Annona cherimola seed extract

Abstract

The present invention relates generally to the field of cosmetics. More specifically, the present invention relates to a skin care cosmetic composition containing a seed extract of the Annona cherimola plant. The present invention is based on the discovery that such seed extract has several beneficial effects on the skin. In particular, Annona cherimola seed extract exerts soothing, soothing, moisturizing, and anti-pruritic effects. Therefore, in another aspect, the present invention provides a cosmetic method for improving the skin appearance or reducing skin dryness in a subject, comprising administering a skin care composition of the present invention. In yet another aspect, the present invention provides a cosmetic method for soothing or calming irritated skin in a subject, comprising administering a skin care composition of the present invention. The present invention also relates to the use of Annona cherimola seed extract for skin care, particularly for soothing or calming irritated skin, or for improving the skin appearance or reducing dryness and pruritus.

Description

[Technical Field]

[0001] The present invention relates generally to the field of cosmetics. More specifically, the present invention relates to a skin care cosmetic composition containing a seed extract of the Annona cherimola plant. The present invention is based on the discovery that the seed extract has several beneficial effects on the skin. In particular, Annona cherimola seed extract exerts soothing, soothing, moisturizing, and anti-pruritic effects. Therefore, in another aspect, the present invention provides a cosmetic method for improving the skin appearance or reducing dryness in a subject, comprising administering a skin care composition of the present invention. In yet another aspect, the present invention provides a cosmetic method for soothing or calming irritated skin in a subject, comprising administering a skin care composition of the present invention. The present invention also relates to the use of Annona cherimola seed extract for skin care, particularly for soothing or calming irritated skin, or for improving the skin appearance or reducing dryness and pruritus. [Background technology]

[0002] There is a strong demand for topical cosmetics that improve skin appearance.These products soften and moisturize the skin, so as to delay the signs of stress and aging.In recent years, many compounds that are commonly used in cosmetics have been replaced with natural ingredients, thereby following the consumer demand for more natural products.

[0003] In particular, plant and herb extracts are now commonly used as additives in cosmetics, e.g., skin care products. Some plant extracts have been found to have significant beneficial effects on the skin. For example, extracts of Epilobium angustifolium have been found to soothe the skin. Other extracts, such as extracts from oat (Avena sativa), are known to relieve itching and irritation. Extracts of the bark of the American cherry (Prunus serotina) have been found to intensely moisturize the skin. Therefore, plant or herb extracts are widely used by the cosmetics industry due to their diverse skin-improving properties.

[0004] Despite the advances made in the cosmetics field in the past in identifying and developing natural ingredients for skin care products, there remains a need for additional natural ingredients that reduce the symptoms of irritated skin, such as itching, dryness, and redness. Summary of the Invention [Means for solving the problem]

[0005] The present invention is based on the discovery that a seed extract, preferably an aqueous seed extract, obtained from the cherimoya plant (Annona cherimola) is highly suitable for use in skin products. Specifically, the inventors have found that Annona cherimola seed extract is effective in soothing and calming human skin. In addition, as shown in the examples described herein, Annona cherimola seed extract enhances cannabinoid receptor 2 activity, inhibits signal transducer and activator of transcription 3 (STAT3) activity, and inhibits the expression of IL-1β, IL-8, and calcitonin gene-related peptide (CGRP). Finally, Annona cherimola seed extract also reduces itching and redness in irritated skin and reduces pain perception.

[0006] In a first aspect, the present invention provides a skin care cosmetic composition comprising an Annona cherimoya plant seed extract as an active ingredient. The seed extract is preferably an aqueous seed extract. The seed extract is present in the skin care composition in an amount sufficient to induce at least one of the above-mentioned skin-improving effects. In general, those skilled in the art of cosmetics can easily add the seed extract in an appropriate amount to the final skin care composition.

[0007] In a preferred embodiment, the seed extract is present in the skin care cosmetic composition in an amount of 0.05 to 25.0% (w / w), which means that the seed extract is present in the skin care cosmetic composition in an amount of about 0.05 to about 25.0% (w / w), about 0.05 to about 20.0% (w / w), about 0.05 to about 15.0% (w / w), about 0.05 to about 10.0% (w / w), about 0.05 to about 8.0% (w / w), about 0.1 to about 25.0% (w / w), about 0.1 to about 20.0% (w / w), about 0.1 to about 1 5.0% (w / w), about 0.1 to about 10.0% (w / w), about 0.1 to about 8.0% (w / w), about 0.5 to about 25.0% (w / w), about 0.5 to about 20.0% (w / w), about 0.5 to about 15.0% (w / w), about 0.5 to about 10.0% (w / w), about 0.5 to about 8.0% (w / w), about 0.1 to about 20.0% (w / w), about 0.5 to about 15.0% (w / w), about 1.0 to about 10.0% (w / w), about 1.0 to about 8.0% (w / w), about 2.0 to about 25.0% (w / w), about 2.0 to about 20.0% (w / w), about 2.0 to about 15.0% (w / w), about 2.0 to about 10.0% (w / w), about 2.0 to about 8.0% (w / w), about 3.0 to about 25.0% (w / w), about 3.0 to about 20.0% (w / w ), about 3.0 to about 15.0% (w / w), about 3.0 to about 10.0% (w / w), about 3.0 to about 8.0% (w / w), about 4.0 to about 25.0% (w / w), about 4.0 to about 20.0% (w / w), about 4.0 to about 15.0% (w / w), about 4.0 to about 10.0% (w / w), or about 4.0 to about 8.0% (w / w). In a most preferred embodiment, the seed extract is present in the cosmetic composition in an amount of about 0.5% to 3% (w / w).

[0008] As used herein, the term "plant extract" refers to a blend of compounds isolated from a plant. Plant extracts can be prepared from one or more parts of a plant, such as the whole plant, plant flowers, seeds, fruits, or leaves. Thus, the term "seed extract" refers to a blend of compounds isolated from plant seeds. Extracts can be obtained by incubating seed material with a solvent according to standard procedures described in the art. The term encompasses crude extracts and extracts that have been subjected to one or more concentration or purification procedures. The seed extract of the present invention can exist in a liquid form, preferably as a solution, or in a solid form, such as a dry powder.

[0009] Seed extracts can be prepared from Annona cherimoya seeds extracted from the fruit of the plant. To prepare the seeds, the fruit is opened and the pulp is removed. The seeds may then be dried before further processing. Drying can be carried out at temperatures between 20°C and 80°C, preferably between 25°C and 70°C, and most preferably between 30°C and 50°C. A drying temperature of 40°C is particularly preferred. The weight loss resulting from drying should be in the range of 1% to 20% (w / w), preferably between 2.5% and 12% (w / w), and more preferably between 5% and 10%.

[0010] Before disintegration, plant materials can be blanched at temperatures between 45°C and 100°C to remove bacterial contamination and improve digestibility. Additionally, blanching reduces the activity of enzymes such as hydrolases, lipases, and oxidases, thereby enhancing the stability and quality of the plant material.

[0011] The seeds are then disrupted before contacting them with the solvent. Disruption can be achieved, for example, by repeated freezing and thawing, or by suitable equipment such as a homogenizer, a high-pressure homogenizer, an ultrasonic homogenizer, a mill, or a grinder. For example, a standard ball mill or a French press can be used. The seeds can be disrupted in frozen or unfrozen form. In addition, the decomposition of the cells of the seed material can also be achieved with enzymes. For this purpose, the seed coat can be peeled and treated with a suitable enzyme that leads to the destruction of the structural components of the cells. Incubation of the seed material with pectinase, collagenase, cellulase, and / or hemicellulase is suitable for effectively decomposing the cells.

[0012] In the next step, the disintegrated seed material is contacted with a suitable solvent for extraction, such as water, an aqueous buffer such as phosphate-buffered saline or citrate buffer, a glycol such as propylene glycol or butylene glycol, or a glycol-water mixture, an alcohol or an alcohol-water mixture, in particular ethanol or an ethanol-water mixture, or methanol or a methanol-water mixture, or glycerin or a glycerin-water mixture.

[0013] The seed extract used in preparing the skin care composition of the present invention is preferably aqueous seed extract, that is, the extract prepared using aqueous solvent, preferably water or aqueous buffer solution.This has the particular advantage that the extract does not contain significant amount of acetogenin.In short, acetogenin is a class of polyketides found in plants of the Annonaceae family.To date, more than 400 acetogenins have been known, and many of them are neurotoxic.Acetogenins are poorly soluble in water, and therefore do not accumulate in aqueous extracts.If seed extract is prepared using organic solvent, acetogenins must be removed from the extract before being formulated into skin care products.

[0014] In a preferred embodiment, the aqueous seed extract used in the skin care composition of the present invention contains no or only low amounts of acetogenins. As a result, the skin care cosmetic composition of the present invention preferably contains less than 0.001% (w / w), more preferably less than 0.0001% (w / w), and even more preferably less than 0.00001% (w / w) of acetogenins. In a particularly preferred embodiment, the skin care composition of the present invention is acetogenin-free.

[0015] In another preferred embodiment, the aqueous seed extract is used in the skin care composition of the present invention, which contains no or only a small amount of phytosterols. As a result, the skin care cosmetic composition of the present invention preferably contains less than 0.001% (w / w), more preferably less than 0.0001% (w / w), and even more preferably less than 0.00001% (w / w) of phytosterols. In a particularly preferred embodiment, the skin care cosmetic composition of the present invention is phytosterol-free.

[0016] Mass spectrometry of the extract prepared according to the examples described below revealed that the extract contained kerimolacyclopeptides A, B, C, D, E, and F, as well as the alkaloids isoboldine, reticuline, collidine, michelarubine, and riboflavin. Therefore, in another preferred embodiment, an aqueous seed extract is used in the skin care cosmetic composition of the present invention, which contains at least one peptide selected from the group consisting of kerimolacyclopeptides A, B, C, D, E, and F. More preferably, an aqueous seed extract containing kerimolacyclopeptides A to F is used. In yet another preferred embodiment, an aqueous seed extract is used in the skin care cosmetic composition of the present invention, which contains at least one alkaloid selected from the group consisting of isoboldine, reticuline, collidine, michelarubine, and riboflavin. More preferably, an aqueous seed extract containing all of these alkaloids is used.

[0017] In a preferred embodiment, the solvent is water. In another preferred embodiment, the solvent is a Soerensen sodium citrate buffer, for example, a buffer containing 0.1 M disodium citrate and 0.1 N HCl, at a pH of 1.2 to 5.0, preferably pH 3.0, from 2.0 to 5.0. In another preferred embodiment, the solvent is a Soerensen sodium citrate buffer, for example, a buffer containing 0.1 M citric acid monohydrate (C6H8O7 x HO) and 0.1 M trisodium citrate dihydrate solution, at a pH of 5.0. In yet another preferred embodiment, the solvent is a Soerensen phosphate buffer, for example, a buffer containing 0.06 M potassium phosphate and 0.06 M disodium phosphate, at a pH of 5 to 8, for example, at pH 5. The ratio of seed material to solvent during extraction can be in the range of 1:100 (w / w) to 50:100 (w / w). Preferably, the ratio of seed material to solvent during extraction is 5:100 (w / w) or more, 10:100 (w / w) or more, 15:100 (w / w) or more, 20:100 (w / w) or more, 25:100 (w / w) or more, 30:100 (w / w) or more, 35:100 (w / w) or more, 40:100 (w / w) or more, 45:100 (w / w) or more, or 50:100 (w / w).

[0018] After extraction, the liquid phase is separated from the solid plant material, for example, by decantation, centrifugation, filtration, or a combination of these methods. For example, a standard decanter apparatus, such as a CA22 apparatus (GEA Westfalia Separator Group GmbH, Oelde, Germany), can be used. The extract mixture obtained from the decanter can be subjected to an additional filtration step, such as membrane filtration. The liquid phase thus obtained is considered an extract according to the present invention.

[0019] The extract can be used directly to formulate the skin care compositions of the present invention. However, to stabilize the extract, it is preferred to keep the extract at ambient temperature for several hours, from 1 to 24 hours, preferably from 8 to 16 hours. The extract may then be subjected to another filtration to remove any remaining solids. For this purpose, a 0.2 μm cellulose acetate membrane may be used.

[0020] The skin care cosmetic composition of the present invention is preferably formulated for topical application, which means that the composition is provided in a form that allows consumers to dispense the composition after applying it to the skin.Preferably, the skin care composition of the present invention is formulated as a liquid, ointment, cream, scrub, lotion, paste, gel, hydrogel, foam or powder.When the composition is formulated as a liquid, the composition can be packaged in a pump dispenser that can spray the liquid onto the skin area to be treated.The skin care composition of the present invention can also be incorporated into a patch that is applied to the skin.

[0021] In a particular preferred embodiment, the composition of the present invention is formulated for topical application to the skin of a subject.The skin to be treated can include facial and body skin, for example, the skin of the neck, chest, back, arm, hand, leg or thigh.According to a preferred embodiment, the skin to be treated with the composition of the present invention is facial skin.According to another preferred embodiment, the skin to be treated with the composition of the present invention is body skin.

[0022] If necessary, the skin care composition can be applied to the area of ​​skin that needs treatment, such as the face or body, at least once a day, twice a day, or even more frequently. When applied twice a day, the first and second applications are preferably separated by at least 6 hours, preferably 8 hours. Typically, the cosmetic composition is applied once in the morning and once in the evening. The composition of the present invention can be used for a long period of time without any adverse side effects. For example, the treatment period can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 12 weeks, at least 24 weeks, or longer. In some embodiments, the treatment is extended to several months, such as 4 months, 6 months, 8 months, 12 months, 18 months, or 24 months.

[0023] In addition to the seed extract, the skin care composition of the present invention will contain at least one dermatologically acceptable carrier. The carrier may be present in the skin care composition in an amount of about 30% to about 99%, preferably about 40% to about 98%, more preferably about 50% to about 97%, more preferably about 60% to about 96%, and even more preferably about 70% to about 95% by weight of the skin care composition. As used herein, a dermatologically acceptable carrier is compatible with both the skin and the other ingredients in the composition. In particular, a dermatologically acceptable carrier does not irritate or otherwise adversely affect the skin. Furthermore, a dermatologically acceptable carrier does not interfere with the activity or solubility or dispersibility of other ingredients in the composition. Suitable dermatologically acceptable carriers include, but are not limited to, aqueous solutions, emulsions, dispersions, and solids. In a preferred embodiment, the dermatologically acceptable carrier is an emulsion. The emulsion may be an oil-in-water emulsion, a water-in-oil emulsion, or a water-in-oil-in-water emulsion. When an emulsion is used as a carrier, the aqueous phase of the emulsion preferably contains Annona cherimoya seed extract. The emulsion may contain one or more anionic, cationic, or nonionic emulsifiers, for example, in an amount of 1% to about 5% based on the weight of the carrier.

[0024] The composition of the present invention can further comprise any additional ingredients that are commonly used in skin care cosmetics.Such ingredients are described in detail in, for example, International Cosmetic Ingredient Dictionary and Handbook, 16th edition (2016).For example, the composition of the present invention can also comprise one or more of the following: antioxidant, binder, pH adjuster, buffer, colorant, thickener, emollient, moisturizer, exfoliant, preservative, plant extract, essential oil, and fragrance.

[0025] For example, the compositions of the present invention may include an exfoliating compound such as urea. Exfoliating compounds useful in the compositions of the present invention include, but are not limited to, urea, alpha-hydroxy acids, and beta-hydroxy acids, as well as their esters, anhydrides, and salts. Suitable hydroxy acids include, for example, urea, glycolic acid, lactic acid, malic acid, mandelic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acids, salicylic acid, and their derivatives. The use of urea is particularly preferred because it also has an additional water-binding effect, enhancing the absorption of other cosmetic ingredients. The skin care compositions of the present invention may include from about 1% to about 5% (w / w) of an exfoliating compound. In some embodiments, the compositions may contain two or more different exfoliating compounds.

[0026] In one preferred embodiment, the skin care composition of the present invention contains a compound that functions as a pH adjuster. Because the composition of the present invention is used on human skin, it typically has a slightly acidic pH to be more compatible with the acidic environment of the skin. The composition can have a pH ranging from about 2.5 to about 6.5, preferably from about 4.0 to about 6.0, and more preferably from about 5.0 to about 6.0 or from about 5.5 to about 6.0. An acidic pH can be achieved by adding an acid, e.g., a carboxylic acid such as an alpha hydroxy acid, to the skin care composition of the present invention. The nature of the acid that can be used in the composition of the present invention is not particularly limited. Suitable acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, and caprylic acid. In a particularly preferred embodiment, the composition contains lactic acid as a pH adjuster. For application to human skin, lactic acid is particularly useful because it is also secreted by skin flora to create a protective acidic environment on the human skin surface.

[0027] The skin care compositions of the present invention can also contain an emollient. Suitable emollients for the skin care compositions of the present invention include, but are not limited to, olive oil, palm oil, soybean oil, sesame oil, rapeseed oil, evening primrose oil, sunflower seed oil, avocado oil, olive oil, coconut oil, castor oil, safflower seed oil, myristyl lactate, isopropyl myristate, polyethylene glycol, isopropyl palmitate, isopropyl stearate, isobutyl palmitate, isocetyl stearate, or cetyl alcohol. The skin care compositions of the present invention can contain about 1% to about 5% (w / w) of an emollient. In some embodiments, the composition can contain two or more different emollients.

[0028] The skin care composition of the present invention can also contain a moisturizing agent to improve skin hydration. Moisturizing agents suitable for use in the composition of the present invention include, but are not limited to, glycerin, polyethylene glycol ethers of glycerin, amino acids such as proline and arginine, sugars and sugar alcohols such as glucose, mannose, trehalose, and polyglycerol sorbitol, 1,3-butylene glycol, propylene glycol, diglycerol, glycerol monopropoxylate, glycogen, sodium hyaluronate, sodium polyaspartate, sodium polyglutamate, sorbeth 20, sorbeth 6, and hydrogenated starch hydrolysates. The skin care composition of the present invention can contain about 1% to about 5% (w / w) of moisturizing agent. In some embodiments, the composition can contain two or more different moisturizing agents.

[0029] The skin care compositions of the present invention preferably contain at least about 30% (w / w) water. More preferably, the skin care compositions contain at least about 35% (w / w) water, at least about 40% (w / w) water, at least about 45% (w / w) water, at least about 50% (w / w) water, at least about 55% (w / w) water, at least about 60% (w / w) water, at least about 65% (w / w) water, at least about 70% (w / w) water, at least about 75% (w / w) water, at least about 80% (w / w) water, at least about 85% (w / w) water, or at least about 90% (w / w) water. An amount of water of at least about 70% (w / w) water in the final skin care composition is most preferred.

[0030] In another aspect, the present invention provides a cosmetic method for improving the appearance of a subject's skin, comprising administering to the subject's skin a composition described elsewhere herein. In yet another aspect, the present invention provides a cosmetic method for reducing dry skin in a subject, comprising administering to the subject's skin a composition described elsewhere herein. In yet another aspect, the present invention provides a cosmetic method for relieving irritated skin, comprising administering to the subject's skin a composition described elsewhere herein. In yet another aspect, the present invention provides a cosmetic method for soothing irritated skin, comprising administering to the subject's skin a composition described elsewhere herein. In a particularly preferred embodiment, the method comprises topical administration of a composition to facial skin. In another particularly preferred embodiment, the cosmetic method achieves the desired effect through one or more of the following activities: stimulating cannabinoid receptor 2, enhancing cannabinoid receptor 2 activity, inhibiting or reducing signal transducer and activator of transcription 3 (STAT3) activity, inhibiting IL-1β expression, inhibiting IL-8 expression, and inhibiting calcitonin gene-related peptide (CGRP) expression.

[0031] In another aspect, the present invention provides the use of a composition described elsewhere herein, i.e., a composition comprising Annona cherimoya seed extract as an active ingredient, for cosmetic skin care. Specifically, the composition is used to relieve irritated skin, soothe irritated skin, improve skin appearance, and / or reduce dryness and itching. Preferably, the use of the seed extract is based on one or more of the following activities: stimulating cannabinoid receptor 2, enhancing cannabinoid receptor 2 activity, inhibiting or reducing signal transducer and activator of transcription 3 (STAT3) activity, inhibiting IL-1β expression, inhibiting IL-8 expression, and inhibiting calcitonin gene-related peptide (CGRP) expression.

[0032] As used herein, all percentages are by weight of the skin care cosmetic composition unless specifically stated otherwise. All ratios are by weight unless otherwise stated. [Brief explanation of the drawings]

[0033] [Figure 1] Extract F230 acts as a cannabinoid receptor 2 agonist. 293T-CB2-CRE-luc cells were treated with extract F230 or the positive control WIN-55,212-2 for 15 minutes. Cells were then incubated with 10 μM forskolin. After 6 hours of incubation, cells were lysed and luciferase activity was measured. The specific transactivation observed for F230 is expressed as [%] of forskolin stimulation. [Figure 2] This shows that extract F230 has STAT3 inhibitory activity. HeLa-STAT3-luc cells were treated with F230 and stimulated with IFN-γ. After 6 hours, the cells were lysed and luciferase activity was measured. Results are expressed as the percentage of inhibition of IFN-γ-induced STAT3 activity (100% activation). Experiments for each F230 concentration were performed in triplicate. [Figure 3]We show that extract F230 effectively reduces capsaicin-induced release of the cytokine IL-1β in co-cultures of keratinocytes and human sensory neurons. [Figure 4] We show that extract F230 effectively reduces capsaicin-induced release of the cytokine IL-8 in co-cultures of keratinocytes and human sensory neurons. [Figure 5] Figure 1 shows that extract F230 reduces capsaicin-induced CGRP expression. Increasing amounts of F230 result in higher levels of CGRP expression inhibition. [Figure 6] It shows that extract F230 significantly reduces skin itching compared to placebo already after 1 minute of application. [Figure 7] Figure 1 shows that extract F230 significantly reduces skin redness compared to placebo after 17 days of application. [Figure 8] Figure 1 shows that extract F230 increases the skin perception threshold compared to placebo. [Example]

[0034] The following examples illustrate certain preferred embodiments of the present invention, however it should be noted that the present invention is not limited to such embodiments.

[0035] Example 1: Preparation of Annona cherimola seed extract An extract of Annona cherimoya was prepared as follows. Fruit from Annona cherimoya was opened and the pulp was removed. The seeds were harvested and dried overnight at 40°C. After drying, the seeds were disintegrated by grinding. The disintegrated seeds were then extracted for 4 hours at 50°C using a sodium citrate buffer solution at pH 6.0 containing 0.1 M citric acid monohydrate (C6H8O7·H2O) and 0.1 M trisodium citrate dihydrate (C6H5O7Na3·2H2O).

[0036] After extraction, the liquid phase was separated from the solid plant material using a Decanter CA22 apparatus (Westfalia Seperator AG). The liquid fraction was held at room temperature for 16 hours for stabilization, followed by a final filtration step using a 0.2 μm cellulose acetate membrane. The extract thus obtained is hereafter referred to as "F230." Mass spectrometry analysis of the F230 extract revealed that it contained kerimolacyclopeptides A, B, C, D, E, and F (see references 1-10) as well as the alkaloids isoboldine, reticuline, collidine, michelarubine, and riboflavin.

[0037] Example 2: Cannabinoid Receptor 2 Agonist Activity Cannabinoid receptor 2 (CB2) is part of a complex regulatory system involved in inflammation. Among other things, the activity of this receptor is involved in thermoregulation, cell growth, host defense, apoptosis, pruritus, pain, and wound healing (Soethoudt et al. (2017), Nat. Commun. 8, 13958; Ibsen (2017), Cannabis and Cannabinoid Research 2:1, 48-60). CB2 activation leads to the suppression of proinflammatory cytokines and the pain-inducing neuropeptide-like substance CGRP (calcitonin gene-related peptide). Therefore, compounds that enhance CB2 activity are potentially useful for alleviating inflammatory skin conditions, such as those associated with atopic dermatitis, psoriasis, and pruritus.

[0038] To evaluate the potential effects of the F230 extract prepared as described in Example 1, 293T-CB2 cells stably transfected with a cDNA encoding human cannabinoid receptor 2 (CB2) were cultured at 1 × 10 in 24-well plates. 5The cells were incubated at a cell density of 1000 cells / ml and transiently transfected with 0.1 μg / ml of CRE-luc, a plasmid containing six consensus cAMP response elements (CRE) linked to firefly luciferase. Transient transfection was performed using Rotifect (Carl Roth GmbH, Karlsruhe, Germany) according to the manufacturer's instructions. Cells were harvested 24 hours after transfection.

[0039] To measure the CB2 agonist activity of F230, 293T-CB2-CRE-luc cells were treated with increasing concentrations of either F230 extract or the positive control WIN-55,212-2 (Sigma Aldrich, Taufkirchen, Germany) for 15 min, followed by treatment with 10 μM forskolin (Sigma Aldrich, Taufkirchen, Germany). After 6 h of stimulation, cells were lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol. Luciferase activity was then measured using an Autolumat LB9501 (Berthold Technologies, Bad Wildbad, Germany) according to the instructions of the luciferase assay kit (Promega, Madison, USA). The background obtained with lysis buffer was subtracted in each experiment, and specific transactivation was expressed as [%] of forskolin stimulation (the latter was set to 100%).

[0040] result The results are shown in Figure 1. The assay results reflect CB2 agonist activity as the inhibition of forskolin-induced CRE-Luc activity. It can be seen that the positive control WIN-55,212-2 acts as a CB2 agonist and partially antagonizes the effect of forskolin. The same effect is observed for F230, but the level of forskolin antagonism increases with increasing amount of F230. As a result, this assay demonstrates that F230 exerts CB2 agonist activity.

[0041] Example 3: STAT3 inhibitory activity Signal transducer and activator of transcription 3 (STAT3) is a human transcription factor encoded by the STAT3 gene. STAT3. During skin inflammation, IFN-γ stimulates STAT3 expression, which in turn induces the expression of additional pro-inflammatory cytokines, thereby contributing to inflammation (Archer et al. (2017), The Journal of Immunology, 198(1 Supplement) 197.4). Inhibition of IFN-γ-stimulated STAT3 expression is thought to reduce the inflammatory immune response in the skin.

[0042] To assess the potential effect of the F230 extract prepared as described in Example 1 on STAT3 activity, HeLa-STAT3-luc cells stably transfected with the plasmid 4xM67 pTATA TK-Luc were used. Cells were plated at 2x10 in 96-well plates the day before the assay. 4 The cells were seeded at a cell density of 100 / ml and then treated with F230 for 15 minutes, followed by stimulation with 25 U / ml of IFN-γ.

[0043] After 6 h, cells were washed twice with phosphate-buffered saline (PBS) and lysed by incubation in 50 μl lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl, 1 mM DTT, 1% Triton X-100 (Sigma Aldrich, Taufkirchen, Germany), and 7% glycerol (Sigma Aldrich, Taufkirchen, Germany) for 15 min at room temperature on a horizontal shaker.

[0044] Luciferase activity was measured using a plate reader Berthold / LB941 (Berthold Technologies) according to the instructions of the luciferase assay kit (Promega, Madison, Wisconsin, USA). Relative luminescence units (RLU) were calculated, and the results were expressed as a percentage of inhibition of IFN-γ-induced STAT3 activity (100% activation). Experiments for each concentration of test item were performed in triplicate wells.

[0045] result The results are shown in Figure 2. As can be seen, F230 antagonizes IFN-γ-induced STAT3 activity. Increasing amounts of F230 result in higher levels of STAT3 inhibition.

[0046] Example 4: Inhibition of IL-1β and IL-8 expression As pro-inflammatory cytokines, IL-1β and IL-8 are involved in numerous skin diseases associated with inflammation, such as psoriasis, atopic dermatitis, neutrophilic skin diseases, and eczema (Jensen (2010); Current opinion in investigational drugs (London, England: 2000); 11(11), 1211-20); Bou-Dargham et al. (2017), Med. Res. Rev., 37:180-216; Amarbayasgalan et al. (2013), Int Arch Allergy Immunol, 160:63-74). Therefore, inhibiting the production of these cytokines helps reduce skin inflammation.

[0047] To investigate whether the F230 extract prepared as described in Example 1 can inhibit the expression of IL-1β and IL-8, a coculture system of keratinocytes and human sensory neurons was used. Human sensory neurons were derived from human induced pluripotent stem cells (hiPSCs). hiPSCs were obtained by transfecting human fibroblasts reprogrammed into neuronal cells. hiPSCs were seeded at a density of 250,000 cells in 6-well plates (Corning GmbH, Kaiserslautern, Germany) coated with a thin layer of Matrigel® in a medium consisting of DMEM-F12 (Pan-Biotech, Aidenbach, Germany) supplemented with 10% knockout serum replacement (Life Technologies, Carlsbad, USA), 0.1 μM retinoic acid (Sigma-Aldrich, Taufkirchen, Germany), 1% P / S (Pan-Biotech), and a cocktail of inhibitors. Cultures were maintained at 37°C and 5% CO2 for 6 days.

[0048] After 6 days of differentiation, hiPS cells were seeded at a density of 20,000 cells in differentiation medium using 96-well plates coated with a thin layer of Matrigel®. After 9 days, the medium was replaced with maturation medium: DMEM-F12 supplemented with 1% N2 (Life Technologies, Carlsbad, USA), 10 ng / mL neurotrophin 3 (NT-3), 10 ng / mL glial cell line-derived neurotrophic factor (GDNF, Pan-Biotech), 10 ng / mL brain-derived neurotrophic factor (BDNF, PanBiotech), and 10 ng / mL nerve growth factor (NGF, Sigma). Commercially available human keratinocytes were thawed and grown at 75 cm in keratinocyte growth medium. 2Keratinocytes were grown in culture flasks. At the end of growth, the cells were dissociated and frozen in liquid nitrogen. On day 14 of neuronal culture, the keratinocytes were thawed again and seeded on top of neurons in 96-well plates at a density of 30,000 cells per well in a medium composed of 2 / 3 maturation medium and 1 / 3 keratinocyte growth medium. The medium was changed every 2 or 3 days. On day 18, the medium was removed and fresh medium was added, either (i) control medium, (ii) medium containing capsaicin, (iii) medium containing capsaicin and capsazepine, or (iv) medium containing capsaicin and three different concentrations of F230. After 1 hour of capsaicin stimulation, the supernatants were removed and assayed for cytokine release. Samples were evaluated by flux cytometry (BD Bioscience) and compared to untreated controls.

[0049] result The results of measuring IL-1β secretion are shown in Figure 3. Incubation with capsaicin strongly induced IL-1β expression. In contrast, treatment of cocultured cells with capsazepine before or during capsaicin activation completely abrogated IL-1β release. Similarly, increasing the amount of F230 resulted in increasing levels of inhibition of IL-1β expression. The results of measuring IL-8 secretion are shown in Figure 4. Incubation with capsaicin strongly induced IL-8 expression, whereas treatment of cocultures with capsazepine before or during capsaicin activation significantly reduced IL-8 release. Similarly, increasing the amount of F230 resulted in increasing levels of inhibition of IL-8 expression.

[0050] Example 5: Inhibition of CGRP Calcitonin gene-related peptide (CGRP) is a member of the calcitonin family of peptides that acts as a neuropeptide. CGRP release is associated with neurogenic inflammation through vasodilation, resulting in erythema and pain. CGRP production is induced by capsaicin. Using the coculture approach described above with human sensory neurons and keratinocytes, we measured the amount of CGRP released into the supernatant after 30 minutes of stimulation with 10 μM capsaicin. ELISA (Antibodies-online) was used to detect CGRP. Results were compared with those of untreated cells.

[0051] result The results are shown in Figure 5. As can be seen, F230 counteracts capsaicin-induced CGRP expression. Increasing amounts of F230 result in higher levels of CGRP expression inhibition.

[0052] Example 6: Reduction of skin itching For in vivo testing, compositions containing F230 and placebo compositions were prepared.

[0053] A composition containing 3% F230 was prepared as follows. A. 237.875g deionized water B. Xanthan gum 2.50g C. Euxyl PE9010 2.00g D.F230 7.5 E. Citric acid (10%) 0.125g

[0054] Components A and B are mixed and dispersed until uniform. Then, components C to E are added in the specified order. Finally, the pH is adjusted to 5.71.

[0055] A placebo composition was prepared as follows. A. 238.00g deionized water B. Xanthan gum 2.50g C. Euxyl PE9010 2.00g D. 7.50g deionized water

[0056] Components A and B were mixed and dispersed until uniform. Components C to D were then added in the specified order. The pH of the composition was adjusted to 5.70.

[0057] To investigate the effect of F230 on skin pruritus, a composition containing 3% F230 was applied to itchy skin. A double-blind study was conducted using 42 healthy volunteers (9 men and 33 women). 22 volunteers were assigned to F230 treatment and 20 volunteers to placebo treatment. Some of the volunteers had a history of dry and itchy skin, including 11 subjects with atopic dermatitis, 8 subjects with type IV allergies, 1 subject with psoriasis, and 1 subject with type II diabetes. 12 volunteers had sensitive skin. 9 volunteers had normal skin. Pruritus was scored at time points t=0, 1 minute, 5 minutes, and 24 hours. The baseline at t=0 was set at 0%. The frequency of pruritus was rated by the study subjects on a 5-point scale before and after use of the test composition.

[0058] result The results are shown in Figure 6. It can be seen that F230 significantly reduced itching compared to placebo already 1 minute after application.

[0059] Example 7: Reduction of skin redness To investigate the effect of F230 on skin redness, a formulation containing 3% F230 was applied to skin with abnormal redness. The volunteers used in Example 6 were used in this study. The formulation was applied twice a day. Redness was scored at t=0 and 17 days after application. The baseline at t=0 was set to 0%. Before and after the test period, trained graders evaluated any erythema caused by abrasion on the subject's skin.

[0060] result The results are shown in Figure 7. It can be seen that redness due to abrasion was reduced by F230 compared to placebo after 2 weeks of application (day 17) compared to baseline (day 1).

[0061] Example 8: Reduction of skin sensitivity To investigate the neurorelaxing effect of F230 on skin sensitivity, a formulation containing 3% F230 was applied to the skin. The volunteers used in Example 6 were used in this study. The formulation was applied once. Skin sensitivity was determined by measuring the current perception threshold (CPT) at 250 Hz and 5 Hz using a Neurometer® CPT / C device (Neurotron Inc., Baltimore, USA) at t=0 (i.e., immediately before application) and 40 minutes after application of the test formulation or placebo. The baseline at t=0 was set to 0%.

[0062] result The results are shown in Figure 8. It can be seen that at both 250 Hz and 5 Hz the perception threshold is much higher after treatment of the skin with F230 compared to placebo.

[0063] References 1) Li C.-M., et al. (1997), Phytochemistry, 45, 521-523, 2) Li C.-M., et al. (1998), Phytochemistry, 47, 1293-1296, 3) Li C.-M., et al. (1998), Phytochemistry, 48, 555-556, 4) Li C.-M., et al. (1997), Phytochemistry, 50, 1047-1052, 5) Morita H., et al. (1999), Tetrahedron, 55, 7509-7518, 6) Wele A., et al. (2005), Phytochemistry, 66, 1154-1157, 7) Wele A., et al. (2002), Tetrahedron, 60, 405-414, 8) Wele A., et al. (2004), J. Natural Prod., 67, 1577-1579, 9)Wele A.,et al.(2005),Phytochemistry,66,693-696、 10)Wele A.,et al.(2005),Phytochemistry,66,2376-2380。

Claims

1. 1. A skin care cosmetic composition comprising an aqueous extract of seeds of the Annona cherimola plant, the aqueous extract comprises at least one peptide selected from the group consisting of kerimolacyclopeptides A, B, C, D, E and F; A skin care cosmetic composition for topical application to the skin of the face or body.

2. The cosmetic composition according to claim 1, wherein the extract is present in the composition in an amount of 0.05 to 25.0% (w / w).

3. The cosmetic composition according to claim 2, wherein the extract is present in the composition in an amount of 0.5 to 5.0% (w / w).

4. The cosmetic composition according to any one of claims 1 to 3, which is formulated as an ointment, cream, lotion, paste, gel, hydrogel, foam or powder.

5. 5. The cosmetic composition of claim 1, further comprising an emollient selected from the group consisting of olive oil, palm oil, soybean oil, sesame oil, rapeseed oil, evening primrose oil, sunflower seed oil, avocado oil, coconut oil, castor oil, safflower seed oil, myristyl lactate, isopropyl myristate, polyethylene glycol, isopropyl palmitate, isopropyl stearate, isobutyl palmitate, isocetyl stearate, or cetyl alcohol.

6. The cosmetic composition according to any one of claims 1 to 5, having a pH in the range of 2.5 to 6.

5.

7. The cosmetic composition according to claim 6, having a pH in the range of 4.0 to 6.

0.

8. 8. The cosmetic composition according to claim 1, comprising a moisturizer selected from the group consisting of glycerin, polyethylene glycol ether of glycerin, amino acids, sugars, sugar alcohols, 1,3-butylene glycol, propylene glycol, diglycerol, glycerol monopropoxylate, glycogen, sodium hyaluronate, sodium polyaspartate, sodium polyglutamate, Sorbeth 20, Sorbeth 6, and hydrogenated starch hydrolysates.

9. 9. The cosmetic composition according to claim 8, wherein the amino acid is selected from the group consisting of proline and arginine, and the sugar and / or sugar alcohol is selected from the group consisting of glucose, mannose, trehalose and polyglycerol sorbitol.

10. 10. The cosmetic composition of any of claims 1 to 9, comprising an exfoliating compound selected from the group consisting of urea, alpha-hydroxy acids and beta-hydroxy acids, and their esters, anhydrides, and salts.

11. A cosmetic method for skin care (excluding methods for treating humans) comprising applying the composition according to any one of claims 1 to 10 to the facial skin of a subject.

12. Use of a composition according to any one of claims 1 to 10 for skin care cosmetics (excluding human treatment).

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