Application of sea-buckthorn stem-leaf-free anhydrous extract or composition containing extract in maintaining and / or improving skin microcirculation

Abstract

The invention discloses application of a sea-buckthorn stem-leaf-free anhydrous extract or a cosmetic composition containing the extract in the aspects of relieving dark circles and / or under-eye puffiness around eyes and / or maintaining and / or increasing skin color gloss.

Description

Technical Field

[0001] The present invention relates to cosmetic and / or dermatological uses of an anhydrous liquid extract of seabuckthorn stemless leaves or a cosmetic and / or dermatological composition comprising the extract for maintaining and / or improving skin microcirculation. Background Art

[0002] The periocular area, bounded above by the upper eyelid and below by the cheekbones, is a key area of ​​focus for the global cosmetic industry as it is the first area of ​​the face to show signs of fatigue and stress.

[0003] The skin around the eyes is anatomically very thin, approximately 0.5 mm thick (three times thinner than the rest of the face), and is therefore extremely sensitive to environmental stressors (UV rays, pollution, alcohol, stress, lack of sleep). As a result, the infraorbital skin is highly susceptible to free radicals and is prone to inflammation.

[0004] So, dark circles and eye bags are manifestations of localized inflammation of the healthy infraorbital area in response to stress (UV rays, pollution, fatigue, etc.).

[0005] The cutaneous microcirculation, especially in the infraorbital area, consists of two parallel vascular plexuses that are less than 1 mm from the skin surface. The superior vascular plexus, located in the papillary dermis, consists of arterioles and venules, with capillary loops extending perpendicular to the skin surface. Capillaries are the site of oxygen and nutrient exchange within the skin tissue, while venules play a role in the leukocyte diapedesis process. The inferior vascular plexus is located at the dermal-subcutaneous junction. It consists of arteries and veins that originate from the adipose tissue and underlying muscles, which pass through the fascia to form ascending arterioles and descending venules and connect with the superficial vascular plexus.

[0006] Microvascular endothelial cells are the major component of dermal blood vessels. Endothelial cells are interconnected by intercellular junctions, forming a barrier between the blood and the surrounding dermal tissue. The endothelial barrier is a dynamic structure that controls the exchange of fluids and solutes, including plasma proteins, and cells, especially leukocytes. Under basal steady-state conditions, intercellular junctions have low permeability to fluids and solutes. In addition, multiple regulatory processes exist within endothelial cells to maintain endothelial barrier function.

[0007] In the inflammatory state caused by various factors (ultraviolet rays, pollution, fatigue, stress), a variety of pro-inflammatory mediators including TNF-α (tumor necrosis factor-α) act on endothelial cells, causing increased vascular permeability, leading to the opening of intercellular junctions, and ultimately causing leukocyte infiltration, causing red blood cells and other nutrients that make up the plasma to leak out. As a result, the skin microcirculation deteriorates, and the accumulation of white blood cells in the skin tissue leads to the formation of eye bags, while the accumulation of red blood cells in the extracellular space causes the skin around the eyes to show purple pigmentation, which is the characteristic of dark circles.

[0008] Hemoglobin, the main component of red blood cells, degrades and releases pigmented degradation products, such as heme (red pigment), which accumulates in the dermis and epidermis. However, the degradation of free heme also leads to the release and accumulation of iron molecules in skin tissue. Free ferrous ions undergo oxidation and produce ROS (reactive oxygen species), which can lead to increased skin oxidation and inflammation. For this reason, ferrous ion chelation is a complementary strategy to heme degradation and can be used to combat the formation of dark circles, while also helping to improve the radiance of the complexion.

[0009] In the context of research on skin microcirculation, the applicant became interested in seabuckthorn.

[0010] Sea buckthorn (Hippophae rhamnoides L., according to the fourth edition of the 2016 Phylogenetic Classification System of Angiosperms) was described by Linnaeus in 1753 as a thorny, dioecious shrub native to temperate regions of Europe and Asia. The species was introduced to Canada by Russian immigrants in the early 20th century. Recommended for use in controlling soil erosion, the species is now grown throughout Canada and is cultivated for its fruit in Saskatchewan, British Columbia, and Quebec.

[0011] Seabuckthorn is a shrub that grows 1 to 5 meters tall and has lanceolate, deciduous leaves with a silvery underside. Its flowers are petalless and very small, light green in color, and bloom before the leaves emerge. The female plant produces fleshy, berry-like fruits 6 to 8 mm in diameter on the branches, which are tightly packed on the branches. The berries ripen in early autumn, when they are a beautiful orange color with a strong flavor. The ripe fruits remain on the branches throughout the winter.

[0012] The morphological characteristics of sea buckthorn vary significantly due to the widely varying climatic conditions in the distribution area of ​​the species. Varietal selection (ease of harvesting, high oil content, disease resistance, etc.), extensive cross-breeding between subspecies and genetic improvement programs have made it impossible to distinguish between the different subspecies today. Only horticultural cultivars such as "Leikora" or "Orange Energy" have clear names.

[0013] Sea buckthorn is widely distributed in the northern hemisphere continents and has several common names, which are often related to its morphology or the environment in which it grows: for example, "argasse", "grisset", "glossy thorn", "purging thorn", "thorny willow", "false buckthorn", "seaberry", "olive" or "Siberian pineapple". In other languages, it is called "sea buckthorn" (English), "Sanddorn" (German) or "espino amarillo" (Spanish).

[0014] In North America, fruit harvesting takes place in the autumn before the leaves fall, due to the high snowfall in winter. Fruit-bearing branches are cut and frozen, and then mechanically sorted to separate the fruit from the branches and leaves. The leaf byproduct is dried and used as a tea substitute.

[0015] The application of seabuckthorn in food, medicine, gardening and ecology can be traced back to ancient times. The ancient Greeks used its leaves and twigs as feed supplements to accelerate the weight gain of horses and make their fur shiny. Its Latin name "hippophae" is derived from "hippos" (meaning "horse") and "phaos" (meaning "luster").

[0016] Seabuckthorn is still widely used in traditional Chinese medicine (including Tibetan medicine), traditional Japanese medicine, and Ayurvedic medicine (India), especially as a tonic for the treatment of various skin and mucous membrane diseases. It is also used to treat digestive disorders, lung inflammation, and dysmenorrhea or irregular menstruation. In particular, in the Tibet Autonomous Region of China, the seeds, fruits or leaves of seabuckthorn can be used to treat edema, promote tissue regeneration, treat inflammation and bacterial infections; in Turkey, the fruits and leaves of seabuckthorn are used as preservatives for wound healing and ulcer treatment.

[0017] In combination with other plants, it is also used to treat certain cardiovascular diseases (especially problems with platelet aggregation), digestive disorders, indigestion, lung inflammation, and menstrual disorders or dysmenorrhea. In fact, almost all parts of sea buckthorn are used in traditional medicine: in addition to berries and seeds, extracts of the leaves and bark are also prepared.

[0018] Sea buckthorn leaf extract is used to prepare teas, tinctures, and other decoctions, such as for the following applications: Prevent and treat colds, tonsillitis, bronchitis, and acute respiratory viral infections (can boost immunity and fight seasonal illnesses); Prevent and treat hypertension, have sedative and antihypertensive effects, and can be used as a blood pressure stabilizer; Treat cardiovascular system diseases; Treat diabetes (has blood sugar lowering effect); Treat inflammatory diseases of joints; Treat liver diseases (it has a liver-protective effect and can promote liver cells to return to normal in inflammatory diseases); Colitis, gastric ulcers, and duodenal ulcers are treated by using an infusion or powder of the dried leaves.

[0019] French patent document FR 2 943 255 describes a Hippophae rhamnoides seed extract rich in fatty acids and sterols obtained by supercritical carbon dioxide extraction, which can stimulate the activity of 5-α reductase and sebum secretion to treat skin hormone imbalance problems associated with menopause, such as loss of skin luster. Its corresponding biological activity was confirmed by the stimulation of 5-α reductase activity by normal human fibroblasts (NHFs), the synthesis of collagen I / normal human fibroblasts, the synthesis of glycosaminoglycans and hyaluronic acid / normal human fibroblasts, and the synthesis of integrins by normal human keratinocytes.

[0020] French patent document FR 2 971 940 describes obtaining aqueous, alcoholic or glycolic extracts of winter branches of sea buckthorn (Hippophae rhamnoides) for their depigmenting effect on the skin, body hair and hair. "Winter branches" refers only to the leafless stems of sea buckthorn, which contain indole compounds. The biological mechanism responsible for this depigmenting effect is based on the stimulation of the biosynthesis of melanin produced by melanocytes.

[0021] The phytochemical components of sea buckthorn leaf extracts described in the prior art vary in both quality and quantity, depending on the extraction process / extraction operating parameters used. For example, Jayashankar et al. described an extraction method using supercritical carbon dioxide and ethanol as a co-solvent in 2012 and 2014, which was carried out at a pressure of 200 bar and a temperature of 50°C, and finally produced an extract containing isorhamnetin and having anti-inflammatory effects on multiple targets including interleukin-6. Enkhtaivan et al. described a methanol extraction method in their 2017 study, which can produce an extract containing aglycone flavonoids (such as isorhamnetin). Tanwar et al.'s research work in 2018 described a method for extracting sea buckthorn leaves using 70% ethanol, which has a changed polarity compared to anhydrous solvents (such as 96% ethanol). This change in polarity will lead to a change in the solvent's extraction capacity, and ultimately produce a sea buckthorn extract with specific phytochemical components. Sadowska et al. (2018) described the use of a complex method to obtain fractionated extracts from Hippophae rhamnoides leaves, stems, and fruits using 80% methanol and different stages of evaporation and partial solubilization of the concentrate, with specific qualitative and quantitative phytochemical compositions.

[0022] Korean patent documents KR 20140148141 and KR 101121590 describe ethanol extracts obtained by extracting seabuckthorn leaves under specific operating conditions, particularly in terms of temperature and extraction time. Therefore, the phytochemical components thereof differ in quality and quantity depending on the operating parameters applied. For example, the extraction described in Korean patent document KR 101121590 is carried out at room temperature by using C1-C4 alcohols, which enables the extraction of isorhamnetin-3-O-glucoside-7-O-rhamnoside, which is the source of therapeutic effects such as cancer prevention.

[0023] Applicants have surprisingly demonstrated that an extract of Hippophae rhamnoides leaves has an effect on skin microcirculation, making it useful for reducing dark circles and / or eye bags around the eyes, and / or for improving the radiance of the skin tone.

[0024] More precisely, according to a first aspect, the present invention aims at the cosmetic and / or dermatological use of an anhydrous extract of Hippophae rhamnoides leaves or a cosmetic composition comprising said extract on healthy skin, aiming at maintaining and / or improving the skin microcirculation, ultimately reducing dark circles and / or eye bags around the eyes, and / or maintaining and / or improving the radiance of the skin complexion.

[0025] As shown below, the applicant has demonstrated that the extract according to the invention is able to: Reduce the expression of vascular cell adhesion molecule-1 (VCAM-1) adhesion protein, and / or Reduce the adhesion of monocytes to endothelial cell membranes, and Increased transendothelial electrical resistance, and / or Increase the expression of the heme oxygenase-1 (HMOX-1) enzyme, and / or Enhanced chelation of ferrous ions, and / or Increase the expression of antioxidant enzymes glutathione peroxidase 2 (GPX2) and / or glutathione peroxidase 3 (GPX3) and / or thioredoxin (TXN), and / or Enhances free radical scavenging activity.

[0026] The vascular cell adhesion molecule-1 (VCAM-1) adhesion protein is the source of extravasation of leukocytes and erythrocytes into the extracellular space by migrating between endothelial cells. This is facilitated by the disruption of endothelial cell junctions, which results in the formation of paracellular spaces through which cells can pass.

[0027] More specifically, leukocytes circulate in the bloodstream, but in order to reach inflamed tissues, they must cross the endothelial barrier. This rapid migration of blood to sites of infection is essential for tissue repair in response to acute inflammation. Leukocyte extravasation is a highly regulated process that involves a complex interaction between leukocytes and endothelial cells, particularly through selectins, integrins, intercellular adhesion molecules (ICAM1), vascular adhesion molecules (VCAM1), junctional adhesion molecules (JAM-1 / A / C), and platelet endothelial cell adhesion molecule (PECAM1).

[0028] The initial stage of the inflammatory response is the reorganization of the endothelial cell surface to capture circulating leukocytes. The release of inflammatory cytokines stimulates the synthesis of adhesion molecules (P-selectin, E-selectin) on the endothelial cell surface, which promotes weak and transient adhesive interactions between leukocytes and endothelial cells locally. The deposition of chemokines on the endothelial surface subsequently triggers the activation of leukocyte integrins (ICAM-β2), which in turn achieve firm adhesion and arrest of leukocytes through interaction with surface receptors (ICAM1, VCAM1).

[0029] In other words, reducing the expression of vascular cell adhesion molecule-1 (VCAM-1) adhesion protein can reduce the inflammatory response.

[0030] Transendothelial resistance can be used to measure the membrane permeability of endothelial cells, which in turn reflects their barrier function. The higher the resistance, the more effective the barrier function.

[0031] In other words, increasing transendothelial electrical resistance is equivalent to enhancing endothelial barrier function.

[0032] Regarding the heme oxygenase-1 (HMOX-1) gene, it encodes an enzyme that can degrade hemoglobin, which is the main component of red blood cells and is also the root cause of dark circle pigmentation.

[0033] As mentioned earlier, the accumulation of red blood cells in the extracellular space can cause purple pigmentation in the skin around the eyes, which is the typical characteristic of dark circles.

[0034] Hemoglobin, the main component of red blood cells, degrades and releases pigmented degradation products such as heme that accumulate in the dermis and epidermis. Free heme is toxic when not complexed. Therefore, its removal is critical. Heme oxygenase type 1 (HMOX-1) catalyzes the degradation of heme to biliverdin. Biliverdin is then converted to bilirubin by biliverdin reductase A. These catabolites help reduce the appearance of dark circles due to their antioxidant properties.

[0035] In other words, increasing the expression of the heme oxygenase-1 (HMOX-1) gene can increase the amount of heme degradation.

[0036] Finally, increasing the transcriptional expression of oxidative defense genes such as glutathione peroxidase 2 (GPX2) and / or glutathione peroxidase 3 (GPX3) and / or thioredoxin (TXN) can reduce oxidative reactions, especially the oxidation of ferrous ions to generate reactive oxygen species (ROS) that accumulate in the extracellular space due to hemoglobin degradation.

[0037] According to the present invention, the extract contains flavonoids, gallic acid derivatives and triterpenoids.

[0038] in particular: Flavonoids include glycosylated flavonols, the preferred ones being isorhamnetin-3-O-glucoside and narcissin; Gallic acid derivatives include gallic acid and ellagic acid; and Triterpenoids include ursolic acid and maslinic acid.

[0039] According to the present invention: The concentration of glycosylated flavonols in the extract ranged from 20 mg / kg to 5 g / 100 g; The concentration of gallic acid derivatives in the extract ranged from 2 mg / kg to 1 g / 100 g; The concentration of triterpenoids in the extract ranged from 80 mg / kg to 7 g / 100 g.

[0040] According to another aspect, the present invention relates to a water-free extract of stemless leaves of Hippophae rhamnoides containing flavonoids, gallic acid derivatives and triterpenoids.

[0041] in particular: Flavonoids include glycosylated flavonols, the preferred ones being isorhamnetin-3-O-glucoside and narcissin; Gallic acid derivatives include gallic acid and ellagic acid; and Triterpenoids include ursolic acid and maslinic acid.

[0042] According to the present invention: The concentration of glycosylated flavonols in the extract ranged from 20 mg / kg to 5 g / 100 g; The concentration of gallic acid derivatives in the extract ranged from 2 mg / kg to 1 g / 100 g; The concentration of triterpenoids in the extract ranged from 80 mg / kg to 7 g / 100 g.

[0043] In another embodiment, the extract of the invention does not contain isorhamnetin and / or isorhamnetin-7-O-glucoside and / or isorhamnetin-3-O-glucoside-7-O-rhamnoside.

[0044] In actual operation, the extract is obtained by a first step of solid extraction / solvent extraction operation, followed by a second step of solid separation / solvent extraction operation, followed by a third step of recovering a liquid or pasty extract in the presence of an anhydrous solvent (preferably selected from the group consisting of 96° ethanol or a mixture of ethanol and supercritical carbon dioxide).

[0045] The present invention also relates to an extract obtainable by the above method.

[0046] The present invention also relates to a cosmetic and / or dermatological composition comprising the extract.

[0047] In particular, solid extraction / solvent extraction can be carried out using different techniques well known to those skilled in the art, such as impregnation, re-impregnation, digestion, dynamic impregnation, decoction, fluidized bed extraction, microwave-assisted extraction, ultrasound-assisted extraction, countercurrent extraction, percolation, re-percolation, leaching, reduced pressure extraction, dialysis, supercritical fluid extraction, subcritical water extraction, and reflux extraction.

[0048] According to another feature, the solid extraction / solvent extraction is carried out from the stemless leaves in fresh, fresh frozen or dried form, and these leaves can also be in whole, pulverized, ground or freeze-ground form.

[0049] Preferably, the leaves are collected during the summer / autumn fruit picking period.

[0050] According to the present invention, the extraction solvent is an anhydrous solvent (i.e. a solvent containing less than 5% water) which is non-polar or medium polar. According to the present invention, the extraction solvent may thus be selected from the group of medium polar solvents comprising alcohols such as ethanol, glycols such as propylene glycol, 1,3-propanediol and butanediol, glycerol, ethyl acetate, low transition temperature mixtures (LTTM) or anhydrous natural deep eutectic solvents (NaDES). It may also be selected from the range of non-polar solvents, such as supercritical carbon dioxide, vegetable oils, C 8 -C 10 Medium chain triglycerides, fatty acid esters (such as octyldodecyl myristate) or 2-methyltetrahydrofuran. These solvents can be used alone or in combination.

[0051] In a preferred embodiment, ethanol (preferably 96° ethanol) or supercritical CO 2 or a mixture of both was used as the extraction solvent.

[0052] In a preferred embodiment, the extraction solvent is 96° ethanol or ethanol and supercritical CO 2 mixture.

[0053] When the extraction solvent is ethanol and supercritical CO 2 When the mixture is ethanol / supercritical CO 2 The weight ratio is preferably between 1:5 and 1:50, more preferably between 1 / 10 and 1 / 20.

[0054] In practice, the plant / solvent ratio used in the extraction step is between 1 / 99 and 80 / 20, preferably between 2 / 98 and 20 / 80.

[0055] When the extraction solvent is ethanol and supercritical CO 2 When the mixture is prepared, the weight ratio of plant / ethanol is preferably between 10 / 90 and 50 / 50, and the weight ratio of plant / supercritical CO 2 The weight ratio of plant / supercritical CO is preferably between 0.5 / 99.5 and 15 / 85. 2 The weight ratio of ethanol is preferably between 2 / 98 and 10 / 90.

[0056] When the extraction solvent is 96° ethanol, the weight ratio of plant / 96° ethanol is preferably between 1 / 99 and 20 / 80.

[0057] When the extraction solvent is ethanol and supercritical CO 2When the mixture is extracted, the extraction operation is carried out at a temperature between 40°C and 60°C (preferably between 45°C and 55°C) and an absolute pressure between 220 and 350 bars (preferably between 270 and 290 bars), and the duration is between 1 and 5 hours (preferably between 2 and 4 hours).

[0058] When the extraction solvent is 96° ethanol, the extraction operation is carried out at a temperature between 60° C. and 90° C. (preferably between 70° C. and 85° C.) and normal pressure for a period of 1 to 5 hours (preferably between 2 to 4 hours).

[0059] According to the invention, the solid extraction / solvent extraction operation is followed by a solid separation / solvent extraction step and then a step of recovering the liquid or pasty phase containing the active substance. This separation can be carried out by any technique known to those skilled in the art, in particular draining, pressing, dewatering, decantation (gravity decantation or centrifugal decantation) or filtration.

[0060] According to another embodiment, the step of recovering the liquid or pasty phase is followed by a concentration step which allows obtaining a concentrated liquid to pasty product according to the concentration factor. In actual operation, the concentration step is carried out by atmospheric evaporation, reduced pressure evaporation or membrane separation.

[0061] After or simultaneously with the concentration step, the extract may be dissolved in a recovery solvent selected from the group consisting of glycols, glycerol, ethanol, anhydrous low transition temperature mixtures (LTTMs), anhydrous natural deep eutectic solvents (NaDESs), medium chain triglycerides, fatty acid esters and vegetable oils.

[0062] Preferably, When the extraction solvent is ethanol and supercritical CO 2 When the mixture is octyldodecyl myristic acid, the recovery solvent is selected from the group consisting of octyldodecyl myristic acid, caprylic / capric triglyceride, vegetable oil and a mixture thereof; more preferably octyldodecyl myristic acid; When the extraction solvent is 96° ethanol, the recovery solvent is selected from the group consisting of 1,3-propylene glycol, propylene glycol, butylene glycol, anhydrous low transition temperature mixtures (LTTMs) and mixtures thereof; preferably 1,3-propylene glycol.

[0063] The extraction solvent is supercritical CO 2 In this embodiment of a mixture of ethanol and ethanol, the ethanol is evaporated and the concentrated plant extract is then dissolved in a recovery solvent, which is preferably octyldodecyl myristate, and its weight proportion is preferably 0.1% to 5%, more preferably between 0.3% and 1%, and in practical operation it is about 0.5% of the weight of the dry plant extract.

[0064] In the embodiment where the extraction solvent is 96° ethanol, the 96° ethanol is evaporated and the plant extract is then dissolved in a recovery solvent, which is preferably a glycol, more preferably 1,3-propylene glycol, preferably in a weight proportion of 0.5% to 5%, more preferably between 1% and 3%, and more preferably about 1.5% by weight of the dry plant extract.

[0065] Finally, for the purpose of aseptic or non-aseptic packaging, the step of dissolving the extract may be followed by one or more filtration steps. Before the final filtration step, additives such as preservatives and antioxidants known to those skilled in the art may be added to the liquid extract to ensure its stability.

[0066] Depending on the nature of the solvent used, it is possible that the extract obtained is too dark in color. In this case, the method for obtaining the extract comprises an additional step of decolorizing the extract, preferably by adsorption, more preferably using activated carbon or decolorizing earth.

[0067] The extract is therefore intended for use in the cosmetic and / or dermatological fields, more preferably in the cosmetic field, in a form suitable for topical application.

[0068] Therefore, the cosmetic and / or dermatological extract or composition containing the extract is in a form suitable for topical use in the cosmetic and / or dermatological fields for reducing dark circles and / or puffiness around the eyes, and / or maintaining and / or improving the radiance of the skin's complexion by maintaining and / or improving the microcirculation of healthy skin.

[0069] The extracts obtainable (more preferably directly) by one of the above-described methods are suitable for use in the cosmetic field, in particular in the form of compositions for use in the cosmetic and / or dermatological fields.

[0070] In practical applications, the extract accounts for 0.1% to 10% by weight of the cosmetic and / or dermatological composition, more preferably 0.5% to 5%.

[0071] The cosmetic and / or dermatological composition according to the invention may be in any form of preparation usually used for topical application to the skin, for example in anhydrous form, oil-in-water emulsion, water-in-oil emulsion, multiple emulsion, silicone emulsion, microemulsion, nanoemulsion, gel, aqueous solution or hydroalcoholic solution form.

[0072] The composition may be more or less fluid, and may be in the form of a white or colored cream, ointment, milky lotion, serum or gel.

[0073] The cosmetic and / or dermatological compositions may contain excipients commonly used in the cosmetic and / or dermatological fields, such as oils, cleansing and / or conditioning surfactants, emulsifiers and co-emulsifiers, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, exfoliants, fragrances, fillers, hydrophilic and lipophilic sunscreens, colorants, neutralizers, penetration enhancers, and polymers. Those skilled in the art are very familiar with these types of excipients.

[0074] In practical applications, the amounts of these various excipients are those conventionally used in the relevant fields under consideration, and the sum of the excipients preferably accounts for 0.01% to 30% of the total weight of the composition.

[0075] Suitable oils and fats that can be cited include mineral oils, oils of animal origin (e.g. lanolin), vegetable oils, synthetic oils (e.g. isopropyl myristate, octyldodecyl alcohol, isostearyl isostearate, decyl oleate, isopropyl palmitate) and silicone oils (cyclomethicone, polydimethylsiloxane). Oils and fats that can be used also include fatty alcohols, fatty acids, waxes and gums, especially silicone elastomers.

[0076] Examples of suitable cleansing and / or conditioning surfactants which may be mentioned are nonionic, anionic, cationic or amphoteric surfactants and mixtures thereof, for example alkyl sulfates, alkyl ether sulfates (e.g. sodium laureth sulfate), alkyl betaines (e.g. cocamidopropyl betaine) or quaternary ammonium salts.

[0077] Examples of suitable emulsifiers and co-emulsifiers include polyglycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid sorbitan esters, fatty alcohol polyethylene glycol ethers, glycerol fatty acid esters, alkyl sulfates, alkyl ether sulfates, alkyl phosphates, alkyl polyglucosides, alkyl polypentosides, and dimethicone copolyols.

[0078] Examples of suitable hydrophilic gelling agents which may be mentioned are carboxyvinyl polymers, acrylic acid copolymers (carbomers), such as acrylate / alkyl acrylate copolymers, polyacrylamides, polysaccharides (such as xanthan gum, guar gum), natural gums (such as cellulose gum and its derivatives), starch and its derivatives, clays and 2-acrylamido-2-methylpropane acid copolymers.

[0079] Examples of suitable lipophilic gelling agents which may be mentioned are modified clays (such as bentonite), metal salts of fatty acids, hydrophobic silica and ethylcellulose.

[0080] Examples of suitable preservatives which may be mentioned are benzoic acid, sorbic acid, propionic acid, salicylic acid, dehydroacetic acid and its salts, benzyl alcohol, ethylhexylglycerin, parabens (parabens) and their salts and esters, triclosan, imidazolidinyl urea, 5-phenoxyethanol, DMDM ​​hydantoin, diazolidinyl urea and chlorphenesin.

[0081] Examples of suitable antioxidants which may be mentioned are chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and its salts, sodium metabisulfite, salicylic acid, ascorbic acid and citric acid and their salts, sodium tartrate, sodium gluconate, carotenoids and tocopherols.

[0082] Examples of solvents that can be used in the cosmetic composition (other than the extraction solvent) include water, ethanol, glycerin, propylene glycol, 1,3-propanediol, butylene glycol, and sorbitol.

[0083] As examples of suitable exfoliants there may be mentioned chemical exfoliants, such as alpha-hydroxy acids (AHA), and physical exfoliants, such as natural or synthetic powders.

[0084] Examples of suitable fillers which may be mentioned are talc, kaolin, mica, sericite, magnesium carbonate, aluminum silicate, magnesium silicate and organic powders (eg nylon powder).

[0085] Examples of suitable colorants which may be mentioned are the lipophilic colorants, hydrophilic colorants, pigments and pearlescent agents customarily used in cosmetic or dermatological compositions, and mixtures thereof.

[0086] Examples of suitable neutralizing agents which may be mentioned are sodium hydroxide, triethanolamine, aminomethyl propanol and potassium hydroxide.

[0087] Examples of suitable penetration enhancers which may be mentioned are alcohols and glycols (eg ethanol, propylene glycol), ethoxydiglycol, alcohols and fatty acids (eg oleic acid), fatty acid esters and dimethyl isosorbide.

[0088] The composition of the present invention may contain other active ingredients in addition to the extracts of the present invention. Examples of suitable active ingredients that may be cited include free radical scavengers, or more generally antioxidants, whitening agents, pigments, emollients, moisturizers, anti-seborrheic agents, anti-inflammatory agents, anti-acne agents, keratolytic and / or desquamative agents, anti-wrinkle agents and toning agents, drainage agents, anti-irritants, soothing agents, vitamins and mixtures thereof, oil control agents, anti-aging active ingredients (such as retinol), healing agents, preservatives and essential oils.

[0089] The present invention and its advantages will become more apparent from the following embodiments given with reference to the accompanying drawings: BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 The ferrous ions (Fe) in the solution containing 1C extract (0.1% to 2% relative to the blank solution (NT solution)) were measured by colorimetry. 2+ ) Photographs of the chelation status being assessed. DETAILED DESCRIPTION

[0091] Example 1: Preparation and Phytochemistry of Supercritical CO and Ethanol Co-solvent Extracts (Extracts 1, 1A, 1B, 1C) analyze Grind the dried seabuckthorn leaves. Add the ground dried seabuckthorn leaves and 96° ethanol in a weight ratio of 1:2 into a supercritical CO 2 extractor. By making supercritical CO 2 A total of 30 kg CO 2 / kg of material is continuously diffused into the extractor for solid extraction / solvent extraction, ethanol / supercritical CO 2 The ratio is 1:15, or plant / solvent (supercritical CO 2 The ratio of CO and ethanol is 3:97, the temperature is set to 50 °C, and the pressure is 285 bar. At the outlet of the extractor, the fluid expands and the gaseous CO 2 The concentrated ethanol extract was collected in liquid form after being released. The ethanol was then evaporated under reduced pressure of 70°C and 50 mbar to obtain a pure concentrated plant extract (labeled as Extract 1). The extract 1 thus obtained was a green-brown paste.

[0092] This concentrated extract 1 can then be dissolved in different solvents as desired.

[0093] Extract 1A: In order to make this concentrated lipophilic extract bioavailable in an aqueous medium for bioefficacy testing, Extract 1 was completely dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 g / 100 mL and stirred at 60° C. for 15 minutes (obtaining Extract 1A). The Extract 1A thus obtained was a green-brown liquid.

[0094] Extract 1B: In order to obtain an oily liquid form that is easy to formulate into cosmetics, Extract 1 was completely dissolved in octyldodecyl myristate (MOD) at a concentration of 0.5 g / 100 g by mechanical stirring at 60°C for 1 hour, and then filtered through a cellulose filter plate with a cutoff threshold between 1.5 and 3 μm (obtaining Extract 1B). The Extract 1B thus obtained was a clear green liquid.

[0095] Extract 1C: In order to obtain an oily liquid extract containing no or almost no green chlorophyll pigment, Extract 1B can be subjected to an additional decolorization step using activated carbon. Under mechanical stirring, 0.8% of powdered activated carbon is added to Extract 1B and stirred at 50°C for 1 hour. The mixture is then filtered through a cellulose filter pad with a cutoff threshold between 2.5 and 4.2 μm (obtaining Extract 1C). The Extract 1C thus obtained is a clear yellow liquid.

[0096] For all extracts, triterpenoids were studied and quantified by high performance liquid chromatography-charged aerosol detector (HPLC-CAD, CAD: charged aerosol detector). The presence of 3 major triterpenoid acids was detected: ursolic acid, oleanolic acid and maslinic acid. These 3 triterpenoid acids were quantified against a calibration curve obtained with an ursolic acid molecular standard. The concentration of these 3 triterpenoid acids in extract 1 was 5%.

[0097] The complete results are shown in Table 1. [Table 1] Table 1: Triterpenic acid components of extracts 1, 1A, 1B, and 1C

[0098] The phenolic compounds were studied and quantified by ultra-high performance liquid chromatography-ultraviolet detection (UHPLC-UV) only for extracts 1 and 1A. The presence of octyldodecyl myristate (MOD) did not allow the analysis of extracts 1B and 1C. The presence of flavonoids such as glycosylated flavonols (7 molecules) and gallic acid derivatives (3 molecules) were detected in these extracts. Their concentrations were determined against calibration curves established with narcissin molecular standards (for glycosylated flavonols) and with ellagic acid (for gallic acid derivatives). The concentrations in extracts 1B and 1C were determined by calculation according to the dilution factor. The concentration in extract 1 was about 1%.

[0099] All the results are shown in Table 2. [Table 2] Table 2: Phenolic compounds of extracts 1, 1A, 1B, and 1C

[0100] Example 2: Preparation of ethanol extracts (Extracts 2, 2A, 2B, 2C) and phytochemical analysisThe dried sea buckthorn leaves were ground. The ground dried sea buckthorn leaves and 96° ethanol were added to the extractor in a weight ratio of 1:9. Solid-liquid extraction was carried out for 3 hours under normal pressure and at the reflux temperature of the mixture by continuous mechanical stirring. At the end of the extraction, solid-liquid separation was achieved by filtering on canvas. The crude liquid extract was then filtered through a cellulose filter plate to a precision of 0.8-0.5 μm, followed by dechlorophyllization by adding 0.2% activated carbon and mechanical stirring for 1 hour at normal temperature. Thereafter, the ethanol was evaporated under reduced pressure of 80°C and 50 mbar to obtain a concentrated pure plant extract (labeled as extract 2). The extract 2 thus obtained was a brown paste.

[0101] This concentrated extract 2 can then be dissolved in different solvents as desired.

[0102] Extracts 2A and 2B: In order to make this concentrated extract with medium polarity bioavailable in aqueous media for bioefficacy testing, Extract 2 was completely dissolved in dimethyl sulfoxide (DMSO) and stirred at 60°C for 15 minutes at two concentrations: 10 g / 100 mL (Extract 2A) and 1.4 g / 100 mL (Extract 2B). The extracts thus obtained were brown liquid (Extract 2A) and clear amber liquid (Extract 2B), respectively.

[0103] Extract 2C: In order to obtain an anhydrous liquid form for easy use by cosmetic formulators, Extract 2 was completely dissolved in 1,3-propylene glycol (PDO) at a concentration of 1.4 g / 100 g by mechanical stirring at 60°C for 1 hour, and then filtered through a cellulose filter pad to a final retention threshold of between 0.3-0.1 μm (Extract 2C). The extract 2C thus obtained was a clear amber-yellow liquid.

[0104] The phytochemical properties of the extracts are shown in Tables 3 and 4.

[0105] The phenolic compounds in the extracts were studied and quantified by ultra-high performance liquid chromatography-ultraviolet detection (UHPLC-UV). Similar to extract 1, the presence of flavonoids of the glycosylated flavonol type (7 molecules) and gallic acid derivatives (3 molecules) were detected in extract 2. Their concentrations were determined against calibration curves established with narcissin molecular standards (for glycosylated flavonols) and with ellagic acid (for gallic acid derivatives). The total concentration of phenolic compounds identified in extract 2 was about 4%.

[0106] All the results are shown in Table 3. [Table 3] Table 3: Phenolic compounds of extracts 2, 2A, 2B, and 2C

[0107] For extract 2, triterpenoids were studied and quantified by high performance liquid chromatography-charged aerosol detector (HPLC-CAD, CAD: charged aerosol detector). As with extract 1, the presence of three major triterpenoid acids was detected, namely ursolic acid, oleanolic acid and hawthorn acid. These three triterpenoid acids were quantified against a calibration curve obtained with an ursolic acid molecular standard. The concentrations in extracts 2A, 2B and 2C were determined by calculation based on the dilution factor. The concentration of these three triterpenoid acids in extract 2 was 1%.

[0108] All the results are shown in Table 4. [Table 4] Table 4: Triterpene acid components in extracts 2, 2A, 2B, and 2C

[0109] Example 3: Effects of extracts 1A and 2A according to the present invention on the synthesis of vascular cell adhesion molecule-1 (VCAM-1) in two-dimensional culture of human skin microvascular endothelial cells under inflammatory conditions.

[0110] Principle of the method: In situ immunolabeling combined with image analysis was used to examine the synthesis of vascular cell adhesion molecule-1 (VCAM-1) in monolayer cultures of skin microvascular endothelial cells under inflammatory conditions and with or without treatment with extract 1A or extract 2A.

[0111] Experimental protocol: Human skin microvascular endothelial cells were pretreated with extract 1A at a final concentration of 0.025% and extract 2A at a final concentration of 0.07% for 18 hours. Endothelial cells were then stimulated with tumor necrosis factor-α (TNF-α) at ​​a concentration of 0.2 ng / mL and simultaneously treated with extract 1A at a final concentration of 0.025% and extract 2A at a final concentration of 0.07% for 6 hours. Untreated cells (NT) were used as controls. Cells were washed with phosphate-buffered saline, fixed, permeabilized with 0.1% Triton for 5 minutes, and saturated with 1% bovine serum albumin (BSA) for 1 hour. Cells were incubated with primary antibody (anti-vascular cell adhesion molecule-1 antibody) overnight, washed with phosphate-buffered saline (PBS) buffer, and then incubated with secondary antibody conjugated to the fluorescent dye Alexa 594 for 1 hour. Fluorescence was read on a Cytation 5 imaging spectrophotometer (Biotek, USA) using appropriate filters. Fluorescence measurements by image analysis were performed using conventional acquisition parameters.

[0112] This study was based on 3 independent experiments (n=3). Statistical analysis was performed using the Mann-Whitney nonparametric t-test to compare the synthesis of vascular cell adhesion molecule-1 (VCAM-1) in untreated (NT) skin microvascular endothelial cell cultures with that in endothelial cells treated with extract 1A or extract 2A.

[0113] result

[0114] The results are expressed as a percentage relative to untreated (NT) cells and are shown in Tables 5 and 6. Values ​​are expressed as the mean ± standard deviation of 3 experiments (n=3). [Table 5] Table 5: Synthesis of vascular cell adhesion molecule-1 in human skin microvascular endothelial cells treated with extract 1A at a concentration of 0.025% under inflammatory conditions (percentage relative to untreated cells). [Table 6] Table 6: Synthesis of VCAM-1 (percentage relative to untreated cells) in human skin microvascular endothelial cells treated with extract 2A at a concentration of 0.07% under inflammatory conditions.

[0115] Compared with untreated cells, the extracts of the present invention can significantly reduce the protein synthesis of vascular cell adhesion molecule-1 (VCAM-1) in skin microvascular endothelial cells under inflammatory conditions (extract 1A reduced by 82% relative to the untreated group, and extract 2A reduced by 55% relative to the untreated group). The extraction solvent alone does not lead to a decrease in the synthesis of vascular cell adhesion molecule-1 (relevant data not listed). Extracts 1A and 2A can reduce vascular permeability under inflammatory conditions, that is, the leakage of white blood cells and red blood cells into the extracellular space, and therefore, extracts 1A and 2A can improve endothelial barrier function.

[0116] Example 4: Effects of the extract 1A and extract 2A of the present invention on reducing the adhesion of adult mononuclear blood cells (MNC) to human skin microvascular endothelial cells in two-dimensional monolayer culture under inflammatory conditions.

[0117] Principle of the method: In situ fluorescence labeling combined with image analysis was used to measure the adhesion of mononuclear blood cells to the membrane of skin microvascular endothelial cells in monolayer cultures treated with or without extract 1A or extract 2A under inflammatory conditions.

[0118] Experimental protocol: Human skin microvascular endothelial cells were pretreated with extract 1A or extract 2A at a final concentration of 0.025% and 0.05%, respectively, for 18 hours. Endothelial cells were subsequently stimulated with 0.2 ng / mL of tumor necrosis factor-α (TNF-α) and simultaneously treated with extract 1A or extract 2A at a final concentration of 0.025% and 0.05%, respectively, for 4 hours. Untreated cells (NT) served as controls. In parallel, MNCs were labeled with calcein for 1 hour before contacting them with previously stimulated and treated endothelial cells. After 1 hour of MNC adhesion, the endothelial cell monolayer was rinsed with phosphate-buffered saline and the number of adherent MNCs was determined by reading fluorescence on a Cytation 5 imaging spectrophotometer (Biotek) with appropriate filters. Fluorescence measurements were performed by image analysis using conventional acquisition parameters.

[0119] This study was based on 3 independent experiments (n=3). The Mann-Whitney nonparametric t-test statistical test method was used to compare the adhesion of MNC to untreated skin microvascular endothelial cell membranes and to endothelial cells treated with Extract 1A or Extract 2A.

[0120] result:

[0121] The results are presented as percentages relative to untreated cells (NT cells), as shown in Tables 7 and 8. The values ​​are expressed as the mean ± standard deviation of 3 experiments (n=3). [Table 7] Table 7: Adhesion of mononuclear blood cells (MNC) in human skin microvascular endothelial cells treated with extract 1A at a concentration of 0.025% (percentage relative to untreated cells) [Table 8] Table 8: Adhesion of mononuclear blood cells (MNC) in human skin microvascular endothelial cells treated with extract 2A at a concentration of 0.05% (percentage relative to untreated cells)

[0122] Compared with the untreated condition, the extracts of the present invention can significantly reduce the adhesion of mononuclear blood cells (MNC) to the membrane of skin microvascular endothelial cells in monolayer culture under inflammatory conditions (extract 1A reduced by 35% compared to the untreated group (NT), and extract 2A reduced by 43% compared to the untreated group (NT). The extraction solvent alone does not lead to a decrease in the number of adherent mononuclear blood cells (relevant data not shown). Extracts 1A and 2A can reduce vascular permeability under inflammatory conditions, that is, the leakage of white blood cells and red blood cells into the extracellular space, so they can promote the improvement of endothelial barrier function.

[0123] Example 5: Effects of the extract 1A and extract 2A of the present invention on the increase of transendothelial electrical resistance (TEER) under inflammatory conditions in two-dimensional culture of human skin microvascular endothelial cells.

[0124] Principle of the method: The in vitro membrane permeability of skin microvascular endothelial cell monolayers treated with or without extract 1A or extract 2A under inflammatory conditions was measured using the transendothelial electrical resistance (TEER) technique. The transendothelial electrical resistance (TEER) is a quantitative technique that measures the integrity of the endothelial cell barrier with the help of two electrodes placed in two chambers: an electric current passes through the cell monolayer and the resistance of the cell barrier is measured in ohms. Thus, under non-inflammatory conditions, the cells adhere to each other and the resistance of the cells is high (enhanced endothelial barrier function), while under inflammatory conditions, the cells separate from each other and the resistance of the cells is low (impaired endothelial barrier function).

[0125] Experimental protocol: Human skin microvascular endothelial cells were pretreated with extract 1A or extract 2A at a final concentration of 0.01% and 0.07% for 24 hours, respectively. Endothelial cells were then stimulated with 0.2 ng / mL of tumor necrosis factor-α (TNF-α) and simultaneously treated with extract 1A or extract 2A at a final concentration of 0.01% and 0.07% for 24 hours, respectively. Untreated cells (NT) were used as controls. The integrity of the endothelial barrier was assessed by measuring the transendothelial resistance (in ohms) of the cells in real time using a calibration system consisting of two electrodes.

[0126] This study was based on 3 independent experiments (n=3). Statistical analysis of transendothelial electrical resistance between untreated skin microvascular endothelial cells (NT) and endothelial cells treated with extract 1A or extract 2A was performed using the Mann-Whitney nonparametric t-test.

[0127] result: The results are presented as percentages relative to untreated cells (NT cells), as shown in Tables 9 and 10. The values ​​are expressed as the mean ± standard deviation of 3 experiments (n=3). [Table 9] Table 9: Transendothelial electrical resistance measured in human skin microvascular endothelial cells treated with extract 1A at a concentration of 0.01% (percentage relative to untreated cells) [Table 10] Table 10: Transendothelial electrical resistance measured in human skin microvascular endothelial cells treated with extract 2A at a concentration of 0.07% (percentage relative to untreated cells)

[0128] Compared with the untreated condition, the extracts of the present invention can significantly increase the transendothelial resistance in the monolayer culture under inflammatory conditions (extract 1A increased by 111% compared to the untreated group (NT), and extract 2A increased by 160% compared to the untreated group (NT). The extraction solvent alone does not increase the transendothelial resistance (relevant data not shown). Extracts 1A and 2A can enhance the endothelial barrier function under inflammatory conditions, so they can promote the reduction of vascular permeability.

[0129] Example 6: Effect of Extract 1A on increasing transcriptome expression of the heme oxygenase-1 (HMOX-1) gene encoding a hemoglobin-degrading enzyme in two-dimensional culture of human skin fibroblasts.

[0130] Principle of the method: Real-time polymerase chain reaction (PCR) was used to measure the expression of heme oxygenase-1 (HMOX-1) gene in human skin fibroblast monolayer cultures treated with or without extract 1A.

[0131] Experimental protocol: Human skin fibroblasts from the eyelids and eye bags were treated with extract 1A at a final concentration of 0.025% for 6 hours. Untreated cells (NT) were used as controls. The cells were rinsed with phosphate-buffered saline and then lysed to extract RNA. After RNA quality was tested and verified, the relative expression of the heme oxygenase-1 (HMOX-1) gene was evaluated by real-time polymerase chain reaction.

[0132] This study was based on 3 independent experiments (n=3). Statistical analysis was performed using the Mann-Whitney nonparametric t-test to compare the expression of the heme oxygenase-1 (HMOX-1) gene in untreated human skin fibroblast cultures and skin fibroblasts treated with Extract 1A.

[0133] result: The expression of heme oxygenase-1 (HMOX-1) gene in human skin fibroblasts treated with or without extract 1A was quantitatively analyzed. The results are presented as a percentage relative to untreated cells (NT cells), as shown in Table 11. The values ​​are expressed as the mean ± standard deviation of 3 experiments (n=3). [Table 11] Table 11: Expression of heme oxygenase-1 (HMOX-1) gene in human skin fibroblasts treated with extract 1A at a concentration of 0.025% (percentage relative to untreated cells)

[0134] Extract 1A significantly increased the expression of the heme oxygenase-1 (HMOX-1) gene in human skin fibroblasts compared to the untreated group (119% increase compared to the untreated group (NT)). The extraction solvent alone did not increase the expression of the heme oxygenase-1 gene (data not shown). Therefore, Extract 1A promotes the degradation of hemoglobin present in red blood cells, which is the root cause of the characteristic pigmentation of dark circles.

[0135] Example 7: Effect of extract 1C on ferrous ions (Fe 2+ )The effect of chelation.

[0136] Principle of the method: In an in vitro test tube, ferrozine was used to evaluate the chelating capacity of extract 1C. Ferrozine reacts with ferrous ions present in the reaction medium to form a dark purple ferrozine-ferrous ion. 2+ ) complex. Quantitative analysis of the complex by spectrophotometry at 562 nm in a medium with a known iron concentration provides information on the amount of unchelated iron and, therefore, the ability of extract 1C to chelate it. Thus, the lighter the color of the solution containing extract 1C, the greater the chelating ability of the tested extract.

[0137] Experimental protocol: Extract 1C was mixed with ferric chloride (FeCl 2 ) solution for 10 minutes. To evaluate the chelating ability of the extract, o-phenanthroline was then added to the mixture over 10 minutes. The absorbance of the solution was read at 562 nanometers using a spectrophotometer. The lower the absorbance of the solution, the more effective the extract 1C is for ferrous ions (Fe 2+ ) has a stronger chelating ability.

[0138] result: The results of each solution containing 0.1% to 2% of extract 1C compared with the untreated (NT) solution are as follows Figure 1 shown. Extract 1C obtained according to the present invention induces chelation of ferrous ions in a dose-effect relationship. The extraction solvent alone does not induce chelation of ferrous ions (relevant data not shown). Therefore, extract 1C can reduce the characteristic pigmentation of dark circles and improve the glossiness of the complexion by chelating ferrous ions accumulated in the extracellular space due to the degradation of hemoglobin, the main component of red blood cells.

[0139] Example 8: The effect of free radical scavenging activity of extract 2B was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH) in vitro.

[0140] Principle of the method: The antioxidant capacity of extract 2B was evaluated by in vitro test tube colorimetry to detect 1,1-diphenyl-2-picrylhydrazyl (DPPH, referring to the name of the reagent). DPPH is a very stable nitrogen free radical characterized by an intense purple color that fades when antioxidant molecules are present to reduce it. The reducing capacity of the extract was quantitatively analyzed by spectrophotometry to measure the change in absorbance after the reaction of extract 2B with DPPH solution.

[0141] Experimental protocol: Extract 2B was exposed to a solution containing DPPH at a concentration range of 0.1% to 2% for 30 minutes. The absorbance of the solution was read at 518 nm using a spectrophotometer. The lower the absorbance of the solution, the stronger the free radical scavenging activity of Extract 2B. This study was based on 3 independent experiments (n=3). Statistical analysis was performed using the Mann-Whitney nonparametric t-test to compare the free radical scavenging activity of the untreated (NT) condition with that of extract 2B.

[0142] result: The results are presented as a percentage relative to the untreated (NT) condition and are detailed in Table 12. The values ​​are expressed as the mean ± SD of 3 experiments (n = 3). [Table 12] Table 12: Anti-radical activity of extract 2B tested in the concentration range of 0.1% to 2% (percentage relative to untreated condition)

[0143] Extract 2B obtained according to the present invention promotes free radical scavenging activity in a dose-effect relationship. The extraction solvent alone does not increase free radical scavenging activity (relevant data not given). Extract 2B has antioxidant capacity, which can reduce the oxidation of iron accumulated in the extracellular space, which is the root cause of the characteristic pigmentation of dark circles and changes in complexion caused by the degradation of hemoglobin, the main component of red blood cells.

[0144] Example 9: Effects of Extract 2C on the transcriptome expression of oxidative defense genes in two-dimensional culture of normal human primary keratinocytes.

[0145] Principle of the method: The principle of the method is the same as that of Example 5, but the genes of interest here are different, namely GPX2, GPX3 and TXN genes encoding glutathione peroxidase 2, glutathione peroxidase 3 and thioredoxin, respectively.

[0146] Experimental protocol:

[0147] Normal human primary keratinocytes were treated with extract 2C at concentrations ranging from 0.1% to 0.5% for 24 hours. Untreated cells (NT) were used as controls. Cells were rinsed with phosphate-buffered saline and then lysed to extract RNA. After RNA quality was tested and verified, the relative expression of GPX2, GPX3, and TXN genes was evaluated by real-time polymerase chain reaction (PCR). This study was based on 3 independent experiments (n=3). Statistical analysis was performed using the Mann-Whitney nonparametric t-test to compare the free radical scavenging activity of the untreated (NT) condition with that of extract 2C.

[0148] result: The results are presented as percentages relative to untreated cells (NT cells) and are listed in Table 13 (GPX2 gene), Table 14 (GPX3 gene) and Table 15 (TXN gene), respectively. The values ​​are expressed as the mean ± standard deviation of 3 independent experiments (n=3). [Table 13] Table 13: Expression of glutathione peroxidase 2 (GPX2) gene in two-dimensional culture of normal human primary keratinocytes treated with extract 2C at concentrations ranging from 0.1% to 0.5% (percentage relative to untreated) [Table 14] Table 14: Expression of glutathione peroxidase 3 (GPX3) gene in two-dimensional culture of normal human primary keratinocytes treated with extract 2C at concentrations ranging from 0.1% to 0.5% (percentage relative to untreated) [Table 15] Table 15: Thioredoxin in 2D cultures of normal human primary keratinocytes treated with extract 2C at concentrations ranging from 0.1% to 0.5% (TXN) gene expression (percentage relative to untreated conditions)

[0149] Extract 2C obtained according to the present invention significantly increases the transcriptome expression of glutathione peroxidase 2 (GPX2) gene, glutathione peroxidase 3 (GPX3) gene and thioredoxin (TXN) gene in normal human primary keratinocyte two-dimensional culture in a dose-effect relationship. The extraction solvent alone does not increase the expression of GPX2, GPX3 and TXN genes (relevant data not shown). Extract 2C has antioxidant capacity, which can reduce the oxidation of iron accumulated in the extracellular space, and the oxidation of iron is the root cause of the characteristic pigmentation of dark circles and changes in complexion gloss caused by the degradation of hemoglobin, the main component of red blood cells.

Claims

1. Use of an anhydrous extract of stemless leaves of Hippophae rhamnoides or a cosmetic composition comprising the extract for alleviating dark circles and / or puffiness around the eyes and / or for maintaining and / or increasing the radiance of skin complexion.

2. The use according to claim 1, characterized in that The extract contains flavonoids, gallic acid derivatives and triterpenoids.

3. The use according to claim 2, characterized in that: The flavonoids include glycosylated flavonols, preferably isorhamnetin-3-O-glucoside and narcissin; The gallic acid derivative comprises gallic acid and ellagic acid; and The triterpenoid compounds include ursolic acid and maslinic acid.

4. The use according to claim 3, characterized in that: The concentration of glycosylated flavonols in the extract is between 20 mg / kg and 5 g / 100 g; The concentration of gallic acid derivatives in the extract is between 2 mg / kg and 1 g / 100 g; and The concentration of triterpenoids in the extract is between 80 mg / kg and 7 g / 100 g.

5. A water-free extract of stemless leaves of seabuckthorn, comprising flavonoids, gallic acid derivatives and triterpenoids.

6. The extract according to claim 5, characterized in that: The flavonoids include glycosylated flavonols, preferably isorhamnetin-3-O-glucoside and narcissin; The gallic acid derivative comprises gallic acid and ellagic acid; and The triterpenoid compounds include ursolic acid and maslinic acid.

7. The extract according to claim 6, characterized in that: The concentration of glycosylated flavonols in the extract is between 20 mg / kg and 5 g / 100 g; The concentration of gallic acid derivatives in the extract is between 2 mg / kg and 1 g / 100 g; and The concentration of triterpenoids in the extract is between 80 mg / kg and 7 g / 100 g.

8. The extract according to any one of claims 5 to 7, characterized in that It can be obtained by a process comprising a first solid extraction / solvent extraction step, followed by a second solid separation / solvent extraction step, and then a third step of recovering an extract in a liquid or paste state in the presence of an anhydrous solvent selected from a mixture of 96° ethanol or ethanol and supercritical carbon dioxide.

9. The extract according to claim 8, characterized in that When the extraction solvent is a mixture of ethanol and supercritical carbon dioxide, the extraction is carried out at a temperature in the range of 40 to 60° C., preferably in the range of 45 to 55° C., at an absolute pressure of 220 to 350 bar, preferably between 270 and 290 bar, for a duration of between 1 and 5 hours, preferably between 2 and 4 hours.

10. The extract according to claim 8 or 9, characterized in that When the extraction solvent is a mixture of ethanol and supercritical carbon dioxide, the plant / ethanol weight ratio is between 10 / 90 and 50 / 50, the plant / supercritical carbon dioxide weight ratio is between 0.5 / 99.5 and 15 / 85, and the plant / supercritical carbon dioxide-ethanol weight ratio is preferably between 2 / 98 and 10 / 90.

11. The extract according to any one of claims 8 to 10, characterized in that When the anhydrous extraction solvent is a mixture of supercritical carbon dioxide and ethanol, the ethanol is evaporated and the extract is dissolved in a recovery solvent selected from the group consisting of octyldodecyl myristate, caprylic and capric triglycerides, vegetable oils and mixtures thereof; preferably octyldodecyl myristate.

12. The extract according to claim 1, characterized in that When the anhydrous extraction solvent is 96° ethanol, the extraction is continuous and is carried out at a temperature ranging from 60 to 90°C, preferably between 70 and 85°C, at atmospheric pressure, for a duration of between 1 and 5 hours, preferably between 2 and 4 hours.

13. The extract according to claim 8 or 12, characterized in that When the extraction solvent is 96° ethanol, the weight ratio of plant / 96° ethanol is between 1 / 99 and 20 / 80.

14. The extract according to any one of claims 8 or 12 to 13, characterized in that When the anhydrous extraction solvent is 96° ethanol, the ethanol is evaporated and the extract is dissolved in a recovery solvent selected from the group consisting of 1,3-propylene glycol, propylene glycol, butylene glycol, anhydrous deep eutectic solvents (LTTMs) and mixtures thereof; preferably 1,3-propylene glycol.

15. The extract according to any one of claims 8 to 14, characterized in that The method further comprises the step of decolorizing the extract, preferably by adsorption, more preferably using activated carbon or bleaching earth.

16. A cosmetic composition comprising the extract according to any one of claims 5 to 15, characterized in that The extract comprises 0.1% to 10% by weight of the composition, preferably between 0.5% and 5%.

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