a 17% reduction in sugar consumption in favor of polysorbate consumption, which increased significantly by 51%.

[0244] Furthermore, after incubation with the active substance according to the invention, Lactobacillus acidophilus released significantly more trigonelline, up to 65%.

[0245] Example 12: Compositions for use in clinical studies

[0246] A clinical study was conducted using a face cream containing 0.4% of the purified preparation from Example 1 containing epigallocatechin gallate 4'-O-α-monoglucoside or 2% ascorbic acid (vitamin C) compared to a placebo cream to evaluate the efficacy on volunteers with dull skin, large age spots (3 mm or more) on the face and with shallow wrinkles. For this study, volunteers applied the cream twice daily for 56 days.

[0247] For the clinical studies described in Examples 14 to 17, the following compositions were used:

[0248] INCI formula

[0249] Placebo: AQUA / WATER, CETHYL ALCOHOL, Glyceryl Stearate, PEG-75 Stearate, CETETH-20, STEARETH-20, Isodecyl Neopentanoate, Phenoxyethanol, 1-2-Hexanediol, Caprylyl Glycol, Dimethicone, Fragrance

[0250] Active ingredients: water (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

[0251] Vitamin C: Water (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

[0252] Composition

[0253]

[0254] *The purified preparation from Example 1 contained 32.6933 mmol / kg = 20.2869 g / kg of epigallocatechin gallate 4'-O-α-monoglucoside, resulting in a final concentration in the test composition of 0.0811 g / l = 0.0081%.

[0255] Example 13: Panels used in clinical studies

[0256] The clinical studies described in Examples 14 to 16 were conducted as follows:

[0257] A single-center study was conducted on 38 Asian volunteers with dull skin and large age spots (3 mm or more) and shallow wrinkles on the face. The volunteers were divided into two groups as follows:

[0258] - Group 1: 20 Asian women, testing the active cream, compared with the placebo cream, mean age 54 ± 5 ​​years.

[0259] - Group 2: 18 Asian women, testing vitamin C cream compared with placebo cream, mean age 53 ± 8 years.

[0260] The studies were performed according to the standard operating procedures of the Bio EC and adhered to the regulations established in the “Guía parainvestigaciones con seres humanos” (Guidelines for Research on Human Beings) and the guidelines of the Scientific Committee on Consumer Safety (SCCS).

[0261] The volunteers applied a cream containing 0.4% of active substance, 2% of ascorbic acid (vitamin C) on one side of their face and a placebo cream on the other side every other day for 56 days.

[0262] During this study, Color For skin color and spot analysis, use Testing biomechanical properties and using DUB SkinScanner The probe (maximum depth of 3.28 mm) determines the dermal thickness.

[0263] Example 14: By Measurement of skin biomechanical properties

[0264] method

[0265] Analysis of the mechanical properties of the skin allows assessment of the functional status of the following tissue structures:

[0266] - Elastic structures, such as elastic fibers, curvature of connective bundles, wrinkles of the stratum corneum; and

[0267] -Structures that exhibit viscosity, such as interstitial fluid, internal adhesions.

[0268] Research Use MPA 580 (Courage & Khazaka). The measuring principle is based on the suction method: negative pressure is created in the device and the skin is sucked into the cylindrical hole (2 mm in diameter) of the probe. Inside the probe, the penetration depth is determined by an optical measuring system. Each suction phase is followed by a relaxation phase.

[0269] In this study, the following procedures were used:

[0270] - Cycle length: 4 seconds

[0271] - Suction: 2 seconds

[0272] -Relax: 2 seconds

[0273] - Negative pressure: 450 mbar

[0274] - Diameter of chamber: 2mm

[0275] -Measurement area: crow's feet

[0276] At the end of each measurement, the skin's resistance to being sucked up by the negative pressure and its ability to return to its original position are displayed as curves. From these curves, parameters can be calculated.

[0277] - During the suction phase, the deformation of the skin due to the negative pressure measures first the elastic resistance and then the viscous component, which together represent the skin firmness.

[0278] - During the relaxation phase, the immediate recovery of the skin measures the pure skin elasticity, whereas the delayed recovery of the skin to its original position measures the viscoelastic component.

[0279] Figure 2 An example of a curve measured on elastic skin is shown. The following parameters are indicated:

[0280]

[0281] The study focused on the R0 or Uf parameter, which represents the amplitude of the skin during the suction phase. At the same pressure, the more pliable the skin, the greater the amplitude. Therefore, R0 (or UF) will evaluate the viscoelastic dispensability of the skin, or in other words, the firmness of the skin.

[0282] These parameters were measured at D0, D28, and D56.

[0283] result

[0284] After 28 and 56 days of application, a reduction in the R0 parameter was observed, indicating an improvement in firmness in the case of epigallocatechin gallate 4'-O-α-monoglucoside compared to D0 (-8.7% at D28 and -8.2% at D56). The results for epigallocatechin gallate 4'-O-α-monoglucoside and ascorbic acid are shown in the following two tables, respectively:

[0285]

[0286] These measurements demonstrated that skin firmness was significantly improved up to 21-fold following application of a cream containing epigallocatechin gallate 4'-O-alpha-monoglucoside relative to a placebo cream.

[0287] As in Figure 3 As can be seen, the effect of epigallocatechin gallate 4'-O-α-monoglucoside after D28 and D56 days was also significantly better than ascorbic acid.

[0288] Example 15: Use Skin color analysis

[0289] method

[0290] Skin color measurement is based on The photos taken by the acquisition system are carried out. It is a 2D acquisition system for taking standardized and multimodal photos of the entire face. The device is equipped with a 24M pixel sensor and also has a specific UV lamp. The acquisition methods are: UV, filter-free photos, cross-polarized photos, parallel polarized photos, standard 45° photos and standard 60° photos.

[0291] Skin tone radiance, pigmentation spots, color (L*, a*, b* parameters) and melanin on spots from the photographs were analyzed using image analysis software.

[0292] The 2D photographs of the face were taken at D0, D28, and D56.

[0293] Uniformity results

[0294] After 28 and 56 days of application of the active cream, a significant increase in the mean pigment uniformity was observed in the case of the active substance by +1.2% and +1.5%, respectively, compared to D0.

[0295] In the case of placebo, a significant increase of 0.4% relative to D0 was shown after 28 and 56 days.

[0296] Thus, relative to placebo, the effect on pigment uniformity was 3.0 times more significant after 28 days and 3.8 times more significant after 56 days for the product containing epigallocatechin gallate 4'-O-alpha-monoglucoside.

[0297] As can be seen from the table below, the results for the active substance were significant compared to D0 and placebo:

[0298]

[0299] For comparison, the results in the case of ascorbic acid are shown in the following table:

[0300]

[0301]

[0302] Thus, vitamin C was only 1.6 times better than placebo at day 28 and 1.8 times better at day 56.

[0303] Glossy results

[0304] As can be seen from the results in the table below, a significant increase in skin radiance was observed after 28 and 56 days of application of an active cream containing 0.4% epigallocatechin gallate 4'-O-α-monoglucoside:

[0305]

[0306] Compared to placebo, significant increases of 4.5-fold and 2.6-fold were observed after 28 and 56 days, respectively.

[0307] For comparison, the results in the case of ascorbic acid are shown in the following table:

[0308]

[0309]

[0310] Pigmentation defect reduction results

[0311] As can be seen from the results in the table below, a clear reduction in pigmentary imperfections was observed after 28 and 56 days of application of the active cream containing 0.4% of epigallocatechin gallate 4'-O-α-monoglucoside:

[0312]

[0313] Thus, treatment with the active substance resulted in a significant reduction of pigmentary defects by -16.1% and -21.7% after 28 and 56 days, respectively, compared to the placebo.

[0314] For comparison, the results in the case of ascorbic acid are shown in the following table:

[0315]

[0316] Example 16: Use Ultrasound measurement of skin scanners

[0317] method

[0318] The DUB Skin Scanner System is a high-frequency, high-resolution ultrasound diagnostic tool for use in dermatology, cosmetics and pharmaceuticals, as well as clinical research.

[0319] 75MHz ultrasound is a commonly used method for non-invasive skin analysis. High-frequency ultrasound (75MHz) can explore the superficial layers of the skin and can penetrate up to 3mm into the skin. Skin ultrasound is based on the direction and measurement of the amplitude of the ultrasound wave group in the skin and on the part reflected by the interface separating two media with different acoustic properties. Ultrasound imaging techniques used to visualize the structure of the subcutaneous body allow the evaluation of the uniformity of the skin, including density and skin thickness.

[0320] Thickness and density were measured three times to obtain the average value for each image.

[0321] For this study, analysis of dermal density was performed at D0, D28, and D56.

[0322] result

[0323] As can be seen from the results shown in the table below, a clear increase in dermal density was observed after 28 and 56 days of application of the active cream containing 0.4% of epigallocatechin gallate 4'-O-α-monoglucoside:

[0324]

[0325] Application of a cream containing 2% ascorbic acid also resulted in a significant increase in dermal density after 28 and 56 days:

[0326]

[0327] Example 17: Age spot removal study using Raman spectroscopy

[0328] Group Description

[0329] A double-blind and placebo-controlled clinical evaluation was conducted on 45 Caucasian female volunteers aged 45 to 75 years. The volunteers presented with brown spots on their hands. All subjects participating in the study gave their informed consent at the beginning of the study.

[0330] Volunteers applied a product containing 0.4% of the purified preparation of Example 1 (containing epigallocatechin gallate 4'-O-α-monoglucoside) or 2% ascorbic acid or a placebo to their hands twice a day (morning and evening) for 56 days, and the anti-freckle efficacy was analyzed by Raman spectroscopy at D0 and D56.

[0331] Composition

[0332] The composition described in Example 12 was used.

[0333] Raman spectroscopy

[0334] The device includes a confocal Raman probe, which is coupled to a dispersive Raman spectrometer. The excitation laser beam is sent to the remote probe via a 5μm core diameter optical fiber, and the Raman signal is transmitted to the spectrometer via a 100μm core diameter optical fiber. The probe is equipped with a 100X long working distance objective lens operating in air.

[0335] The piezoelectric device allows the collection of Z Raman profiles by ensuring axial measurements from the surface down to a defined depth in the skin. A color video camera integrated in the probe allows visualization of the skin surface. The camera can also be used to control the focus of the laser on the skin.

[0336] The spectrometer is equipped with a 1024 x 256 element CCD (coupled charge detector) cooled by the Peltier effect, and a grating with 830 grooves / mm, which allows to cover 550 to 3700 cm in a single acquisition. -1 Large spectral range, spectral resolution is about 6cm -1 The excitation source was a 660 nm laser diode, and the power at the sample was set to 30 mW, in compliance with radiation protection standards. To protect against laser radiation, protective glasses were used.

[0337] The 660 nm excitation was chosen as the best compromise between parasitic fluorescence generation and the sensitivity of the CCD camera in the whole spectral range. For skin characterization, measuring high wavenumber vibrations is important to obtain information on water content. For data acquisition, the device was controlled by Labspec 5 software.

[0338] The Z-profile consists of a depth scan through the skin. Raman spectra were collected at different focal points, from the skin surface Z = 0 μm to 30 μm (below the skin surface), with a step size of 3 μm.

[0339] The measurement depths are indicated in the table below, with the corresponding acquisition time, resulting in a time of 1 min for a complete profile.

[0340] Depth(μm) Collection time(s) Depth(μm) Collection time(s) -10 4 12 4 -6 4 15 4 -3 4 18 4 0 4 21 4 3 4 25 4 6 4 30 4 9 4 0 4

[0341] A total of 519 Raman profiles were recorded: 2 profiles per condition and for each region (unstained and brown spot regions). Each profile contained 14 spectra, resulting in 7266 spectra recorded.

[0342] These measurements are used to evaluate a spectral signature of skin background fluorescence. This signature is related to the degree of skin lightening: a decrease in this signature indicates a positive skin lightening effect, while a stable or increasing signature means that the product has no skin lightening effect.

[0343] Data analysis

[0344] Matlab 7.2 (The MathWorks Inc., USA) was used for preprocessing of the spectral data. Abnormal profiles (poor S / N (signal-to-noise) ratio, fluorescence, incomplete profiles with large zero offsets, saturation, etc.) were excluded from the database. Each non-abnormal profile was submitted to background correction, which allowed the Raman signal of the skin to be cleaned up. These background corrections included smoothing the spectrum with a 9-mm Savitzky-Golay filter, peak correction, baseline correction, and normalization of the intensity of the entire wavenumber range with a vector function.

[0345] In order to preserve skin background fluorescence, no baseline correction was performed, which allowed the extraction of skin fluorescence markers that correlated with skin lightness.

[0346] After these preprocessing steps, the corrected data are used to determine the spectrum corresponding to the stratum corneum surface.

[0347] result

[0348] In this clinical study, spectral signatures of skin background fluorescence were measured, which correlated with the degree of skin lightening after application of a product containing 0.4% epigallocatechin gallate 4'-O-α-monoglucoside (purified preparation from Example 1) or 2% ascorbic acid or placebo to the hands. Skin properties were evaluated using Raman spectroscopy after 28 and 56 days of twice daily application.

[0349] The results are shown in the following two tables:

[0350]

[0351] As can be seen above, both epigallocatechin gallate 4'-O-α-monoglucoside and placebo resulted in similar decreases in melanin values ​​after 28 days (-10.2 and -14.3, respectively). However, after 56 days, the decrease was more pronounced in the case of epigallocatechin gallate 4'-O-α-monoglucoside (-20.4), while for placebo, melanin values ​​remained roughly the same (-11.03).

[0352] On the other hand, ascorbic acid even caused a slight increase in melanin detection values ​​after 28 days (+2.25 compared to D0), which was reversed after 56 days (-17.52).

[0353] Thus, it was demonstrated that epigallocatechin gallate 4'-O-α-monoglucoside resulted in a significant reduction in the background fluorescence level (i.e., melanin detection value) between the non-spotted control skin and the spotted skin compared to the placebo at D56. The significant reduction in the color difference between the non-spotted control skin and the spotted skin showed a reduction in pigmented spots. In addition, it showed a reduction in the color difference between age spots and the surrounding skin, resulting in improved skin uniformity.

[0354] Finally, a significant difference between ascorbic acid and epigallocatechin gallate 4'-O-α-monoglucoside was also observed in favor of epigallocatechin gallate 4'-O-α-monoglucoside.

[0355] Example 18: Clinical Evaluation on African Skin

[0356] Group Description

[0357] A clinical study was conducted on 85 female volunteers aged 19 to 47 years, who were divided into three groups: active substance, placebo and aglycone. The study was conducted in South Africa on female volunteers with dark skin type (phototype IV or above) 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 applied a cream containing 0.4% of the purified preparation from Example 1 (containing 0.1% (v / v) of epigallocatechin gallate 4'-O-α-monoglucoside) ("active substance") or 0.01% of EGCG aglycone or no active substance (placebo) twice a day, and the product was applied to the entire face for 56 days. After 14, 28 and 56 days, the L* parameter measurement method was used to measure the skin's skin color. Measuring skin color changes.

[0358] INCI formula

[0359]

[0360] *The purified preparation from Example 1 contained 32.6933 mmol / kg = 20.2869 g / kg of epigallocatechin gallate 4'-O-α-monoglucoside, resulting in a final concentration in the test composition of 0.0811 g / l = 0.0081%.

[0361] use Skin color evaluation with L* parameters

[0362] Skin color was evaluated by means of a Chromameter CR400 measuring skin color, and the parameter L* (lightness) was recorded. The greater 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 sub-position for each time interval to serve as a specific pigmentation motion color measurement component in the study. Skin color was analyzed at D0, D14, D28 and D56.

[0363] Statistical analysis

[0364] For all in vivo studies, the Shapiro-Wilk test was used to verify that the raw data followed the Gaussian law. In the case of normally distributed data, the means were compared using unpaired or paired t-Student tests. In the case of non-normally distributed data, the Wilcoxon (paired) and Kruskal-Wallis tests followed by the Mann-Whitney U (unpaired) test were used for paired and unpaired data, respectively.

[0365] Results were considered significant for p<0.1 (indicated by #), p<0.05 (indicated by *), p<0.01 (indicated by **), and p<0.001 (indicated by ***).

[0366] Results: Evaluation of skin color in areas of uneven skin tone

[0367] First, focus on uneven skin areas to compare the performance of each product.

[0368] It was found that 0.4% of the active substance according to the invention showed a significant increase in L* after 14 days, with a time-dependent effect showing +1.3%, +4.7% and +6.3% after 14, 28 and 56 days respectively. These increases are the result of a progressive whitening activity.

[0369] On the other hand, placebo and aglycone showed opposite effects, with a slight decrease in L*, showing no whitening activity.

[0370] Results: Evaluation of overall skin color using 15 measurements on half the face

[0371] Then, use 15 The measurement results analyze the whitening performance on half face.

[0372] The data show that in the presence of the active substance according to the invention, L* increased significantly up to +6.8%, with a significant effect after 14, 28 and 56 days.

[0373] Interestingly, the aglycone and placebo did not show similar performance and had little to no whitening effect at the time points tested.

[0374] During clinical evaluations it was shown that the better bioaccumulation of the active substances according to the invention observed during skin permeation analysis was closely correlated with better clinical whitening performance.

Claims

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

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

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

4. The process according to any one of claims 1 to 3, wherein the medium does not contain any dimethyl sulfoxide or any ether of ethylene glycol (glyme).

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 a dextransucrase.

7. The method according to any one of claims 1 to 6, wherein the concentration of the 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. The process according to any one of claims 1 to 7, wherein the monoglucoside is 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 relative to the sum of di- and tri-glucosides.

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

10. The method according to any one of claims 1 to 9, wherein the 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 the total glucosides formed.

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

12. A method for providing a radiant skin complexion and / or age spot removal, wherein: Epigallocatechin gallate 4'-O-alpha-monoglucoside is topically applied to the skin.

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

14. The method according to claim 12 or 13, wherein the ratio of epigallocatechin gallate 4'-O-α-monoglucoside to other glucosides of epigallocatechin gallate 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. A method according to any one of claims 12 to 14, wherein A cosmetic composition comprising epigallocatechin gallate 4'-O-alpha-monoglucoside and a cosmetically acceptable carrier is topically applied to the skin.

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

17. The method according to claim 15 or 16, wherein the cosmetic composition comprises one or more additional cosmetic active substances selected from the group consisting of: lighteners, dark spot reducers, skin tone conditioners, moisturizers, moisturizers, UV protection active substances and / or anti-aging active substances.

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