Methods for obtaining aqueous extracts of tea, compositions containing such extracts, and their cosmetic uses.

Abstract

This invention relates to a method for obtaining aqueous extracts of tea leaves (Camellia sinensis), particularly fresh tea leaves, Pu-erh tea, matcha, or green tea. The invention also relates to tea extracts rich in small RNAs, sugars, phenolic compounds, organic acids, catechins, and theanine, as well as cosmetic compositions comprising such extracts and their cosmetic uses for the care of the skin, scalp, and appendages, particularly for protecting the skin from external aggressors and oxidation, combating signs of skin aging, enhancing photoprotection, brightening the skin, and improving skin hydration.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and in particular to natural-derived active ingredients used in the preparation of cosmetic formulations to improve the appearance of or protect the skin.

[0002] This invention relates to methods for obtaining aqueous extracts of different types of tea, particularly fresh tea, Pu-erh tea, matcha, or green tea, as well as tea extracts rich in small RNA, sugars, phenolic compounds, organic acids, theine, and theanine; cosmetic compositions containing such extracts; and their cosmetic uses for the care of the skin, scalp, and appendages, and more particularly for protecting the skin from external aggressors and oxidation, combating signs of skin aging, enhancing photoprotection, brightening the skin, and improving skin hydration. Background Technology

[0003] The genus *Camellia* comprises a large number of different species belonging to the family Theaceae. Botanists estimate that there are between 100 and 250 species in this genus. *Camellia* is native to eastern and southern Asia, from the Himalayas to Japan and Indonesia.

[0004] The most well-known and widely used species is *Camellia sinensis*, commonly referred to as the tea plant. It comprises three botanical varieties: *Camellia sinensis* var. *assamica*, *Camellia sinensis* var. *sinensis* (Yunnan), and *Camellia sinensis* var. *Cambodiensis*. Another species, *Camellia assamica*, is also known as the tea plant, but its more refined cultivation limits its use.

[0005] Camellia sinensis species are used in the food industry for their leaves in the production of various teas. Processing the leaves of Camellia sinensis, depending on the specific growing conditions, imparts unique characteristics to the tea. Teas are distinguished, in particular, by their color and sensory properties.

[0006] Once harvested, the leaves undergo processing specific to the tea type, which determines the final degree of oxidation of the leaves. The different key steps are withering (softening the tea leaves to facilitate drying), rolling (to extract essential oils), fixing (using heat to stop the oxidation of the leaves), drying (using hot air circulation to reduce humidity levels), and fermentation (placing the leaves in an oxygen-deficient environment to accelerate the growth of microorganisms).

[0007] In this application, green tea is defined as tea (Camelliasinensis) whose natural oxidation is rapidly stopped after the leaves are picked. To obtain green tea, the tea leaves are withered, rolled to extract the juice, and then heated to prevent the oxidation process. Two possible heating methods exist: the Chinese method, which uses a copper basin placed over a fire; or the Japanese method, which involves spraying the leaves with steam (thus preserving their properties). Preventing oxidation maximizes the content of molecules of interest such as catechins and theanine.

[0008] In this application, matcha is defined as green tea from Japan, obtained by turning tea leaves (Camelliasinensis) into powder (matcha refers to "tea powder"). To obtain matcha, producers cultivate the leaves in shady locations to slow down photosynthesis, thus slowing the conversion of theanine into tannins. This technique gives matcha its unique fruity and vegetable flavor. Green tea is obtained by steaming the leaves to stop oxidation. Once dried, the leaves are crushed using a stone mill to obtain a very fine, bright green powder. Matcha is very rich in polyphenols, a class of antioxidants with recognized health benefits.

[0009] In this application, Pu-erh tea is defined as a post-fermented green tea, also known as raw Pu-erh in Asia or original Pu-erh in the West. Pu-erh tea originated in Yunnan, China. It is made from the leaves of the Camellia sinensis var. assamica. Before being pressed into tea cakes or bricks, the tea leaves undergo initial oxidation at the place of origin. Then, through microbial means, the tea cake undergoes moderate fermentation, which can be accelerated in a humid environment. Subsequently, the tea cake is partially oxidized during the natural aging process.

[0010] Many tea extracts described in the literature are rich in phenolic compounds, typically catechin polyphenols. On the other hand, extracts commercially available or described in the prior art rarely claim to be rich in compositions of different types of plant molecules other than polyphenols. In fact, most tea extraction methods described in the prior art use organic solvents, primarily due to their ability to extract polyphenols, mainly catechin polyphenols (WO2006 / 111666, KR2016021734A, and KR2018125828A). The described extracts are rich in polyphenols and flavonoids but do not contain phenolic acid molecules because they are not extracted using this type of technique. In fact, phenolic acids are primarily extracted in polar solvents, preferably water. Furthermore, other plant compounds known for their beneficial effects, such as amino acids, organic acids, proteins, sugars, and oligonucleotides like small RNAs, are not optimally extracted using this method.

[0011] Cosmetic users now desire formulations that are as natural as possible and as effective as, or even more effective than, synthetic products. In this invention, the prepared extracts are 100% natural to meet consumer demand.

[0012] Furthermore, despite the abundance of anti-aging cosmetics already on the market, the demand for new, effective cosmetic ingredients from natural sources continues to grow.

[0013] One problem this invention aims to solve is to provide a novel aqueous tea extract that meets current cosmetic market requirements in terms of naturalness standards, while still possessing significant biological benefits and being non-toxic.

[0014] In fact, excessively high concentrations of phenolic compounds, such as catechins, can lead to phototoxicity and limit the stability of the extract over time. The extracts described in this invention have concentrations of polyphenols and other plant molecules of interest consistent with their good efficacy. They also exhibit significant stability over time and are non-toxic.

[0015] Another problem this invention aims to solve is to provide a novel aqueous tea extract rich in compounds known to be effective on the skin, such as sugars, phenolic compounds, organic acids, catechins and theanine, as well as small RNAs, obtained by the method described above.

[0016] The inventors have developed a novel tea extract that is particularly rich in small RNAs, sugars, phenols and organic compounds, organic acids, catechins, and theanine by means of a method that avoids the disadvantages of prior art methods, such as the use of detergents and solvents with potential toxicity in cosmetics.

[0017] The resulting extracts can be used in cosmetics for the care of the skin, scalp, and appendages, to obtain a variety of benefits, particularly for protecting the skin from external aggressors and oxidation, for combating signs of skin aging, for enhancing photoprotection, for brightening the skin, for improving skin hydration, for enhancing barrier function, or even for soothing the skin. Summary of the Invention

[0018] First, the present invention relates to a method for obtaining an aqueous extract of tea leaves from the Chinese species of Camellia (Camellia sinensis), wherein the tea leaves are selected from fresh tea, Pu-erh tea, matcha, or green tea, and the method comprises the following steps:

[0019] a) Contact the tea leaves or tea powder with water, and then optionally crush them;

[0020] b) Add phytic acid;

[0021] c) Adjust the pH to a value between 10 and 11;

[0022] d) Keep the mixture under stirring for at least 1 hour at a temperature between 40 and 80°C;

[0023] e) The mixture obtained in c) will be purified to remove residual solid plant material and the filtrate will be collected;

[0024] f) Adjust the pH to a value between 6 and 8;

[0025] g) Treat the mixture with powdered activated carbon;

[0026] h) Filter to remove the activated carbon and collect the filtrate;

[0027] i) Treat the filtrate with polyvinylpyrrolidone (PVPP);

[0028] j) Filter to remove the polyvinylpyrrolidone and collect the filtrate;

[0029] k) Check the pH of the filtrate and, if necessary, readjust the pH to a value between 6 and 6.5.

[0030] Secondly, the present invention relates to an aqueous crude extract of fresh tea leaves, Pu-erh tea, matcha, or green tea, said aqueous crude extract being rich in small RNA of up to 150 nucleotides in length, rich in sugars, rich in phenolic compounds, and rich in organic acids, and free of DNA, said aqueous crude extract being obtainable by the method of the present invention, said aqueous crude extract having a dry weight of 5 to 30 g / kg and containing 0.5 to 10 g / kg of sugars; 0.050 to 2 g / kg of organic acids; 0.050 to 5 g / kg of phenolic compounds, said phenolic compounds including 0 to 200 mg / kg of catechins, 0.020 to 2 g / kg of catechins, and 0.020 to 2 g / kg of theanine; and 50 to 350 mg / kg of low molecular weight RNA of up to 150 nucleotides in length.

[0031] The present invention also relates to diluted aqueous extracts of fresh tea leaves, Pu-erh tea, matcha, or green tea, said diluted aqueous extract being rich in small RNA of up to 150 nucleotides in length, rich in sugars, rich in phenolic compounds, and rich in organic acids, and free of DNA, said diluted aqueous extract being obtainable by the method of the present invention, said diluted aqueous extract having a dry weight between 4 and 20 g / kg, said diluted aqueous extract comprising, by total weight of said extract, the following concentrations: 0.2 to 5 g / kg of sugars; 0.030 to 1 g / kg of organic acids; 0.030 to 3 g / kg of phenolic compounds, said phenolic compounds comprising 0 to 120 mg / kg of catechins, 0.010 to 1 g / kg of catechins, and 0.010 to 1 g / kg of theanine; and 10 to 250 mg / kg of low molecular weight RNA of up to 150 nucleotides in length.

[0032] Third, the present invention relates to a composition comprising an effective amount of at least one diluted aqueous extract of tea as an active ingredient and a physiologically acceptable medium, said aqueous extract of tea being selected from extracts of fresh tea, Pu-erh tea, matcha or green tea, or any mixture thereof, according to the present invention.

[0033] Fourth, the present invention relates to the cosmetic use of the compositions according to the invention for the care of skin, scalp and appendages, more specifically for protecting skin from external damage and oxidation, for combating signs of skin aging, for enhancing photoprotection, for brightening skin, and for improving skin hydration. Attached Figure Description

[0034] The invention and its advantages will be better understood by reading the following description of the accompanying drawings and non-limiting embodiments, wherein:

[0035] Figure 1The evaluation of the total sugar content in different tea extracts obtained according to Examples 1 and 2 is shown by colorimetry.

[0036] Figure 2 The evaluation of polyphenol content in different tea extracts obtained according to Examples 1 and 2 is shown by colorimetry.

[0037] Figure 3 The evaluation of the organic acid content in different tea extracts obtained according to Examples 1 and 2 is shown by HPLC-MS.

[0038] Figure 4 The evaluation of the theanine, catechin, and catechin content in different tea extracts obtained according to Examples 1 and 2 is shown by HPLC-UV.

[0039] Figure 5 The evaluation of hyaluronidase inhibition of different tea extracts obtained according to Examples 1 and 2 is shown by measuring turbidity.

[0040] Figure 6 The assessment of reactive oxygen species levels in skin biopsies treated with monosodium glutamate stress and different Pu-erh tea extracts obtained according to Examples 1 and 2 is presented. Detailed Implementation

[0041] limited

[0042] Unless otherwise stated, all terms used herein have their most widely known meanings. For the purposes of this invention, the following terms are defined as follows:

[0043] The term "tea extract" refers to all types of treatments applied to the leaves of the Chinese species of Camellia.

[0044] The term "tea" refers to the leaves of all forms of the Chinese species of Camellia (Camellia sinensis); whole or powdery, dried or fresh.

[0045] The terms "small RNA" or "low molecular weight RNA," or "small RNA of up to 150 nucleotides in length," refer to low molecular weight, non-coding RNA (ribonucleic acid) of up to 150 nucleotides in length, such as all types of single-stranded and / or double-stranded small non-messenger RNA, such as microRNA, interfering RNA, introns, small nuclear RNA, or even any fragment of RNA extracted from plants using the methods described in this invention. In the extracts according to the invention, small RNA is in the form of a complex mixture of many RNAs whose size is primarily between 25 and 150 nucleotides. Therefore, synthetically produced and thus non-natural RNAs are not included in this definition.

[0046] The term "organic acid" refers to α-hydroxy acids (or AHAs), which are carboxylic acids derived from sugars found in tea, such as lactic acid, malic acid, citric acid, tartaric acid, succinic acid, and uronic acid.

[0047] "Phenolic compounds" refer to plant-derived molecules having an aromatic ring containing at least one phenolic group with one or more hydroxyl groups, such as phenolic acids, flavonoids like catechins or their derivatives, tannins, or any other polyphenols. Phenolic compounds are known to be potent antioxidant molecules both in plants and for cosmetic applications. These secondary plant metabolites are also produced in plant defense mechanisms against biotic or abiotic stresses.

[0048] The term "carbohydrates" refers to all types of sugars found in plants, such as monosaccharides like glucose and fructose, as well as oligosaccharides and polysaccharides. Typically, polysaccharides contain more than 10 monosaccharide units, while oligosaccharides contain 3 to 10 monosaccharide units.

[0049] The term "plant molecules of interest" refers to all molecules present in the tea extract of the present invention, particularly small RNAs of up to 150 nucleotides in length, sugars, phenolic compounds, organic acids, catechins, and theanine.

[0050] When describing a range of values, the boundaries of that range should be understood to explicitly include all intermediate values ​​within that range. For example, a range of values ​​between 1% and 10% should be understood to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all decimal values ​​between 1% and 10%.

[0051] Unless otherwise specified, numerical percentages refer to weight percentages, that is, the weight of the compound relative to the total weight of the intended mixture.

[0052] The compositions described herein may “contain” essential compounds or optional components, “compose of” or “essentially consist of” them.

[0053] The phrase "consistently made of" means that the composition or component may include additional ingredients, provided that the additional ingredients do not alter or introduce new features of the composition or use described in this application.

[0054] The term "topical application" refers to applying or smearing the extract of the present invention or a composition containing it onto the surface of the skin or mucous membrane.

[0055] The term "skin" refers to the skin of the face, especially the eye area and mouth, nose, forehead, neck, hands, and the whole body.

[0056] The term "scalp" refers to the skin covering the skull, including hair follicles and the spaces between the follicles.

[0057] The term "appendages" refers to hair on the head and body (produced by hair follicles) as well as nails, which are rich in keratin.

[0058] The term "skin brightening" refers to reducing the intensity of skin color, which is related to the amount of melanin in the epidermis, either by acting on pigmentary disorders such as age spots or senile freckles, or by uniformly or locally reducing the pigmentation of the skin.

[0059] The term "effective amount" refers to the minimum amount of the extract of the present invention required to obtain at least one desired bioactivity or to protect the skin from external damage and oxidation, combat signs of skin aging, enhance photoprotection, brighten the skin, and improve skin hydration, and that amount is non-toxic.

[0060] The term "skin hydration" refers to the water content and distribution in the upper layer of the epidermis.

[0061] The term "improvement of skin hydration" refers to any improvement in the external appearance of the skin caused by dehydration, such as dryness, tightness, and discomfort, regardless of whether the changes are related to internal or external factors, such as adverse environmental conditions.

[0062] The term "signs of skin aging" refers to all changes in the external appearance of the skin caused by aging, such as wrinkles and fine lines, cracks, eye bags, dark circles, withering, and loss of skin elasticity, firmness, and / or tone. However, it also includes all internal changes in the skin that do not systematically lead to the altered external appearance, such as thinning of the skin, or all internal degeneration of the skin following environmental stresses such as pollution and sun radiation, including ultraviolet radiation.

[0063] The term "signs of skin aging" also refers to pigmented disorders, such as senile lentigines or solar lentigines.

[0064] The term "external harm" refers to solar radiation, including visible light, ultraviolet light, and infrared light radiation, and pollution, which may originate from the ambient atmosphere outside or inside a residence, and includes particles of different sizes (PM10 is 10 μm, PM2.5 is 2.5 μm, or ultrafine particles less than 100 nm) and several chemical elements (volatile organic compounds, polycyclic aromatic hydrocarbons, heavy metals, etc.).

[0065] The phrase "improves the appearance of the skin" refers to a more refined skin texture, greater radiance, and a more even skin tone.

[0066] The term "physiologically acceptable" refers to a medium or solvent that is suitable for contact with the outer layer of skin or mucous membranes without toxicity, irritation, excessive allergic reactions or similar intolerances, and is proportionate to a reasonable benefit / risk ratio.

[0067] It should be understood that this invention relates to mammals, and more particularly to humans.

[0068] Extraction method

[0069] Classical RNA extraction protocols use solvents unsuitable for cosmetic applications (Zumbo, P. 2014, "Phenol-chloroform Extraction," 2014). These methods aim to obtain completely purified nucleic acids (RNA, DNA, or small RNA), free from any other molecules of concern such as secondary metabolites, vitamins, sugars, peptides, etc., which can have beneficial effects on the skin and thus possess cosmetic interest.

[0070] Also known is document FR2831168, which describes a method for obtaining plant extracts rich in nucleic acids (DNA and RNA). This method uses cellulase.

[0071] Also known from the prior art are patent documents EP1723958 and WO03101376, which describe compositions for local application comprising synthetic double-stranded RNA oligonucleotides having a known sequence of length from 12 to 40 nucleotides and having siRNA (short interfering RNA) function.

[0072] Also known is document FR 1502361 (also published under number WO2017084958), which describes a method for obtaining an aqueous extract of plants rich in low molecular weight ribonucleic acid (RNA) for use in the preparation of cosmetic compositions. The method uses EDTA at a concentration between 2 and 15 mM.

[0073] Using phytic acid instead of EDTA has the following advantages: Unlike EDTA, phytic acid is a natural molecule found in the outer shells of seeds such as grains and legumes. Therefore, the use of phytic acid can yield 100% naturally derived tea extracts while maintaining good extraction efficiency of plant molecules present in the plant. It also improves the extraction efficiency of small RNAs and other compounds present in tea leaves, such as sugars, phenolic compounds, or organic acids such as tartaric acid, malic acid, or citric acid.

[0074] Therefore, the present invention relates first to an extraction method for obtaining an aqueous extract of leaves from all types of tea of ​​the Camellia sinensis species.

[0075] More specifically, the present invention relates to an extraction method for obtaining aqueous extracts of fresh tea leaves, Pu-erh tea, green tea, or matcha.

[0076] The extraction method of the present invention can obtain extracts rich in plant molecules with cosmetic benefits, such as small RNAs up to 150 nucleotides in length, sugars, phenolic compounds, organic acids, theanine, and theanine, while avoiding the use of solvents that are not considered cosmetic solvents.

[0077] The method of the present invention has a reduced impact on the environment.

[0078] The method of the present invention can be applied to fresh tea leaves, or to tea leaves that have been processed in various ways to conform to the names of matcha, green tea, or pu-erh tea.

[0079] In the first step a) of the method, tea leaves are mixed with water. The water used is distilled water, demineralized water, or water rich in mineral salts and / or trace elements. Distilled water is preferred.

[0080] Tea can come in different forms: fresh, dried, whole leaves, or powder.

[0081] Preferably, in step a), the fresh or dried tea leaves are ground in water. Grinding the tea leaves is a mechanical action that promotes better extraction. For tea leaves that are already in powder form, such as matcha mixed directly with water, grinding is unnecessary.

[0082] In step a), the ratio of plant material to water is 3% to 20% by weight, more preferably 3% to 10% by weight.

[0083] In step b), phytic acid is added to the tea-water mixture from step a).

[0084] In the presence of phytic acid, which is conducive to the complete destruction of cell membranes and nuclear membranes, the extraction process continues with alkaline lysis.

[0085] Phytic acid is an excellent chelating agent that forms ionic bridges between pectin molecules surrounding cellulose microfibers via complexation with divalent ions such as calcium ions, leading to weakening and disruption of the pectin-cellulose membrane in plant cells. This results in the release of cell contents during extraction. The phytic acid treatment step in an alkaline medium is essential for enriching the extract with low molecular weight RNA and ensuring better extraction yields of other plant molecules of interest, namely sugars, phenolic compounds, organic acids, and theanine and theanine.

[0086] Phytic acid is a molecule that is naturally found in the outer shell of seeds such as cereals and legumes. Phytic acid exists as a calcium salt or sometimes as a magnesium salt, and it plays an important role in plants, for example, it is a major source of phosphorus.

[0087] Preferably, the phytic acid used is a sodium salt form of phytic acid powder. Preferably, it is used at a concentration between 1 and 10 mM, more preferably between 1 and 5 mM, and even more preferably at a concentration of 3 mM.

[0088] A concentration of 3 mM is also optimal for better yields of other compounds of interest, such as sugars, phenols, organic acids, theanine, and theanine.

[0089] In step c), the pH is adjusted to an alkaline value between 10 and 11 by adding sodium hydroxide (NaOH). It is essential that the pH be alkaline during step c), between 10 and 11. Preferably, this pH is adjusted to a value between 10.3 and 10.8. In fact, this level of pH is related to the effects of phytic acid, which leads to the disruption of cell membranes, including the nuclear membrane, the lysis of plant cells, and the denaturation of DNA (the separation of the two strands of the double helix).

[0090] pH monitoring showed that at the end of step c), it remained alkaline and stable between 9 and 11.

[0091] Extraction step d) is preferably carried out at a temperature between 40 and 80°C for at least 1 hour. Preferably, the step lasts for 1 hour. Advantageously, the step is carried out at a temperature between 60 and 80°C. Even more preferably, the step is carried out at 80°C. During this step, the mixture is advantageously placed under moderate stirring.

[0092] In step e), the mixture obtained in d) is purified to remove residual solid tea leaves and recover the soluble fraction constituting the aqueous crude extract of the present invention. Any method known to those skilled in the art can be used to perform such a purification step. Preferably, the mixture is filtered directly on a filter having a porosity greater than or equal to 30 μm to facilitate collection of the filtrate. Alternatively, the mixture obtained in c) can be centrifuged at low speed, for example at 4000 g for at least 10 minutes, to deposit residual plant material as a granular precipitate and recover the aqueous crude extract in the supernatant.

[0093] In step f), the pH is adjusted to a value between 6 and 8.

[0094] In step g), powdered activated carbon is added to the mixture. Preferably, a mixture of two different types of activated carbon is used, and even more preferably, 0.25% by weight of each of the two types is used to facilitate a synergistic effect on the decolorization of the extract. The activated carbon is selected based on its ability to deplete the polyphenol extract, which primarily determines the color of the extract. This step is preferably continued for 30 minutes, with stirring at a temperature between 40 and 50°C for optimal results.

[0095] In step h), the mixture obtained in step g) is purified by removing activated carbon particles using a filter with a porosity greater than or equal to 30 μm to obtain a clear filtrate.

[0096] In step i), the filtrate obtained in step h) is contacted with powdered polyvinylpyrrolidone (PVPP) to maximize the removal of phenolic compounds, such as high molecular weight polyphenols and tannins, contained in the mixture. These phenolic compounds are not retained by the activated carbon treatment step and cause discoloration of the extract and may interfere with its stability. Preferably, a final concentration of PVPP between 5 and 30 g / L is used. Even more preferably, a final concentration of 10 g / L of PVPP is used.

[0097] This step can last 10 to 30 minutes, preferably 10 minutes, with stirring at a temperature between 40 and 50°C to obtain the best results.

[0098] According to the method of the present invention, particularly steps g) to i), it is possible to obtain an extract containing little or no catechins, epicatechins, or epigallocatechins. Only the extract obtained from Pu-erh tea contains low concentrations of catechins. Thin-layer chromatography analysis shows that the tea extracts of the present invention do not contain epicatechins or epigallocatechins.

[0099] In step j), the PVPP particles are removed by using a filter with a porosity greater than 25 μm, followed by filtration with a filter with a porosity of 0.8 μm to clarify the mixture obtained in step i).

[0100] Then, in step k), the pH of the mixture obtained in step j) is adjusted to a value between 6 and 6.5. The crude extract according to the invention is then obtained.

[0101] The pH can be adjusted by adding hydrochloric acid (HCl) solution or any other equivalent acid compatible with cosmetic use, such as citric acid. The pH adjustment step in step k) of the method of the invention is essential to obtain a stable extract without precipitating the phytochemicals of interest. In fact, this pH is optimal for maintaining suspensions of low molecular weight RNA and other plant molecules of interest, such as sugars, phenolic compounds, organic acids, theanine, and theanine.

[0102] In fact, pH levels below 6 can typically lead to the precipitation of nucleic acids, resulting in the precipitation of low molecular weight RNA up to 150 nucleotides in length, or even the precipitation of certain sugars, polyphenols, and proteins. The pH adjustment step in step i) of the method of the present invention is essential to achieve optimal stability of the extract.

[0103] The extract obtained in step k) can be diluted using a physiologically acceptable solvent for cosmetic use to stabilize the product and enhance its shelf life over time. The diluted extract is then obtained.

[0104] The extract is diluted to a dry weight of 4 to 20 g / kg, and its pH is adjusted to a value between 5.8 and 6.5, preferably between 6.0 and 6.5. This step improves the product's stability over time.

[0105] Secondly, this invention relates to aqueous extracts of tea obtained from fresh tea, Pu-erh tea, matcha, or green tea, said aqueous extract being rich in small RNAs up to 150 nucleotides in length, rich in sugars, rich in phenolic compounds, rich in organic acids, rich in catechins, and rich in theanine, and free of DNA, said aqueous extract being obtainable by the methods described above. Such extracts do not contain DNA (deoxyribonucleic acid).

[0106] This invention also relates to an aqueous extract of tea obtained from fresh tea, Pu-erh tea, matcha, or green tea, said aqueous extract being rich in small RNAs up to 150 nucleotides in length, rich in sugars, rich in phenolic compounds, rich in organic acids, rich in catechins, and rich in theanine, said aqueous extract being obtained directly by the method described above. This extract does not contain DNA (deoxyribonucleic acid).

[0107] By using the method of the present invention according to steps a) to k), an aqueous concentrated crude extract of tea in an amber to dark amber color is obtained, the aqueous concentrated crude extract having a dry weight of 5 to 30 g / kg, the aqueous concentrated crude extract containing 0.5 to 10 g / kg of sugars; 0.050 to 2 g / kg of organic acids; 0.050 to 5 g / kg of phenolic compounds, the phenolic compounds including 0 to 200 mg / kg of catechins, 0.020 to 2 g / kg of theanine and 0.020 to 2 g / kg of theanine; and 50 to 350 mg / kg of low molecular weight RNA with a length of up to 150 nucleotides. However, due to the various treatments of tea leaves, the obtained extract can exhibit significant variability depending on factors such as the location or year of harvest, season, climatic conditions, biological stress, etc.

[0108] The extract thus obtained can then be diluted in a physiologically acceptable solvent for cosmetic use, so that the concentration of the extract can be adjusted to between 4 and 20 g / kg dry weight.

[0109] Physiologically acceptable solvents include water, glycerol, ethanol, propylene glycol, butanediol and their natural forms, dipropylene glycol, ethoxylated or propoxylated diethylene glycols, cyclic polyols, or any mixture of these solvents. Ethanol, butanediol, propylene glycol, and glycerol exist as synthetic solvents or plant-derived solvents, the latter being described as natural solvents.

[0110] Therefore, the obtained extract can be mixed with 30% to 50% plant-derived butylene glycol, 30% to 50% plant-derived propylene glycol, or 30% to 70% plant-derived glycerin.

[0111] Preferably, the extract obtained by the method of the present invention is diluted in butanediol such that the diluted extract contains 30% or 50% of the final butanediol concentration.

[0112] Such so-called diluted extracts have a dry weight of 4 to 20 g / kg and the following concentrations: 0.2 to 5 g / kg of sugars; 0.030 to 1 g / kg of organic acids; 0.030 to 3 g / kg of phenolic compounds, including 0 to 120 mg / kg of catechins, 0.010 to 1 g / kg of catechins and 0.010 to 1 g / kg of theanine; and 10 to 250 mg / kg of low molecular weight RNA with a length of up to 150 nucleotides.

[0113] A non-limiting example is a diluted green tea extract, which more specifically contains 1.4 g / kg of sugars, 95.5 mg / kg of organic acids, 32 mg / kg of phenolic compounds, and 77 mg / kg of low molecular weight RNA with a length of up to 150 nucleotides.

[0114] Therefore, the extracts of the present invention contain a wide range of plant molecules that can exhibit beneficial effects on the skin without the risk of skin irritation or other health damage.

[0115] For example, sugars actively participate in the hydration of the epidermal layer and thus resist external aggression without causing any undesirable effects. The tea extract of this invention particularly contains both monosaccharides and polysaccharides.

[0116] Camellia sinensis, a member of the Theaceae family, produces catechins, potent natural insecticides. Caffeine is also known as caffeine, 1,3,7-trimethylxanthine, or methylthecobalamin. In addition to its well-known effects on the nervous and cardiovascular systems, catechins also have beneficial effects on the skin, particularly on skin laxity.

[0117] This plant also produces theanine, a non-protein amino acid commonly found in tea. Theanine is known for its relaxing activity on the brain, allowing for the reduction of mental and physical stress.

[0118] The tea extract of this invention also contains phenolic compounds, such as polyphenols, phenolic acids, and flavonoids such as catechins; however, molecules of this family have been partially removed by activated carbon and PVPP treatment steps. This relative depletion of phenolic compounds enhances the product's stability over time and yields a non-phototoxic extract.

[0119] In one particular embodiment of the invention, the diluted aqueous extract according to the invention contains up to 1.5 g / kg of phenolic compounds, including up to 120 mg / kg of catechins.

[0120] In another specific embodiment of the invention, the diluted aqueous extract according to the invention contains up to 1.2 g / kg of phenolic compounds, including up to 30 mg / kg of catechins.

[0121] In another specific embodiment of the invention, the diluted aqueous extract according to the invention contains up to 100 mg / kg of phenolic compounds and does not contain catechins.

[0122] In another specific embodiment of the invention, the diluted aqueous extract according to the invention contains up to 50 mg / kg of phenolic compounds and does not contain catechins.

[0123] In a particular embodiment of the invention, the diluted aqueous extract according to the invention contains up to 3 g / kg of phenolic compounds and does not contain catechins.

[0124] Third, the present invention relates to cosmetic compositions comprising an effective amount of at least one aqueous tea extract as an active ingredient and a physiologically acceptable medium, wherein the at least one aqueous tea extract is selected from extracts of fresh tea, Pu-erh tea, matcha, green tea, or any mixture thereof, and wherein the at least one aqueous tea extract is obtained according to the present invention.

[0125] Advantageously, the tea extract of the present invention is added in a physiologically acceptable medium at a concentration of 0.05 to 5% by weight relative to the total weight of the composition, preferably at a concentration of 0.1 to 2.5% by weight relative to the total weight of the composition.

[0126] The compositions of the present invention are formulated for application by any suitable route, particularly oral or topical, and the formulation of the compositions is adapted by those skilled in the art.

[0127] Preferably, the compositions of the present invention are in a form suitable for topical application. Therefore, such compositions must contain a physiologically acceptable medium, i.e., a medium compatible with the skin and appendages, and cover all suitable cosmetic forms, without posing any risk of discomfort during their application.

[0128] The compositions used to carry out the present invention may be in particular in the form of aqueous, hydroalcoholic or oily solutions or gels, oil-in-water, water-in-oil or multiple emulsions; they may also be in the form of suspensions or powders suitable for use on skin, mucous membranes, lips and / or hair.

[0129] Such compositions can be more or less viscous and can also have the appearance of a cream, lotion, liquid, emulsion, serum, ointment, gel, paste, balm, or foam. They can also be in solid form, such as sticks, or can be applied to the skin as an aerosol.

[0130] Examples of commonly used physiologically acceptable media in the envisioned application areas are adjuvants required for formulation, such as solvents, thickeners, gelling agents, diluents, emulsifiers, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, esters, and vegetable oils or butter, etc.

[0131] In all cases, those skilled in the art will ensure that such adjuvants and their ratios are selected in such a manner that the advantageous properties sought in the compositions according to the invention are not impaired. For example, such additives may each correspond to 0.01 to 20% by weight of the total weight of the composition. When the compositions of the invention are emulsions, the fatty phase may be 2 to 90% by weight, preferably 5 to 30% by weight, relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the compositions are selected from those conventionally used in the field under consideration. For example, they may be used in proportions of 0.3 to 30% by weight, relative to the total weight of the composition.

[0132] According to another advantageous embodiment of the invention, the aqueous tea extract of the invention can be encapsulated or contained in a cosmetic vector such as liposomes or any other nanocapsule or microcapsule used in the cosmetic field, or adsorbed onto powdered organic polymers, mineral supports such as talc and bentonite.

[0133] Advantageously, in addition to the active ingredients of the present invention, the compositions of the present invention may also contain at least one other active agent having cosmetic effects similar to and / or complementary to those of the present invention.

[0134] For example, additional active agents may be selected from: anti-aging agents, firming agents, brightening agents, moisturizers, draining agents, microcirculation promoters, exfoliants, desquamating agents, extracellular matrix stimulants, energy metabolism activators, antibacterial agents, antifungal agents, soothing agents, free radical scavengers, UV protectants, anti-acne agents, anti-inflammatory agents, anesthetics, temperature inducers, cooling inducers, and weight loss agents.

[0135] Such additional surfactants can be selected from the following:

[0136] - Vitamin A, especially retinoic acid, retinol, retinyl propionate and retinyl palmitate;

[0137] -Vitamin B3, and especially nicotinamide and tocopheryl nicotinate;

[0138] - Vitamin B5, Vitamin B6, Vitamin B12, Panthenol;

[0139] - Vitamin C, especially ascorbic acid, ascorbate glucoside, ascorbate tetrapalmitate, ascorbate magnesium phosphate and sodium;

[0140] - Vitamins E, F, H, K, PP, and Coenzyme Q10;

[0141] - Metalloproteinase inhibitors or TIMP activators;

[0142] -DHEA, its precursors and derivatives;

[0143] - Amino acids such as arginine, ornithine, hydroxyproline, hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine, methionine and its derivatives, and N-acylated amino acid compounds;

[0144] - Natural or synthetic peptides, including dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides, and their lipophilic derivatives, isomeric derivatives, and derivatives complexed with other substances such as metal ions (e.g., copper, zinc, manganese, magnesium, etc.). Examples include commercially known peptides with the following names: CHRONOGEN TM ,LAMINIXYLIS TM PEPTIDE Q10 TM COLLAXYL TM (Patent FR2827170, ), PEPTIDE VINCI 01 TM (Patent FR2837098, ), PEPTIDE VINCI 02 TM (Patent FR2841781, ), ATPeptide TM (Patent FR2846883, ) or through ATPeptide TM The synthetic peptide with the sequence Arg-Gly-Ser-NH2 is being sold.

[0145] - Artemia salina extract, as GP4GTM (FR2817748, )Sale;

[0146] - Plant peptide extracts, such as flax extract (Lipigenin) TM Patent FR2956818 Extracts from soybeans, spelt wheat, grapevines, rapeseed, flax, rice, corn, and peas;

[0147] - Yeast extract, such as Dynagen TM (Patent FR2951946, ) or Actontine TM (Patent FR2944526, );

[0148] -Dehydroacetic acid (DHA);

[0149] - Phytosterols of synthetic or natural origin;

[0150] - Salicylic acid and its derivatives, α- and β-hydroxy acids, silanols;

[0151] -Amino sugars, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine;

[0152] - Extracts of polyphenols, isoflavones, and flavonoids, such as grape extract, pine extract, and olive extract;

[0153] - Lipids, such as ceramides or phospholipids; animal-derived oils, such as squalene or squalane; vegetable oils, such as sweet almonds, dried coconut kernels, castor beans, jojoba, olives, rapeseed, peanuts, sunflower seeds, wheat germ, corn germ, soybeans, cotton, alfalfa, poppies, squash, evening primrose, millet, barley, rye, safflower, passion fruit, hazelnuts, palm oil, almonds, avocados, calendula; ethoxylated vegetable oils, shea butter, cocoa butter, babassu oil;

[0154] -All UV protectants and sunscreens;

[0155] - Cyclic AMP and its derivatives, adenylate cyclase activators and phosphodiesterase inhibitors, Centella asiatica extract, asiaticoside and asiaticoic acid, methylxanthines, theophylline, theobromine, trichomoniasis, esculin and esculoside, ACE inhibitors, Val-Trp peptide, neuropeptide Y inhibitors, enkephalin, ginkgo extract, dioscorea extract, rutin, wintergreen tea extract, guarana extract, oligosaccharides, polysaccharides, carnitine, ivy extract, fucoidan extract, hydrolyzed extract of prunella vulgaris, hydrolyzed extract of cockscomb, smooth fruit elm (Anogeissus leiocarpus) extract, cassava leaf extract, palmitoylcarnitine, carnosine, taurine, elderberry extract, algal extracts such as palmaria palmata extract.

[0156] - Hyaluronic acid or sodium hyaluronate and all its fractions and promoters;

[0157] - Menthol and menthol lactate, and other active ingredients with a refreshing effect;

[0158] - Aloe vera, bisabolol, allantoin, and other active ingredients with soothing effects;

[0159] -Vanillyl butyl ether and other active ingredients with heating effects;

[0160] - Benzoyl peroxide and other anti-acne active ingredients.

[0161] Fourth, the present invention relates to the cosmetic use of compositions comprising the tea extract of the present invention, said tea extract being derived from fresh tea, Pu-erh tea, matcha or green tea, or any mixture thereof, said use being for the care of skin, scalp and appendages, more particularly for protecting skin from external aggressors and oxidation, for combating signs of skin aging, for enhancing photoprotection, for brightening skin, and for improving skin hydration.

[0162] In one particular embodiment, the invention also relates to the cosmetic use of a composition comprising a tea extract of the invention obtained from fresh tea, matcha, or green tea, or any mixture thereof, for the care of the skin, scalp, and appendages, more particularly for protecting the skin from external aggressors and oxidation, for combating signs of skin aging, for enhancing photoprotection, for brightening the skin, and for improving skin hydration.

[0163] The skin is an organ composed of multiple layers (dermis, epidermis, and stratum corneum) that covers the entire surface of the body and ensures protective functions against external aggressors, sensory aggressors, immune aggressors, metabolic aggressors, and thermoregulatory aggressors, or even acts as a barrier to limit dehydration.

[0164] In particular, the stratum corneum acts as a protective physical barrier, often referred to as the "skin barrier function." This function is crucial for tissue homeostasis and protection against external environmental influences.

[0165] The appearance of the skin can be altered by internal changes (internal aging, disease, and hormonal changes such as pregnancy) or external factors (environmental factors such as pollution, sunlight, pathogens, temperature changes, etc.). All such changes affect not only the skin but also keratinous appendages such as body hair, eyelashes, eyebrows, nails, and scalp hair.

[0166] Example

[0167] The embodiments of the method according to the present invention are described below by way of example.

[0168] Example 1: Preparation of fresh tea, Pu-erh tea, matcha, or green tea (Camellia) according to the method of the present invention Small RNA-rich extract of *Sinensis*

[0169] In the first step a) of preparing the extract, matcha (Japanese green tea in powder form), pu-erh tea (tea from Yunnan in the form of compressed whole leaves), green tea (tea from China in the form of dried leaves) and fresh tea (leaves picked directly from the tree) were used, all of which were from the Camellia species Camelliasinensis.

[0170] Weigh the green tea or Pu-erh tea so that it constitutes 3% of the raw materials involved in the method, or 30g / kg. The amount of distilled water added is 968g.

[0171] Weigh the fresh tea leaves to make them 6% of the raw materials involved, or 60g / kg, since the water content of the fresh tea leaves is half the fresh weight of the leaves. The amount of distilled water added is 938g.

[0172] The mixture of tea leaves and water is ground in a blender to allow for better extraction of plant compounds because there is a larger exchange surface between the solid material, the tea, and the water extract solution.

[0173] The dried matcha leaves, already in powder form, are added directly to a mixture containing 968g of distilled water and pre-homogenized 2g / L or 3mM of phytic acid.

[0174] In step b), 2 g / L (i.e., the final concentration of 3 mM) of phytic acid is added to the mixture of water and finely ground tea leaves (except for matcha).

[0175] In step c), the pH is adjusted to 10.5 for optimal extraction and to allow enrichment of extracts containing low molecular weight RNA as well as extracts containing various plant molecules.

[0176] In step d), the mixture is heated at 80°C for 1 hour with moderate stirring.

[0177] In step e), the mixture is filtered through a filter with a porosity of 30 μm to separate the solids from the filtrate. Then, a three-stage sequential filtration is performed using a filter with reduced porosity to clarify the plant extract until it is filtered to a porosity of 3 μm.

[0178] In step f), the average pH of the filtrate collected in step e) is higher than 9 and is adjusted to between 6 and 8 to achieve the optimal pH for subsequent activated carbon treatment.

[0179] Step g) involves treating the extract with a duo of two types of activated charcoal to decolorize it, particularly by removing polyphenols. 2.5 g of each type of activated charcoal is added per liter of the extract. The treatment is carried out at a temperature between 40 and 50°C for 30 minutes.

[0180] Step h): The activated carbon is removed by passing the extract through a filter with a porosity of 30 μm.

[0181] In step i), a second decolorization treatment is performed on the filtrate obtained in g) by adding powdered polyvinylpyrrolidone (PVPP). 10 g / L of PVPP is added to the extract to remove some of the tannins contained therein. The treatment is carried out at a temperature between 40 and 50°C for 10 minutes.

[0182] In step j), sequential filtration is then performed on a filter with reduced porosity to remove PVPP from the plant extract; the first 30 μm filtration allows PVPP to be retained, followed by continuous filtration through a porosity not exceeding 0.8 μm to clarify the extract.

[0183] In step k), check the pH and then adjust the pH to a value between 6 and 6.5 using a citric acid or hydrochloric acid solution.

[0184] A water-based crude extract with a dry weight between 5 and 30 g / kg was obtained.

[0185] The extract obtained in step k) is diluted with 30% butylene glycol. These solvents are physiologically acceptable for cosmetic use, allowing for product stability and increasing its shelf life over time. The diluted extract is then obtained, and the pH is adjusted to between 6.0 and 6.5.

[0186] The resulting diluted extract had a dry weight between 4 and 20 g / kg.

[0187] The tea extract thus obtained according to the method described in this embodiment is referred to as PSR (Plant small RNA) tea in the tables or figures presented in this invention.

[0188] Example 2: Preparation of tea (Camellia sinensis) using so-called conventional extraction methods ) Extracts:

[0189] For comparative purposes, tea extracts obtained by so-called conventional or classical extraction were prepared. Such extraction methods were chosen because they are optimal for extracting different types of phenolic compounds and other polar molecules such as sugars and amino acids, making them a good reference method for comparison with the method of the present invention applied in Example 1.

[0190] In the first step of preparing 1 kg of extract, the same teas as in Example 1 were used: matcha (Japanese green tea in powder form), pu-erh tea (tea from Yunnan in the form of compressed whole leaves), green tea (tea from China in the form of dried leaves), and fresh tea (leaves picked directly from the tree), all of which were from Camellia sinensis species.

[0191] Weigh dried or powdered tea leaves such as matcha, pu-erh tea, and green tea so that they account for 3% of the raw materials used in this method, i.e., 30g / kg, which are mixed with 970g of distilled water.

[0192] Fresh tea leaves were mixed with 940g of distilled water. The fresh tea leaves, due to their natural water content, comprised 6% of the raw materials used, or 60g / kg. A grinding step was applied to the fresh tea, green tea, and Pu-erh tea. The extraction pH was not adjusted and remained between 5 and 7.

[0193] Then, while stirring, the mixture is heated at 45°C for 1 hour.

[0194] The mixture is then filtered through a filter with a pore size of 30 μm to separate the solids from the filtrate. Sequential filtration is then performed using a filter with reduced porosity to clarify the plant extract until it is filtered to a pore size of 1 μm.

[0195] At the end of this step, activated charcoal treatment is performed to decolorize the extract, particularly by removing polyphenols. 2.5 g of each type of activated charcoal is added per liter of extract. The treatment time is 30 minutes, the temperature is between 40 and 50°C, and the pH is between 6 and 8. 30 μm filtration is then performed to remove the activated charcoal from the extract.

[0196] A second decolorization treatment was then performed by adding powdered polyvinylpyrrolidone (PVPP). 10 g / L of PVPP was added to the extract to remove some of the tannins present in the extract. The treatment was carried out at a temperature between 40 and 50°C for 10 minutes. The extract was then subjected to sequential filtration through a porosity-reducing filter to clarify it until a filtration thickness of 0.2 μm was achieved.

[0197] Check the pH, then adjust it to between 6 and 6.5 using citric acid, hydrochloric acid, or baking soda solution.

[0198] An aqueous crude extract with a dry weight between 1 and 5 g / kg was obtained. The method implemented in Example 2 was intended to prepare a control extract to obtain comparative analytical data related to different tea extracts obtained by the method of the present invention. The results obtained are shown in the accompanying drawings and in the application text.

[0199] Example 3: Characteristics of the tea extract obtained according to Example 1

[0200] The crude extract of fresh tea, made directly from fresh leaves harvested from the tree, had a dry weight of 6.5 g / kg. Physicochemical analysis showed that the obtained extract had the following concentrations: 2 g / kg total sugars, 120 g / kg total organic acids, 31 mg / kg total phenolic compounds, and 120 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0201] The extract is then diluted with a physiologically acceptable cosmetic solvent, ensuring better stability and preservation of the extract over time; the amount of solvent can be 30% to 70%.

[0202] The extract was diluted with plant-derived butylene glycol to obtain a final concentration of 30% butylene glycol and 70% tea extract. The extract thus diluted had a dry weight of 4.3 g / kg and contained the following concentrations: 1.4 g / kg total sugars, 83 mg / kg total organic acids, 48 ​​mg / kg total phenolic compounds, and 74 mg / kg low molecular weight RNA of up to 150 nucleotides in length.

[0203] The crude extract of green tea, made from dried leaves from China, had a dry weight of 10.2 g / kg. Physicochemical analysis showed that the obtained extract had the following concentrations: 1.5 g / kg total sugars, 137 mg / kg total organic acids, 1.3 g / kg total phenolic compounds, and 110 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0204] The extract is then diluted with a physiologically acceptable cosmetic solvent, ensuring better stability and preservation of the extract over time; the amount of solvent can be 30% to 70%.

[0205] The extract was diluted with plant-derived butylene glycol to obtain a final concentration of 30% butylene glycol and 70% tea extract. The extract thus diluted had a dry weight of 7.1 g / kg and the following concentrations: 1.1 g / kg total sugars, 96 mg / kg total organic acids, 931 mg / kg total phenolic compounds, and 77 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0206] The crude extract of matcha, prepared from crushed leaves from Japan, had a dry weight of 14.2 g / kg. Physicochemical analysis showed that the obtained extract had the following concentrations: 2.4 g / kg total sugars, 179 mg / kg total organic acids, 2 g / kg total phenolic compounds, and 87 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0207] The extract is then diluted with a physiologically acceptable cosmetic solvent, ensuring better stability and preservation of the extract over time; the amount of solvent may be 30% to 70%.

[0208] The extract was diluted with plant-derived butylene glycol to obtain a final concentration of 30% butylene glycol and 70% tea extract. The extract thus diluted had a dry weight of 10.2 g / kg and contained the following concentrations: 1.7 g / kg total sugars, 125 mg / kg total organic acids, 1420 mg / kg total phenolic compounds, and 61 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0209] The crude extract of Pu-erh tea, made from compressed whole leaves from Yunnan, had a dry weight of 13.8 g / kg. Physicochemical analysis showed that the obtained extract had the following concentrations: 3.3 g / kg total sugars, 137 mg / kg total organic acids, 193 mg / kg total phenolic compounds, and 250 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0210] The extract is then diluted with a physiologically acceptable cosmetic solvent, ensuring better stability and preservation of the extract over time; the amount of solvent may be 30% to 70%.

[0211] The extract was diluted with plant-derived butylene glycol to obtain a final concentration of 30% butylene glycol and 70% tea extract. The extract thus diluted had a dry weight of 8 g / kg and the following concentrations: 1.1 g / kg total sugars, 137 mg / kg total organic acids, 1176 mg / kg total phenolic compounds, and 177 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0212] Example 4: Quantification of different components of the diluted tea extract obtained in Examples 1 and 2

[0213] The dry weight of each type of extract was measured according to the method of the present invention or according to conventional methods. The dry weight represents the weight of the extract after 12 hours of steaming at 105°C, after which water and volatile compounds have evaporated.

[0214] Compared to tea extracts obtained through conventional extraction, the dried extracts of various tea extracts obtained by the method of this invention have higher dry weights. Therefore, when the method of Example 1 is applied to extract different types of tea, the extraction yield is higher; see Table 1.

[0215] Table 1

[0216]

[0217] The total sugar content in different tea extracts obtained by the method of the present invention was determined by spectrophotometry relative to the total sugar content in extracts obtained by conventional methods, wherein the spectrophotometric determination was an adaptation of the determination method described by Dubois et al. (1956) (Dubois et al., “Colorimetric method for the determination of sugars and related substances”, Anal. Chem, 1956, 28(3), 350-356). This type of analysis involves dissolving the raw material in concentrated sulfuric acid and then reacting it with phenol to form a colored complex. The absorbance of the complex was read at 490 nm on a spectrophotometer. The sugar content was determined using a standard glucose curve.

[0218] Analysis shows that for all types of tea—fresh tea, green tea, matcha, and Pu-erh tea—the method described in this invention yields the highest concentration of sugars compared to conventional extraction methods. (See [link to relevant documentation]). Figure 1 .

[0219] The total polyphenol content of the tea extract according to the present invention, relative to the total polyphenol content of conventional tea extracts, was determined by Folin-Ciocalteu spectrophotometry (Singleton et al, Analysis of total phenols and other oxidative and antioxidant substrates using the Folin-Ciocalteu reagent, 1999, 299:152). Polyphenolic compounds present in the sample react with the Folin-Ciocalteu reagent; oxidation of the reagent yields a blue color. The absorbance of the sample was read at 760 nm on a spectrophotometer. The total polyphenol concentration was expressed as gallic acid equivalents using a gallic acid standard curve. Analysis showed that for all types of tea—fresh tea, green tea, matcha, and Pu-erh tea—the polyphenol concentration extracted by the method described in this invention was the highest compared to conventional extraction methods. See [link to relevant documentation]. Figure 2 .

[0220] The organic acids contained in the tea extract according to the present invention were characterized and quantified relative to those contained in conventional tea extracts. High-performance liquid chromatography (HPLC) combined with a mass detector was used. All samples were separated using an Agilent 1260 HPLC system (Agilent Technologies) on an EC 150 / 4.6 Nucleoshell RP 18plus-5 μm column (150 × 4.6 mm) (Macherey Nagel: 763236.46). The flow rate was 0.3 mL / min. The mobile phase consisted of a solution of 0.01% formic acid (HCOOH) (A) and acetonitrile (B). A gradient program facilitated elution, as described in Table 2.

[0221] Table 2

[0222]

[0223] The column temperature was maintained at 25 °C, and the injection volume was 5 μL. Detection was performed using an ACQUITY Qda mass spectrometer detector (WATERS) with a negative-mode electrospray ionization source. The ion source was set at a capillary voltage of 0.8 kV and a probe temperature of 600 °C. For each compound as described in Table 3, M / z and cone voltage were used as targets.

[0224] Table 3

[0225] compound Mass (m / z) Tapered hole voltage (V) tartaric acid 149.0 10 malic acid 132.9 3 Citric acid 191.0 10 lactic acid 88.9 5 Succinic acid 116.95 15 Glucuronic acid 193 10

[0226] Organic acids were identified by comparing the retention time and mass spectrometry peak of the sample with standards. Quantitative estimation of the organic acids was performed based on the peak area compared to the standard area.

[0227] Quantitative and identification analysis of organic acids in the different tea extracts obtained according to Examples 1 and 2 by HPLC-MS showed that the PSR tea extract and the conventional extract contained different types of organic acids, mainly citric acid, lactic acid, malic acid and succinic acid, as shown in Table 4. Tartaric acid and uronic acid were not detected in either the PSR tea extract or the conventional extract.

[0228] Table 4

[0229]

[0230] The analysis also shows that, compared to conventional extraction methods, these organic acids are extracted to a greater extent using the method of the present invention, such as in... Figure 3 As shown in the image.

[0231] The theanine content in the tea extract according to the present invention was determined relative to that in conventional tea extracts. Tea leaves contain this unique amino acid, which accounts for 50% of the total amino acids in tea. It is a non-protein amino acid. High-performance liquid chromatography (HPLC) combined with a UV detector was used for analysis. All samples were separated using an Agilent 1200 HPLC system (Agilent Technologies) via a 250 mm × 4.6 mm × 5 μm Uptisphere C18-2 column (Interchim). The flow rate was 0.4 mL / min. The mobile phase consisted of 0.1% aqueous phosphoric acid (H3PO4) solution (A) and acetonitrile (ACN) (B). Elution was facilitated by the following gradient program, see Table 5:

[0232] Table 5

[0233]

[0234] The column temperature was maintained at 30°C. The injection volume was 5 μL, and the detection wavelengths were set at 196 nm and 210 nm using a UV detector. Theanine standards were purchased from Sigma-Aldrich. Theanine identification was performed by comparing the retention time and UV peak of the sample with those of the standards. Quantitative estimation of theanine was performed based on the peak area compared to the area of ​​the standards.

[0235] HPLC-UV analysis, which can quantify and identify theanine in various tea extracts obtained according to the method of the present invention or according to so-called conventional methods, indicates that the tea extracts obtained by the method of the present invention contain theanine. However, as in Figure 4 As shown, less theanine was observed in the extract obtained according to the present invention compared to extracts obtained by conventional extraction.

[0236] Theine and caffeine are the same molecule, and theine is characterized by being an alkaloid present in large quantities in tea leaves. The theine content of the tea extract according to the present invention was also determined relative to that of conventional tea extracts. All samples were separated using an Agilent 1100 HPLC system (Agilent Technologies) on a 100 mm × 4.6 mm × 2.6 pm Uptisphere CSevolution C18-AQ column (Interchim). The flow rate was 0.8 ml / min. The mobile phase consisted of an aqueous solution of 0.1% TFA (trifluoroacetic acid) (A) and methanol (B). Elution was facilitated by the following gradient procedure, see Table 6:

[0237] Table 6

[0238]

[0239] The column temperature was maintained at 25°C. The injection volume was 5 μL, and the detection wavelength was set to 272 nm using a UV detector. Theanine standards were purchased from Sigma-Aldrich. Theanine identification was performed by comparing the retention time and UV peak of the sample with those of the actual standards. Quantitative estimation of theanine was performed based on the chromatographic peak area compared to the area of ​​the standard.

[0240] HPLC-UV analysis, which can quantitatively identify catechins in various tea extracts obtained by the method according to the invention or by so-called conventional methods, shows that all types of tea extracts obtained by the method of the invention or by so-called conventional extraction methods contain catechins. The results indicate that, as in... Figure 5 As shown, the extract obtained by the method of the present invention contains fewer catechins than the tea extract obtained by conventional extraction methods.

[0241] Quantification of catechins was also performed. Due to the presence of two asymmetric carbons, catechins exist in several stereoisomers. The most common isomers in nature are (+)-catechin and (-)-epicatechin. The other two enantiomers are much less common, and their presence appears to be related to enzymatic reactions or thermal treatment. All samples were separated using an Agilent 1100 HPLC system (Agilent Technologies) on a 100 mm × 4.6 mm × 2.6 pm Uptisphere CS evolution C18-AQ column (Interchim). The flow rate was 0.8 mL / min. The mobile phase consisted of an aqueous solution of 0.1% TFA (trifluoroacetic acid) (A) and methanol (B). Elution was facilitated by the following gradient program, see Table 7:

[0242] Table 7

[0243]

[0244] The column temperature was maintained at 25°C. The injection volume was 20 μL, and the detection wavelengths were set to 255 nm, 280 nm, 290 nm, and 324 nm using a UV detector. Catechin standards were purchased from Sigma-Aldrich. Catechin identification was performed by comparing the retention time and UV spectral peaks of the sample with those of the actual standards. Quantitative estimation of catechins was performed based on the peak area of ​​the sample concentration relative to the area of ​​the standard.

[0245] HPLC-UV analysis, which can quantify and identify catechins in various tea extracts obtained by the method according to the invention or by so-called conventional methods, indicates that the tea extracts obtained by the method of the invention or by conventional extraction methods contain little or no catechin-type polyphenols, such as in Figure 4 As shown in the diagram, only the extract from Pu-erh tea contains some catechins. The absence or low amount of such molecules in the extract is specifically explained by the implementation of a stage involving activated charcoal and PVPP treatment, which removes these compounds. This result was also confirmed by the implementation of thin-layer chromatography, allowing visualization of various molecules such as catechins, epicatechin, and epigallocatechin. Tea extracts obtained by either the method of this invention or by conventional methods do not contain such molecules. This confirms the effectiveness of the activated charcoal and PVPP treatment.

[0246] Low molecular weight RNA was quantified and its size measured using miniaturized electrophoresis techniques on a microfluidic chip specifically designed for the analysis of nucleic acids such as low molecular weight RNA (Bioanalyzer). (Agilent). This type of method allows for the determination of the size and concentration of nucleic acids contained in the extract.

[0247] The quantitative results obtained by this Bioanalyser demonstrate that the tea extract obtained by the method described in this invention allows for the extraction of low molecular weight RNA from different types of tea, as shown in Table 8. In contrast, no low molecular weight RNA (nd) was detected in conventional tea extracts.

[0248] Table 8

[0249]

[0250] Example 5: Evaluation of the tea extracts from Examples 1 and 2 in terms of their activity against hyaluronidase inhibition in in vitro tests. Take the item:

[0251] principle:

[0252] The purpose of this test is to demonstrate, through a turbidity measurement experiment, the inhibitory effect of the extracts from Examples 1 and 2 on the activity of an enzyme, such as hyaluronidase. Hyaluronidase catalyzes the degradation of hyaluronic acid (a glycosaminoglycan highly present in the dermis of the skin and possessing moisturizing and anti-aging properties) into monosaccharides or disaccharides and smaller hyaluronic acid fragments. Hyaluronic acid has the ability to react with acidic albumin solutions, thus forming a haze that can be measured at 600 nm using a spectrophotometer. Therefore, the inhibition of enzyme activity is measured by analyzing the turbidity level of the sample after it has been contacted with the enzyme and its substrate.

[0253] plan:

[0254] The enzyme was incubated with the extracts obtained according to Examples 1 and 2 to represent a final concentration of 1% in the reaction mixture at 37°C in a pH 7 buffer for 20 minutes, ideal conditions for enzyme equilibration. Hyaluronic acid, the substrate of the enzyme, was added to the mixture at a concentration of 0.3 mg / ml. The mixture was slowly homogenized and then incubated precisely at 37°C for 45 minutes. The remaining hyaluronic acid in the mixture was then contacted with an acidic albumin solution and homogenized. The reaction was allowed to proceed for 10 minutes, after which the transmittance was read at 600 nm on a spectrophotometer. The data were compared with those obtained using a negative inhibition control, which corresponds to conditions where the enzyme had degraded 100% of the substrate, and therefore 100% enzyme activity.

[0255] result:

[0256] The tea extract prepared according to Example 1 showed a hyaluronidase inhibition percentage of 67.9% (for the weakest, fresh tea) to about 80% (for the strongest, matcha and pu-erh tea). The tea extract prepared conventionally according to Example 2 showed an inhibition percentage of 1.7% (weakest, green tea) to 57.7% (strongest, pu-erh tea).

[0257] in conclusion:

[0258] Tests used to evaluate the inhibitory effect of the extract on hyaluronidase activity in vitro have shown that the tea extract prepared according to Example 1 exhibits a significantly stronger inhibitory effect on the enzyme compared to the extract prepared according to Example 2, as shown in... Figure 5 As shown in the image.

[0259] Example 6: Compared with the conventional Pu-erh tea extract prepared according to Example 2, the Pu-erh tea extract prepared according to Example 1... Tea extract on the effects of sodium glutamate stress on skin biopsies live Assessment of reactive oxygen species.

[0260] principle:

[0261] The purpose of this study is to demonstrate the effect of the Pu-erh tea extract prepared according to the present invention on reducing reactive oxygen species generated by monosodium glutamate (MSG) stress. Glutamate is known as an excitatory neurotransmitter in the central nervous system. In the skin, glutamate signal transduction occurs in pain nerve endings and mechanoreceptor fibers.

[0262] Thioredoxins (TRXs) are a class of small proteins involved in redox reactions, which are known to occur in all organisms. The main function of TRX-interacting proteins (TXNIPs) is to bind and inactivate TRX, leading to increased accumulation of reactive oxygen species (ROS) and apoptosis. TXNIP expression is closely related to the level of ROS production.

[0263] plan:

[0264] Human skin biopsies were cultured in a medium containing 100 mM monosodium glutamate and treated either with PBS (phosphate-buffered saline) for 48 hours as a stress control, or with 0.5% (v / v dilution) and 1% (v / v dilution) Pu-erh tea extract prepared according to Example 1 for 48 hours, or with the same concentration of conventional Pu-erh tea extract prepared according to Example 2 for 48 hours. As a non-stress control, skin biopsies were cultured in a conventional medium and treated with PBS. After treatment, the biopsies were fixed for histological analysis and embedded in paraffin. After deparaffining, the antigen sites were demasked by microwave heating in citrate buffer at pH 6 and saturated with 5% bovine serum albumin for 30 minutes. Then, rabbit monoclonal anti-TXNIP antibody was applied at a 1:100 ratio for 1 hour and 30 minutes in a humidified chamber. The sections were rinsed in phosphate buffer and then treated in a light-protected humidified chamber using Alexa. The slide was incubated with anti-rabbit secondary antibody for 1 hour. Finally, the slide was rinsed again in phosphate buffer and then mounted on a slide for examination under an Eclipse E600 microscope (Nikon). Images were taken using a QImaging Retiga 2000R Fast1394 camera and analyzed using Q-Capture Pro 7 software (QImaging).

[0265] result:

[0266] Compared to unstressed skin biopsy tissue, application of monosodium glutamate (MSG) stress resulted in a +65% increase in reactive oxygen species (ROS). When skin biopsy tissue was treated with 0.5% (v / v dilution) and 1% (v / v dilution) of the extract of this invention, ROS were significantly reduced by -28% and -29%, respectively, compared to untreated stressed explants. Conventional Pu-erh tea extract tested under the same conditions showed even smaller reductions, at -7% and -18%, respectively. Results in... Figure 6 The values ​​are shown below. Mean ± sem; n = 10. Statistical analysis using Student's t-test: ns: not significant; ***: highly significant.

[0267] in conclusion:

[0268] The Pu-erh tea extract prepared according to the present invention exhibits antioxidant activity against reactive oxygen species. This activity was found to be higher than that obtained using conventional Pu-erh tea extracts.

[0269] Example 7: Rich Cream ) Preparation

[0270] Table 9

[0271]

[0272] Preparation method:

[0273] 1. Homogenize phase A in the main container and begin heating at 75-80℃;

[0274] 2. At 30°C, add phase B and homogenize it while heating;

[0275] 3. In a separate beaker, prepare phase C and heat to 75-80℃ until homogeneous;

[0276] 4. At 75°C, add phase C to the main container and homogenize for 10 minutes;

[0277] 5. Allow the temperature to decrease, and add phase D at 65°C. Mix thoroughly until homogenized for 10 minutes;

[0278] 6. Premixed phase E, which is then added to the main container;

[0279] 7. Add phase E at 60°C. Mix thoroughly until homogenized for 10 minutes;

[0280] 8. Add phase F at 35°C and mix thoroughly;

[0281] 9. Premixed phase G, which is then added to the main container;

[0282] 10. Add phase G at 35°C. Mix thoroughly until homogenized;

[0283] 11. In a separate beaker, prepare phase H: Natrosol TM Sprinkle into water at room temperature and homogenize while heating to 60°C;

[0284] 12. Add phase H at 30°C. Mix thoroughly until homogenized;

[0285] 13. Stop at 25℃.

[0286] Therefore, the composition is a white buttercream with a pH between 4.90 and 5.40 and a viscosity (D0) of 160,000 to 210,000 cps (Brookfield RVT / axis D / 5RPM / 1 min / 25°C).

[0287] Example 8: Anti-aging mask formula

[0288] Table 10

[0289]

[0290] Preparation method:

[0291] 1. At 25°C, phase A in the main container will be homogenized;

[0292] 2. At 25°C, sprinkle in phase B and mix thoroughly until homogeneous;

[0293] 3. At 25°C, add phase C and mix thoroughly until homogeneous;

[0294] 4. Premix phase D in a separate beaker and add it to the main container at 25°C;

[0295] 5. At 25°C, add phase E to the main container and mix thoroughly;

[0296] 6. Premix phase F and add it slowly. Mix thoroughly until homogeneous;

[0297] 7. Premix phase G in a separate beaker and add it to the main container until homogeneous;

[0298] 8. Stop at 25℃.

[0299] Therefore, the composition is in the form of a creamy gel with a shimmering green effect, a pH between 5.30 and 5.80, and a viscosity (D0) of 70,000 to 100,000 cps (Brookfield RVT / axis C / 5RPM / 1 min / 25°C).

[0300] Example 9: Slurry formulation

[0301] Table 11

[0302]

[0303] Preparation method:

[0304] 1. Add water to the main container and begin mixing using the hi-lo propeller blade;

[0305] 2. Add the remaining ingredients one at a time, stirring between each addition.

[0306] Therefore, the composition exists in the form of a smooth, translucent slurry with a pH between 5.75 and 6.25 and a viscosity (D0) of 1,100 to 1,400 cps (Brookfield RVT / shaft 3 / 20 rpm / 25°C / 1 minute).

Claims

1. A method for obtaining an aqueous extract of tea leaves from the species Camellia sinensis, wherein the tea leaves are selected from fresh tea, Pu-erh tea, matcha, or green tea, the method comprising the following steps: a) Contact the tea leaves or tea powder with water, and then optionally crush them; b) Add phytic acid; c) Adjust the pH to a value between 10 and 11; d) Keep the mixture under stirring for at least 1 hour at a temperature between 40°C and 80°C; e) The mixture obtained in d) will be purified to remove residual solid plant material and the filtrate will be collected; f) Adjust the pH to a value between 6 and 8; g) Treat the mixture with powdered activated carbon; h) Filter to remove the activated carbon and collect the filtrate; i) Treat the filtrate with polyvinylpyrrolidone; j) Filter to remove the polyvinylpyrrolidone and collect the filtrate; k) Check the pH of the filtrate and, if necessary, readjust the pH to a value between 6 and 6.

5.

2. The method according to claim 1, characterized in that, In step a), the tea leaves are brought into contact with water at a ratio of 3% to 20% by weight of plant material / water.

3. The method according to claim 1, characterized in that, In step a), the tea leaves are brought into contact with water at a ratio of 3% to 10% by weight of plant material to water.

4. The method according to claim 1, characterized in that, In step b), phytic acid at a concentration between 1 and 10 mM is used for treatment.

5. The method according to claim 1, characterized in that, In step b), phytic acid at a concentration between 1 and 5 mM is used for treatment.

6. The method according to claim 1, characterized in that, In step e), at least one filtration is performed on a filter having a porosity greater than or equal to 30 μm.

7. The method according to claim 1, characterized in that, In step j), sequential filtration is performed using a filter with a porosity greater than 25 μm, followed by a filter with a porosity of 0.8 μm.

8. The method according to claim 1, characterized in that, Following step k), the extract is then diluted in a physiologically acceptable solvent and its pH is adjusted to a value between 5.8 and 6.5 to obtain a diluted extract with a dry weight between 4 and 20 g / kg.

9. The method according to claim 1, characterized in that, Following step k), the extract is then diluted in a physiologically acceptable solvent and its pH is adjusted to a value between 6.0 and 6.5 to obtain a diluted extract with a dry weight between 4 and 20 g / kg.

10. The method according to claim 8, characterized in that, The physiologically acceptable solvent is selected from water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diethylene glycols, cyclic polyols, or any mixture of these solvents.

11. An aqueous crude extract of tea, said tea being obtained from fresh tea, Pu-erh tea, matcha, or green tea, said aqueous crude extract being rich in small RNAs having a length of up to 150 nucleotides, rich in sugars, rich in phenolic compounds, and rich in organic acids, and free of DNA, said aqueous crude extract being obtained by the method according to claim 1, characterized in that... The aqueous crude extract has a dry weight of 5 to 30 g / kg and contains 0.5 to 10 g / kg of sugars; 0.050 to 2 g / kg of organic acids; 0.050 to 5 g / kg of phenolic compounds, including 0 to 200 mg / kg of catechins, 0.020 to 2 g / kg of catechins and 0.020 to 2 g / kg of theanine; and 50 to 350 mg / kg of low molecular weight RNA having a length of up to 150 nucleotides.

12. A diluted aqueous extract of tea leaves, said tea leaves being derived from fresh tea, Pu-erh tea, matcha, or green tea, said diluted aqueous extract being rich in small RNAs having a length of up to 150 nucleotides, rich in sugars, rich in phenolic compounds, and rich in organic acids, and free of DNA, said diluted aqueous extract being obtained by the method according to claim 8, characterized in that... It has a dry weight between 4 and 20 g / kg and, based on the total weight of the extract, contains 0.2 to 5 g / kg of sugars; 0.030 to 1 g / kg of organic acids; 0.030 to 3 g / kg of phenolic compounds, including 0 to 120 mg / kg of catechins, 0.010 to 1 g / kg of catechins and 0.010 to 1 g / kg of theanine; and 10 to 250 mg / kg of low molecular weight RNA having a length of up to 150 nucleotides.

13. A composition comprising an effective amount of at least one diluted extract according to claim 12 as an active ingredient and a physiologically acceptable medium, said extract being selected from extracts of fresh tea, Pu-erh tea, matcha, or green tea, or any mixture thereof.

14. The composition according to claim 13, wherein, The active ingredient is present at a concentration between 0.05% by weight and 5% by weight based on the total weight of the composition, and in a physiologically acceptable medium.

15. The composition according to claim 13, wherein, The active ingredient is present at a concentration between 0.1% by weight and 2.5% by weight of the total weight of the composition, and in a physiologically acceptable medium.

16. The composition according to claim 13, characterized in that... The composition is formulated for topical application to the skin, appendages, and scalp, wherein the appendages are hair, body hair, and nails on the head.

17. The non-therapeutic cosmetic use of the composition of claim 13 for the care of skin, scalp and appendages, wherein the appendages are hair, body hair and nails on the head.

18. The non-therapeutic cosmetic use of the composition according to claim 13, for protecting the skin from external damage and oxidation, for combating signs of skin aging, for brightening the skin, and for improving skin hydration.

19. The composition according to claim 13, used for enhancing light protection.

Images