Pseudostellaria heterophylla root extract and use thereof

Abstract

The present invention relates to a root extract of Pseudostellaria heterophylla comprising: - one or more phenolic components, - one or more pseudostellarins, - one or more heterophyllins including heterophyllin B, wherein heterophyllin B represents at least 0.5% by weight relative to the total weight of the dry extract. The invention further relates to the cosmetic use of the said extract.

Description

[0001] Pseudostellaria heterophy I la root extract and use thereof

[0002] Field of the invention

[0003] The present invention relates to a root extract of the plant Pseudostellaria heterophylla, a composition comprising a root extract of Pseudostellaria heterophylla, a method for its preparation and the use thereof. The present invention also relates to a cosmetic use of a composition comprising a root extract of the plant Pseudostellaria heterophylla for combating the signs of aging, for improving the global appearance of the skin. Furthermore, the present invention also refers to a cosmetic use of heterophyllin B obtainable from the root extract of the plant Pseudostellaria heterophylla as an antiaging agent, particularly as antiwrinkle agent.

[0004] Background of the invention

[0005] Skin is the human body's first barrier which protects the organs from attack by the external environment. However, excessive chemical and physical stimulations deteriorate the normal functions of the skin and induce aging thereof.

[0006] Skin aging is a multi-factorial process drives by both intrinsic (e.g.: time, genetic factors, hormones) and extrinsic (e.g., UV-exposure, pollution) factors. Characteristics of intrinsic or chronological aging include visible signs such as thin and dry skin, fine wrinkles, decreased elasticity, aberrant pigmentation. Among which epidermal thinning causes, a decreased of proliferation and renewal capacity of basal keratinocytes is currently reported. During aging, the extracellular matrix undergoes structural alterations and degradation, leading to the increase of wrinkles and loss of elasticity of the skin.

[0007] Retinoids are well known active agents for cosmetic purposes for preventing aging, for the treatment of acne and psoriasis, and other skin diseases.

[0008] Vitamin A and its derivatives, particularly retinol, are substances that slow the aging process most effectively. In keratinocytes, retinol stimulates the cellular activity, promotes their proliferation, strengthens the epidermal protective function, and reduces transepidermal water loss.

[0009] Retinoids - retinol, retinaldehyde, and retinyl palmitate for examples - are known to be responsible of many beneficial effects such as skin elasticity improvement, inhibition of matrix metalloproteinases and enhances synthesis of tissue inhibitors of metalloproteinases, reduction of skin pigmentation and diminution of stimulated melanocytes. Moreover, skin damage caused by ultraviolet radiations can be partially reversed by the application of retinoic acid.

[0010] Several clinical studies have demonstrated that retinol formulations resulted in significant improvement in fine wrinkles combined with in vitro assays on matrix metalloproteinase, collagenase secretion by fibroblasts.

[0011] Although retinol is one the most reference skin antiaging agent in cosmetic field, it also known to provoke irritations, erythema, pruritus, peeling, stinging, or burning, and sensitivity on the skin, all constitute drawbacks. Therefore, to avoid or limit such drawbacks, retinol is usually stabilized and / or encapsulated in delivery systems such as liposomes for example.

[0012] Pure bakuchiol was the first active molecule alternative to retinoids such as retinol. Bakuchiol - a meroterpene found mainly in the seeds of Psoralea corylifolia - has been demonstrated to exhibit an anti-inflammatory, antioxidant, anti-aging and anti-acne activities. Mechanistically, bakuchiol is reported to target several cellular pathways similar to those targeted by retinoids, including numerous retinoid-responsive genes and upregulation of collagen and extracellular matrix synthesis enzymes, without effect on genes encoding the retinoic acid receptors (RAR-B and RAR-G). All these regulations lead to visible effects on skin, namely improvement in wrinkles, pigmentation, and firmness.

[0013] Pseudostellaria heterophylla, also called hai er shen (Kid Ginseng, Child Ginseng), tai zi shen (Crown Prince Ginseng), or False Starwort, is an adaptogen herb belonging to the family Caryophyllaceae and it is used in traditional Chinese medicines and herbalism. The root of Pseudostellaria heterophylla contains two types of cyclopeptide - also called cyclic peptides or ring peptides - which are pseudostellarins and heterophyllins. The root of Pseudostellaria heterophylla further contains polyphenols, sugars, polysaccharides, this latter being considered as one of the main bioactive components within the whole extract. Both the nature and the amount of the aforementioned components are significantly affected by many factors, such as the area where the plant is growing, its origin and its variety, as well as the extraction process performed using the roots. Therefore, the nature of the active components as well as their amount may differ even between the same organ of the same plant species.

[0014] The root extract of Pseudostellaria heterophylla contains heterophyllins A, B, D, J and pseudostellarins A to H. (Hu et al., Chinese Medicine, 14:21 , 2019). The alcoholic root extract of Pseudostellaria heterophylla contains heterophyllins A, B, D, wherein the heterophyllin B content can reach roughly 175 mg per gram of the dried root in form of ground powder. The alcoholic root extract further contains pseudostellarins A to F and G (Hua et al., Molecules, 21 , 1538, 2016). According to the ecological environments, both the quantity and quality of the active components within a wild and a cultivated Pseudostellaria heterophylla have reportedly shown significant differences. In fact, the methanolic root extract of Pseudostellaria heterophylla contains pseudostellarins A to E and G (Chao Han et al., Journal of Chromatography B, 862, 125-131 , 2008), while methanolic root extract of the same plant originating from another region contains heterophyllins A to J and pseudostellarins A to H (Pomilio et al., Current Organic Chemistry, 10, 2075-2121 , 2006). The root of Pseudostellaria heterophylla extracted with ethyl acetate contains heterophyllins A, B, D J and pseudostellarins A to G, fatty acids, and amino acids (Lu et al., Frontiers in Pharmacology, 11 , 850, 2020).

[0015] The use of the root extracts of Pseudostellaria heterophylla is known in the cosmetic field.

[0016] For example, the patents CN-B 107929127 and CN-B 109528804 disclose that radix of Pseudostellaria heterophylla, extracted with an apolar solvent, contains cyclic peptides. The radix extracts obtained are useful for the treatment of the skin. CN-A 104138342 discloses ethanolic extract of the radix of Pseudostellaria heterophylla containing cyclic peptides useful for protecting DNA, in maintaining skin moisture thanks to the polysaccharides and as anti-inflammatory. CN-A 105106280 discloses a hydromethanolic extract of the root of Pseudostellaria heterophylla titrated in pseudostellarin B for inhibiting the tyrosinase activity. JPH-A 07324095 discloses pseudostellarins A to G obtained after solid resin fractionation from root extract of Pseudostellaria heterophylla for inhibiting the tyrosinase activity. More generally, purified pseudostellarins A to E and G are known to inhibit tyrosinase activity and the melanin formation (Morita et al., Tetrahedron, 50, 33, 1994).

[0017] The aforementioned extracts of Pseudostellaria heterophylla have several technical drawbacks. As the root of Pseudostellaria heterophylla contains a high amount of both polyphenols and polysaccharides, the related biological effects are predominantly linked to these components. Polyphenols may be chemically unstable over the time and often lead to the dark coloration of the final cosmetic active ingredient containing the extract that may be undesirable in the formulated cosmetic products.

[0018] Because cosmetic consumers are constantly seeking an efficient product to counteract skin aging, it is highly desirable to make available a retinoid-like natural product - the most accepted by end-consumers - meaning a product having an equivalent efficacy as a retinol, but without side effects and easy to formulate at low cost.

[0019] Therefore, it is desirable to have at disposal a root extract of Pseudostellaria heterophylla having lower content of both polysaccharides and polyphenols before processing, but rich in cyclopeptides, this later being considered more stable, expected to be more efficient biologically even at lower proportions.

[0020] It is also desirable to provide a cyclopeptide-based root extract of Pseudostellaria heterophylla having different and / or improved biological effects on the skin than those known in the art, causing any irritation on the skin, stable over the time, and useful, for example, in skin care products.

[0021] There is still an unmet need in providing a root extract of Pseudostellaria heterophylla having a regular high amount of cyclopeptides content that may be ensured over the time and may be stable regardless of the season as well as the geography origin of the plant.

[0022] In general, peptides are known to exhibit beneficial effects, even at low concentration, useful for numerous cosmetic applications. Among all forms of peptides, the cyclic form appears - compared to linear peptides - advantageously to be more efficient, likely thanks to its chemical stability over the time as well as its tridimensional structure that is less accessible for enzymes resulting to a weak degradation. Moreover, cyclic peptides pass more easily through the dermis than linear ones. Hence, having a composition essentially based on cyclic peptides, from natural origin, exhibiting an equivalent efficacy like retinol but without side effects, is highly desirable for cosmetic purposes.

[0023] Summary of the invention

[0024] A first aspect of the invention relates to a root extract of Pseudostellaria heterophylla comprising:

[0025] - one or more phenolic components,

[0026] - one or more pseudostellarins,

[0027] - one or more heterophyllins, including heterophyllin B, wherein the heterophyllin B represents at least 0.5% by weight relative to the total weight of the dry extract.

[0028] Another aspect of the present invention refers to a method for preparing a root extract of Pseudostellaria heterophylla comprising the steps of: a. performing a solid / liquid extraction of a root, optionally dried, of Pseudostellaria heterophylla, by contacting the root with a polar solvent for obtaining a solution, b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution for obtaining a powder, c. performing a fractionation step on: i. the concentrated solution obtained in step b); or ii. the powder solubilized in a polar solvent obtained in step b); d. recovering the cyclopeptides containing fraction obtained in step c). Another aspect of the invention relates to a cosmetic or pharmaceutic composition comprising:

[0029] - a root extract of Pseudostellaria heterophylla as an active ingredient,

[0030] - at least one further cosmetically and / or pharmaceutically acceptable ingredient other than a root extract of Pseudostellaria heterophylla, wherein the composition is a composition for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampooing, and a spray; and / or wherein the composition is a nutraceutical composition which is administered orally.

[0031] The present invention also refers to a cosmetic active ingredient comprising the root extract of Pseudostellaria heterophylla as defined above in a cosmetically acceptable vehicle.

[0032] The present invention also refers to the cosmetic use of a composition comprising a root extract of Pseudostellaria heterophylla as an active ingredient, or a composition as defined above for combating and / or preventing the signs of aging on the skin and the hair follicles, for improving the global appearance of the skin, in particular for improving the firmness, the elasticity, the radiance, the brightness, the hydration of the skin, for decreasing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

[0033] A further aspect of the present invention refers to a non-therapeutic method for preventing and / or for delaying the signs of aging in the skin of a subject by applying, onto the skin on need, an effective amount of a composition comprising a root extract of Pseudostellaria heterophylla as an active ingredient, or a composition comprising thereof as defined above.

[0034] Another further aspect of the present invention refers to the cosmetic use of heterophyllin B obtainable (or obtained) from a root extract of Pseudostellaria heterophylla for preventing and / or reducing the appearance of wrinkles or fine lines, in particular, for promoting the synthesis of collagen, for decreasing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

[0035] Brief description of figures

[0036] In Figure 1 , the content of pro-collagen I released by normal human dermo-fibroblasts (NHDF) treated or not 3 days with the root extract of Pseudostellaria heterophylla of the present invention, or with retinol, or with bakuchiol was measured by ELISA test. Results are expressed in percentage of variation compared to control conditions.

[0037] In Figure 2, the content of IL-6 released by normal human dermo-fibroblasts (NHDF) treated or not 5 days with the root extract of Pseudostellaria heterophylla of the present invention, or with retinol, or with bakuchiol was measured by ELISA test. Results are expressed in percentage of variation compared to control conditions.

[0038] In Figure 3, the extracellular melanin content was dosed in B16-F1 cells treated or not with kojic acid - positive control - or with the root extract of Pseudostellaria heterophylla of the present invention, at different concentrations (0.3%, 0.15% and 0.05%). The released melanin was measured by optical density (OD). Results are expressed in percentage of variation compared to a non-treated condition.

[0039] Figures 4 shows the ability of the root extract of Pseudostellaria heterophylla of the present invention to improve skin complexion by increasing the brightness - according to the measurement of parameter L* Figure 4A - and decreasing the redness - according to the measurement of parameter a* Figure 4B - after 28 days of topical application. For both parameters (L* a*), the part of volunteers with improvement is indicated for placebo and a root extract Pseudostellaria heterophylla of the present invention below the corresponding graph.

[0040] Figure 5 shows the improvement of skin mechanical properties. Figure 5A demonstrates the ability of the root extract of Pseudostellaria heterophylla of the present invention to decrease the sagging of skin after 28 days of topical application. Figure 5B shows the improvement of firmness of the skin measured by the decrease of R0 cutometer parameter after 28 and 56 days of topical application, with enhancement over time. For both parameters (sagging and firmness), the part of volunteers with improvement is indicated for placebo and the root extract Pseudostellaria heterophylla of the present invention below the corresponding graph.

[0041] Figures 6 shows the comparison of the effects of the root extract of Pseudostellaria heterophylla of the present invention with the ones of bakuchiol regarding the brightness after 28 days of topical application (Figures 6A), the sagging after 28 days of topical application (Figures 6B) and the firmness after 28 days and 56 days of topical application (Figures 6C). For all parameters, the part of volunteers with improvement is indicated for the root extract of Pseudostellaria heterophylla of the present invention and bakuchiol below the corresponding graph.

[0042] Detailed description of the invention

[0043] A first aspect of the present invention relates to a root extract of Pseudostellaria heterophylla comprising:

[0044] - one or more phenolic components,

[0045] - one or more pseudostellarins,

[0046] - one or more heterophyllins, including heterophyllin B, wherein the heterophyllin B represents at least 0.5% by weight relative to the total weight of the dry extract.

[0047] As used throughout the present disclosure, the root extract of Pseudostellaria heterophylla of the present invention is also designated as “the extract” or “root extract” or by its abbreviation “PHE” for Pseudostellaria heterophylla extract.

[0048] As used herein, the term “extract” may be any substance made by extracting the root of Pseudostellaria heterophylla. In accordance with the present disclosure, the extract is a solvent-based extract obtained by solvent extraction of the said plant. As used herein, extracting is achieved by using at least one solvent, preferably a polar solvent, followed by a fractionation method. Accordingly, the extract is obtained by performing a well-known solid / liquid solvent extraction method followed by a fractionation step wherein a chromatographic solid column may be used for obtaining the final extract.

[0049] As used herein, the term “cyclopeptide” or “cyclic peptide”, in singular or plural, may be understood interchangeably in the broadest sense in the art. It means peptides having at least one ring generated from an acyclic peptide by formation of an amide or ester bond, by a disulfide link, or by a new carbon-carbon, carbon-nitrogen, nitrogenoxygen, or carbon-sulfur bond. In particular, the cyclopeptides of the present invention may be selected from the group consisting of heterophyllins and pseudostellarins.

[0050] In an embodiment, the one or more pseudostellarins may be selected from the group consisting of pseudostellarins A to E and G. In a preferred embodiment, pseudostellarins may be selected from at least two or more from the group consisting of pseudostellarins A to E and G. More preferably, pseudostellarins may be selected from at least three or more from the group consisting of pseudostellarins A to E and G. In particular, pseudostellarins may be a combination of pseudostellarins A to E and G.

[0051] In an embodiment, the one or more heterophyllins may be selected from the group consisting of heterophyllins A, C and J, preferably two or more thereof, in particular a combination thereof.

[0052] In a preferred embodiment, the heterophyllin B may represent at least 1 % by weight, more preferably, at least 1 .5% by weight, even more preferably at least 2% by weight, in particular at least 2.5% by weight relative to the total weight of the dry extract. Advantageously, the heterophyllin B may represent between 1 % to 8%, preferably between 1 .5% to 7%, more preferably between 2% to 6%, in particular between 2.5% to 5% by weight relative to the total weight of the dry extract.

[0053] In a preferred embodiment, the amount of the one or more phenolic components, including polyphenols, is less than 45% by weight relative to the total weight of the dry extract. Advantageously, the one or more phenolic components, including polyphenols may range between 5% to 15%, preferably between 7.5% to 12.5% by weight relative to the total weight of the dry extract. Advantageously, the one or more phenolic components may be, for example, non-glycosylated polyphenols. It is to be noticed that a root extract of Pseudostellaria heterophylla extracted with a polar solvent such as water or hydroethanol contains polyphenols between 45% to 99% by weight relative to the total weight of the dry extract. Therefore, such a high content of phenolic components, mainly polyphenols, is responsible at least partly, of the dark coloration of the final cosmetic active ingredient containing the extract. In a preferred embodiment, the total cyclopeptides content may range between 3.5% to 45%, preferably from 5% to 42.5%, more preferably from 7.5% to 40%, in particular from 10% to 37.5% relative to the total weight of the dry extract.

[0054] According to the invention, heterophyllin B may represent approximatively between 2 to 55% by weight relative to the weight of the total cyclopeptides.

[0055] In a preferred embodiment, the extract may further comprise polysaccharides representing less than 0,1 %, preferably less than 0,01 %, more preferably less than 0,001 %, approximately 0% by weight over the total weight of the dry extract.

[0056] In an embodiment, the extract may have a dry matter content of at least 1 g / kg (weight / weight of the extract), preferably at least 1 .5 g / kg, even more preferably at least 2 g / kg, meaning the dry matter content within the concentrated solution obtained in step a) or within the powder obtained in step b).

[0057] The extract according to the present invention may be in form of liquid, pasty or solid at standard conditions (room temperature of approximately 20°C, normal pressure of approximately 1013 hPa). In this context, the terms “liquid” and “fluid” may be understood interchangeably. Preferably, the extract may be first obtained in liquid form. Then, the one or more solvents may optionally be removed to obtain a solid form of the extract. A liquid extract can also be concentrated or condensed, for example under reduced pressure, using but not limited to a vacuum concentrator or a rotary vacuum evaporator. Additionally, after the concentration, the extract may be dried. Then, preferably, the one or more solvents are partly or totally removed. Optionally, a liquid or pasty extract may be obtained. Optionally, the extract may also be re-diluted with one or more cosmetically acceptable solvents for obtaining a solvent-based extract. Optionally, the extract may also be purified by any means (e.g., crystallization, chromatographic means, etc.).

[0058] An extract may be optionally designated as “tincture” or “absolute”. The solvent used for the preparation of the extract may be an aqueous solvent (water or a buffer), or it may be a combination of an aqueous solvent and an alcohol. An aspect of the present invention refers to a cosmetic active ingredient comprising the root extract of Pseudostellaria heterophylla as defined above in a cosmetically acceptable vehicle selected from the group consisting of water, aqueous buffer, alcohols, glycols, ethyl lactate, isopropyl myristate, triglycerides, tri-ethyl citrate, dicaprylyl ether, glyceryl isostearate, glyceryl stearate, ethyl acetate or a mixture thereof. The alcohols may be preferably selected from methanol, ethanol and isopropanol. The glycols may be preferably selected from pentylene glycol and glycerol.

[0059] In an preferred embodiment, heterophyllin B may represent at least 10 mg / kg, preferably at least 20 mg / kg, more preferably at least 25 mg / kg, in particular at least 30 mg / kg, in particular at least 45 mg / kg by weight relative to the total weight of the cosmetic active ingredient.

[0060] In a further preferred embodiment, heterophyllin B may represent between 10 to 500 mg / kg, preferably 20 to 450 mg / kg, more preferably between 25 to 430 mg / kg, in particular between 40 to 400 mg / kg relative to the total weight of the cosmetic active ingredient.

[0061] Accordingly, the ready-to-use root extract of Pseudostellaria heterophylla according to the invention may be either dried, or frozen or freeze-dried, or fresh. Preferably, the ready-to-use root extract of Pseudostellaria heterophylla may be in the form of a cosmetic active ingredient comprising said root extract solubilized in a mixture of glycerokwater in a volume ratio from 95 / 5 to 5 / 95, preferably from 80 / 20 to 20 / 80.

[0062] As used herein, the term “dry extract” may be understood in the broadest sense as an extract according to the present invention as dry matter, i.e. , without the solvent. The dry extract may optionally be physically present as dry matter. It will, however, be directly understood that, in calculations referring a percentage referred to dry matter, it does not have to be dry matter present in physical form, but the dry extract may also be present in dissolved form. Then, in the calculation, the mass of the solvent is mathematically subtracted from the total mass. Dry matter (in physical form) may be obtained by a suitable extraction method, followed by a step of drying the extract obtained. The drying may be performed by any method suitable for this purpose known in the art. Drying means removing the one or more solvents used for extraction. Removing the one or more solvents may be performed by any means. For example, it may be achieved by subjecting the extract to a hot and dry atmosphere and / or placing the extract on a heated plate in order to evaporate the solvent. For example, drying may be achieved by evaporation in a vacuum and / or at elevated temperature (e.g., in a rotary (vacuum) evaporator) or by crystallization of the solid material of interest. Alternatively, or additionally, drying may be achieved by atomisation or by lyophilization.

[0063] Another aspect of the present invention refers to a method for preparing a root extract of Pseudostellaria heterophylla comprising the steps of: a. performing a solid / liquid extraction of a root, optionally dried, Pseudostellaria heterophylla, by contacting the root with a polar solvent for obtaining a solution, b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution for obtaining a powder, c. performing a fractionation step on: i. the concentrated solution obtained in step b); or ii. the powder solubilized in a polar solvent obtained in step b); d. recovering the cyclopeptides containing fraction obtained in step c).

[0064] Advantageously, the method of the invention may be conducted by starting from a dry extract, preferably in powder form, of the root of Pseudostellaria heterophylla obtained after steps a) and b) being performed. Such dry extract may optionally be available commercially or may be partly or completely prepared from a root of Pseudostellaria heterophylla. In such a case, the method according to the invention further comprises the steps c) and d) wherein the powder in step ii) may be solubilised in a polar solvent selected from a water or a mixture of water and an alcohol having a proportion of alcohol between 1 to 20% (v / v), preferably between 1.5 to 15% (v / v), in particular between 2 to 10% (v / v). In a preferred embodiment, the polar solvent used in step ii) is a water. Advantageously, the root of Pseudostellaria heterophylla in powder form may be solubilized in a polar solvent at a proportion between 5 to 20%, preferably between 10 to 40%, in particular between 15 to 30% (weight or powder / volume solvent). In other words, a solubilisation at 5% means putting 5 g of powder in 1 L of polar solvent. After the solubilisation step, the liquid may be filtrated. It is important to noticed that the amount of the heterophyllin B of at least 0.5% by weight over the total weight of the dry extract is completely different from the amount of the heterophyllin B within the alcoholic root extract of Pseudostellaria heterophylla of the prior art. Indeed, the calculation mode is different so that the amount of the heterophyllin B of the present invention is calculated over the total weight of the dry extract, and not over the total weight of the dried root from which the extraction is to be performed. Typically, the heterophyllin B content within the concentrated solution obtained in step a) or within the powder obtained in step b) may be at least 200 mg / g, preferably at least 250 mg / g, more preferably at least 300 mg / g of heterophyllin B over the total weight of the dry matter. Advantageously, the heterophyllin B content within the concentrated solution obtained in step a) or within the powder obtained in step b) may represent 200 mg / g to 1000 mg / g, preferably 250 mg / g to 900 mg / g of heterophyllin B over the total weight of the dry matter.

[0065] As used herein, the term “polar solvent” may be understood in the broadest sense as generally understood in the art. Preferably, it means solvents for use in the well-known solid / liquid extraction method. Accordingly, the solid / liquid extraction is conducted at ambient pressure (i.e., 980 to 1200 hPa), performed at ambient temperature (i.e., 18 to 25°C). Preferably, polar solvents may be selected from water, alcohols, or a mixture thereof at any volume proportions.

[0066] In an embodiment, alcohols may be selected from ethanol, methanol, propanol, butanol, pentanol, phenol, glycerol, 1 ,3-butylene glycol, propane diol, or a mixture of two or more thereof. Preferably, alcohols may be selected from methanol, ethanol, propanol and glycerol.

[0067] In a preferred embodiment, the polar solvent used in step a) may be a water / alcohol mixture containing between 10 and 90% (v / v), or between 20 and 80% (v / v), or between 30 and 70% (v / v) of alcohol. Accordingly, the preferred polar solvent may be a mixture of water / ethanol at the ratio of 30 / 70% (v / v) of ethanol, or at the ratio of 40 / 60% (v / v) of ethanol.

[0068] Advantageously, the step a) may further comprise a step of separating the solid material from the solution, which may be a crude solution.

[0069] According to the invention, the fractionation step is known in the art. In an embodiment, the fractionation, also known as purification, may be performed for example by means of a chromatographic resin column using a convenient eluent solvent, or by a liquid / liquid separation.

[0070] As used herein, “chromatographic resin” relates to the chromatographic media present in the chromatographic column and responsible for the separation of the starting components present in the extract. Accordingly, the chromatographic resin may be packed in a packed bed or in an expanded bed. Different types of chromatographic resins, for example made of porous polymer resin, may be used. It is usual that the chromatographic column may be washed with a water before conducting the fractionation step of the extract containing solution.

[0071] An example of chromatography column that may be used for executing the fractionation step is Diaion™ HP20 resin.

[0072] In an embodiment, the eluent solvent may be the same as the polar solvents as previously described. In a preferred embodiment, the eluent solvent may be selected from water, or a mixture of water / alcohol wherein the alcohol representing 60 to 98% (v / v), preferably 70 to 95% (v / v), more preferably 80 to 92% (v / v) of the mixture. More preferably, the eluent solvent may be selected from water or a mixture of water / ethanol wherein ethanol may represent between 10 to 90 to 90 to 10% (v / v).

[0073] In an embodiment, the fractionation step may be conducted by means of a chromatographic resin column wherein the eluent solvent may be a mixture of water / alcohol. Advantageously, the water / alcohol eluent may comprise an alcohol representing between 5 to 95% (v / v). Preferably, alcohol may be ethanol or methanol. According to an embodiment, the step c) may be conducted by c.1 ) eluting with a mixture of water / alcohol wherein alcohol may represent 5 to 20% (v / v), and further c.2) eluting with a mixture of water / alcohol wherein alcohol may represent 80 to 95% (v / v).

[0074] According to a preferred embodiment, eluting in c.1 ) may be conducted with a mixture of ethanol / water containing 5% (v / v) of ethanol, preferably 10% (v / v) of ethanol, more preferably 20% (v / v) of ethanol.

[0075] According to an embodiment, eluting in c.2) may be conducted with a mixture of ethanol / water containing 80 to 95% (v / v) of ethanol, preferably 85 to 90% (v / v) of ethanol.

[0076] Advantageously, each elution in c.1 ) and c.2) may be performed during 30 to 120 min, preferably 45 to 90 min. The collected eluate constitutes the cyclopeptide-based fraction of the extract, which may be resuspended in an hydroalcoholic solvent, preferably in a mixture of glycerol / water at any volume proportion.

[0077] Another aspect of the invention relates to a cosmetic or a pharmaceutic composition comprising:

[0078] - a root extract of Pseudostellaria heterophylla as described above, as an active ingredient,

[0079] - at least one further cosmetically and / or pharmaceutically acceptable ingredient other than a root extract of Pseudostellaria heterophylla, wherein the composition is a composition for topic use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampooing, and a spray; and / or wherein the composition is a nutraceutical composition which is administered orally.

[0080] Another aspect of the present invention relates to a cosmetic composition comprising (or consisting of) a root extract of Pseudostellaria heterophylla as described above for use in combating and / or preventing the signs of aging on the skin and the hair follicles, for improving the global appearance of the skin, in particular for improving the firmness, the radiance, the brightness, the hydration of the skin, for decreasing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

[0081] As used herein, the terms “signs of skin aging” means any modification of the outer appearance of the skin caused by chronological aging, such as wrinkles and fine lines, creases, bags under the eyes, dark circles, sagging, loss of elasticity and / or skin tone, dulling or lack of radiance, pigment deficiencies, but also any internal modification of the skin which is not manifested systematically in the form of a modified external aspect, such as a reduction and loss of density of the dermis, thickening of the stratum corneum. The skin may also thicken and become rough. The signs of aging also include any degradation of the skin following exposure to UV rays, such as the premature appearance of fine lines around the eyes and the mouth, and expression lines on the face, different areas of pigmentation, such as freckles and solar lentigines, irregularities in the complexion, generalized loss of tone of the skin, loss of colour and thinning of the lips.

[0082] Notably, the experimental studies provided in the present disclosure demonstrated that the compositions comprising (or even consisting of) a root extract of Pseudostellaria heterophylla of the present invention may be used as an active ingredient for cosmetic and / or pharmaceutical purposes, in particular for skin care and hair care.

[0083] In a preferred embodiment, the root extract of Pseudostellaria heterophylla according to the present invention is useful for the obtention of the following effects: a. improving the complexion of the skin, b. reinforcing and / or restoring the epidermal barrier function, c. increasing the synthesis of collagen and improving the extracellular matrix components, d. increasing retinoic acid receptor gamma transcription, e. improving skin cell regeneration and epidermal differentiation, f. regulating oxidative stress in skin cells, g. improving skin defense, h. reinforcing the homeostasis of the skin, i. improving skin soothing, j. counteracting the hyperpigmentation on the skin, k. improving the skin microbiota, l. combination of two or more thereof.

[0084] Herein, the extract according to the invention may optionally be considered as an (or optionally even the sole) active ingredient. As used herein, the term “active ingredient” may be understood in the broadest sense as a component that may exhibit a desired and intended activity, either alone or together with one or more other ingredients such as one or more carriers that are themselves inactive or other active ingredient that may optionally act synergistically. Preferably, the active ingredient of the present invention is a cosmetically active ingredient.

[0085] The cosmetic or pharmaceutic composition may be used for any purpose and in any form. In a preferred embodiment, the composition is a composition for topic use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampooing, and a spray, or wherein the composition is a nutraceutical composition which is administered orally.

[0086] The composition may also be or may be comprised in a product selected from the group consisting of emulsions, gels, ointments, tonics, liquid soaps, bar soaps, bath oils, shower oils, massage oils, makeups, scalp treatments, aftershaves, shaving products, deodorants, shower gel, shampoos, and combinations of two or more thereof.

[0087] Optionally a cosmetic or pharmaceutical composition may also be a nutraceutical composition which can be administered orally to a subject. Then, the cosmetic or pharmaceutical composition may optionally be included in a food product, such as a food supplement. Then, the composition may typically have a systemic effect. A nutraceutical composition according to the present invention may be formulated, with acceptable carriers, in any form suitable for oral administration such as, e.g., tablets, capsules, granules, powder, solutions, emulsions, or suspensions. The cosmetic or pharmaceutic composition may comprise the extract of the present invention in any content. In a preferred embodiment, the composition comprises less than 20% by weight, referred to the total weight of the composition, of an extract of the present invention. In a preferred embodiment, the composition comprises 0.00001 to 15% by weight, more preferably 0.0001 to 10% by weight, even more preferably 0.0005 to 8% by weight, in particular 0.001 to 5% by weight, referred to the total weight of the composition, of the extract according to the present invention

[0088] The at least one further cosmetically and / or pharmaceutically acceptable ingredient other than the extract of the invention may be any cosmetically and / or pharmaceutically acceptable ingredient. In a preferred embodiment, the cosmetically and / or pharmaceutically acceptable ingredient is or comprises at least one cosmetically and / or pharmaceutically acceptable carrier.

[0089] As used herein, the terms “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient”, “cosmetically acceptable carrier”, “cosmetically acceptable excipient”, “carrier” and “excipient” may be understood interchangeably in the broadest sense as any substance that may support the cosmetic and / or pharmacological acceptance or usability of the composition according to the present invention containing a root extract of Pseudostellaria heterophylla. Preferably, neither the extract of Pseudostellaria heterophylla nor a composition containing such at least one extract according to the present invention is toxic when applied to the tissue.

[0090] The composition ready to use preferably may be a liquid formulation, in particular a composition suitable for topic and oral administration. The storage form of the composition may also be liquid, but may also be a dried form (e.g., a powder such as a powder comprising or consisting of a root extract of Pseudostellaria heterophylla), or may be a paste or syrup or the like. Optionally, a dried form, paste or syrup may be dissolved or emulsified prior to being administered to the skin of interest.

[0091] A cosmetically and / or pharmaceutically acceptable carrier may exemplarily be selected from the list consisting of an aqueous buffer, saline, water, alcohols, vegetable oils, mineral oils, polymers, or combination of two or more thereof. Optionally, a cosmetically and / or pharmaceutically acceptable carrier may contain one or more cosmetically and / or pharmaceutically acceptable additives.

[0092] In a preferred embodiment, such cosmetically and / or pharmaceutically acceptable additives may be selected from the group consisting of fragrances / perfumes, dyes, pigments, emulsifiers, lubricants, chelating agents, acidity regulators, antimicrobial agents, preservatives, antioxidants, and combinations of two or more thereof.

[0093] Moreover, the composition of the present invention may contain, in addition to the root extract of Pseudostellaria heterophylla, one or more other known active ingredients, working either synergistically and / or complementarily with the said extract of Pseudostellaria heterophylla without affecting its activity or its indented use as mentioned above.

[0094] The composition according to the present invention may be cosmetic product or may be comprised in a cosmetic product. The composition may be for any use. Any administration routes are suitable that lead to the desired purpose as claimed. Administration may be local or systemic administration. Preferably, administration is local administration. Administration may be topic administration, transdermal administration, oral administration, administration by mean of injection (e.g., intravenous (i.v.), intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injection), or nasal administration. For example, the composition according to the present invention may be formulated, with acceptable carriers in any suitable form for topical, oral, rectal, transmucosal, transnasal, intestinal, enteral and parenteral administrations.

[0095] In a preferred embodiment, the composition is a composition for topic use. In a preferred embodiment, the composition is a composition for topic use which is administered topically to a subject. In a preferred embodiment, the composition is a composition for topic use which is administered topically to a part of the skin such as, e.g., to the face, scalp, or any part of a body. Skin may be all types of skin, including compromised skin, dry skin, healthy skin, normal skin and oily skin. As indicated above, the root extract of Pseudostellaria heterophylla or the composition of the present invention may be used for any purpose. Preferably, it is used for a cosmetic and / or pharmaceutical purpose.

[0096] The extract of the present invention or a composition comprising thereof may have a local and / or a systemic effect. In a preferred embodiment, when administered locally and / or topically, it (mainly or completely) has a local effect.

[0097] A subject having the tissue of interest, in particular skin, scalp and hair, may be subjected from or may be of risk of developing a disorder and / or damage as laid out above.

[0098] As used in the context of the present invention, the term “subject” may be understood in the broadest sense as any living being. A subject preferably is a human or animal, more preferably a human or mammal, in particular a human being.

[0099] Another aspect of the present invention relates to a composition comprising (or consisting of) a root extract of Pseudostellaria heterophylla as described above for use in a method for combating and / or preventing the signs of aging on the skin and the hair follicles, for improving the global appearance of the skin, in particular for improving the firmness, the elasticity, the radiance, the brightness, the hydration of the skin, for decreasing or preventing skin inflammation, for preventing and / or alleviating acne- prone skin, or for two or more thereof.

[0100] Another aspect of the present invention relates to a cosmetic use of heterophyllin B for combating and / or preventing the signs of aging on the skin and the hair follicles, in particular for reducing wrinkles and fine lines, for improving the firmness and / or the elasticity of the skin, for counteracting the irregular hyperpigmentation in skin, for alleviating skin inflammation, or for two or more thereof.

[0101] A further aspect of the present invention relates to a non-therapeutic method for combating and / or preventing the signs of aging on the skin by topically applying an effective amount of heterophyllin B onto the skin on need, as an active ingredient or a composition comprising thereof, wherein preventing signs of aging on the skin may be for reducing wrinkles and fine lines, for improving the firmness and / or the elasticity of the skin, for counteracting the irregular hyperpigmentation in skin, for alleviating skin inflammation, or for two or more thereof.

[0102] Advantageously, heterophyllin B of the present invention may be purified from a root extract of Pseudostellaria heterophylla or may be purchased commercially. In such a case, heterophyllin B may be obtained synthetically.

[0103] Another aspect of the present invention pertains to a cosmetic composition comprising heterophyllin B for its use defined above, wherein heterophyllin B may represent between 0,00001 to 2%, preferably 0,00005 to 1 ,5%, more preferably between 0,0001 to 1 % by weight relative to the total weight of the composition, and wherein the composition may further comprise a cosmetically acceptable carrier.

[0104] Advantageously, the heterophyllin B according to the invention may be formulated in a cosmetically acceptable vehicle in the same way as for the context of the root extract of Pseudostellaria heterophylla.

[0105] Throughout this specification and the claims, unless the context requires otherwise, the open-worded terms such as “comprise”, “contain”, “include”, etc., and variations such as "comprises", "contains", “includes”, “comprising”, “containing”, “including”, etc., may be understood to imply the inclusion of a stated component, member, integer or step or group of components, members, integers or steps, but not the exclusion of any other components.

[0106] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually as well as in combination with each other recited herein. All methods and procedural steps, including product-by-process steps described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as", “for example”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0107] More generally, it should be understood that the aspects and embodiments may be combined in any manner and in any number to additional embodiments. Any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. Although the methods and materials described herein are preferred, other methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention as well.

[0109] All documents cited or referenced herein are hereby incorporated by reference and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0110] The following examples and figures are intended to illustrate further embodiments of the present invention without limiting its scope.

[0111] Examples

[0112] 1 . Preparation of the root extract of Pseudostellaria heterophylla a. Solid / liquid extraction:

[0113] Root of Pseudostellaria heterophylla were collected and cleaned, dried and grinded. The crude powder was extracted under reflux in hydroethanolic solvent containing 60% ethanol (v / v), 7 times for 2 hours. The solution obtained was diluted 6 times, concentrated under reduced pressure at 50°C and then spray dried for obtaining a dried powder having 8.3 g / L of dry matter (w / v of solvent). 200 g of this dried powder was solubilized in 1 L of a demineralised water, stirred at 500 rpm for 1 h at room temperature, and centrifugated. The supernatant was filtrated to getting rid of the insoluble material and filtered for obtaining a supernatant. b. Fractionation step:

[0114] A chromatography resin column (Diaion® HP20, Thermo Fischer Scientific) was previously washed with ethanol and the with water. Then, the supernatant was loaded through the resin column at flowrate of 6 mL / min (6.4 BedVolume / mn). The column resin was eluted by passing 190 mL of a mixture of water / ethanol at 80 / 20 (v / v) of ethanol at a flowrate of 3 mL / min (3.2 BedVolume / mn). The related fraction was collected.

[0115] The resin column was further eluted with 380 mL of a mixture of water / ethanol having 90% (v / v) of ethanol at a flowrate of 3 mL / min (3.2 BedVolume / mn) for obtaining the cyclopeptide-rich root fraction of Pseudostellaria heterophylla, which contains:

[0116] - polyphenols, less than 900 ppm

[0117] - 641 mg / L of cyclopeptides: pseudostellarins A to E and G, wherein heterophyllin B represented 291 mg / L of solvent, meaning heterophyllin B represents 3.5% by weight over the weight of the dry extract.

[0118] - heterophyllins A, C and J

[0119] The cyclopeptide-rich root fraction of Pseudostellaria heterophylla obtained was mixed with 50 mL of demineralized water and then concentrated under rotary evaporator at 50°C under reduced pressure until a final mass of maximum 50 g. Glycerine and water were added in the ratio of 80 / 20 (v / v) respectively. The hydro glycerinated extract was further clarified by filtration at 1 pm (hereinafter referred as “PHE”), so that PHE is the cosmetic active ingredient according to the invention. 2. In vitro experiments and results

[0120] A transcriptom ic analysis was conducted after one day of treatment of skin explants with a formulation containing PHE. Results are expressed in relative expression ratio between the control group and the treated group.

[0121] All data were expressed as mean ± standard deviations. The statistical significance of the data was determined by Student t-tests with Minitab software as all data were normally distributed. Significance is expressed as following: ***: p<0.001 ; **: p<0.01 ; *: p<0.05; ns: p>0.05. a. PHE improves epidermal barrier function and extracellular matrix integrity

[0122] Methods: Upon receiving of the abdominal biopsy, the adipose tissue was removed, skin explants were then cut into pieces (2 cm x 2 cm). The tested compounds - PHE and positive controls - were then topically applied (5 mg / cm2) or not (control) on the surface of the skin explants before its incubation for 24 hours in an humidified incubator at 37°C and 5% CO2.

[0123] All experimental conditions were performed in n=3.

[0124] At the end of incubation, the skin explants were washed in phosphate buffered saline (PBS) solution and placed in RNAIater at 4°C for 24 hours and then frozen at -20°C until RNA extraction. Samples were mechanically homogenized using a Precellys® Evolution homogenizer (Bertin Instrument) and total RNA was extracted from each sample using TriPure Isolation Reagent® according to the supplier’s instructions.

[0125] The amount and quality of RNA were evaluated using capillary electrophoresis (Bioanalyzer 2100, Agilent technologies).

[0126] PCR (Polymerase Chain Reaction) were performed using the « LightCycler® » system (Roche Molecular System Inc.) according to the supplier’s instructions. Results:

[0127] Table 1 shows a significant upregulation of the transcription of some genes related to retinoid signalling pathways.

[0128] The p450 cytochromes are hemoproteins responsible for the oxidative metabolism of a wide variety of molecules of endogenous origin. They enable the detoxification and elimination of xenobiotics, preventing the accumulation of potentially toxic substances in the body.

[0129] In retinoid metabolism, p450 cytochromes such as CYP1A1 and CYP1A2 are involved in the synthesis of retinoic acid, but also in the inactivation and / or degradation into polar metabolites of certain retinoid species not used by the cell.

[0130] Recent generation of retinoids aims to focus specifically on RAR-B or RAR-G to avoid side effects - mainly cutaneous inflammation - of retinoids. Thanks to its selectivity against RAR-G. Trifarotene, the last generated retinoid, shows a strong efficacy to improve retinoid-modulated pathways involved in epidermal differentiation, acne prone-skin resolution. Moreover, the targeting of RAR-G demonstrates an antiinflammatory property and depigmenting activity.

[0131] Results demonstrated a significant increase in RAR-G transcription after PHE treatment, a key player in signalling for retinoic acid effect on acne, pigmentation and inflammation.

[0132] Table 2 shows significant increased transcription of genes involved in the mitochondrial biology such as mitofusin-1 (MFN1 ), in the regulation of oxidative stress generated by ROS thanks to the superoxide dismutase 2 gene (SOD2), and in the proliferative ability of cell by the positive regulation of PCNA (Proliferating Cell Nuclear Antigen). These results highlight the beneficial effect of PHE to regulate the proliferation and the oxygen active species generation. As it ages, the epidermis becomes thin, due in part to a lack of cell renewal and an excessive generation of ROS, which exacerbate skin aging and inflammation.

[0133] Table 3 shows positive regulations of several genes involved in skin barrier function and epidermal differentiation process. Keratin 1 (KRT1 ) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. Their upregulation precedes expression of filaggrin and cornified envelope proteins. This led to the hypothesis that a subset of keratins and the associated protein filaggrin are crucial for barrier function.

[0134] In the outermost layer of the stratum granulosum, Loricrin is involved in the formation of cornified envelope beneath the plasma membrane, participating to the cornified envelope mechanical resistance. It was demonstrated an increase of CALML5 (Calmodulin-like protein 5) which is also involved in terminal differentiation of keratinocytes as well as desmocollin 1 (DSC1 ) and desmoglein (DSG1 ), which constitute the adhesive proteins of the desmosome cell-cell junctions and are required for cell adhesion and desmosome formation.

[0135] Kallikreins (KLKs) are essential proteins involved in skin desquamation by degrading corneodesmosmes that are junctional structures allowing adhesion of corneocytes. Corneodesmosmes are composed of one extracellular protein, corneodesmosin (CDSN), and two transmembrane proteins, desmoglein 1 (DSG1 ) and desmocollin 1 (DSC1 ). Degradation of these proteins by KLKs allows for proper skin desquamation and is required for skin homeostasis.

[0136] The interior of corneocytes consists mainly of keratin filaments aggregated by filaggrin (FLG) protein. FLG degradation products account in part for the water-holding capacity and maintenance of acidic pH of the stratum corneum, both crucial for epidermal barrier homoeostasis by regulating activity of multiple enzymes that control desquamation, lipid synthesis and inflammation.

[0137] By increasing keratins and adhesion proteins such as DSC1 and DSG1 , PHE shows its ability to improve skin barrier function. By increasing kallikreins, useful for skin desquamation and epidermal renewal, PHE shows its ability to reinforce skin homeostasis.

[0138] Table 4 shows that PHE increases significantly the transcription of genes involved in extracellular matrix composition and dermis remodelling.

[0139] It was observed that PHE upregulates genes encoding collagen subunits (COL1A1 , COL3A1 , COL4A2) which are known to be retinoid-responsive genes. Moreover, it was demonstrated an upregulation of the elastin gene (ELN).

[0140] TIMP1 encodes a natural inhibitor of the matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, the encoded protein is able to promote cell proliferation in a wide range of cell types and may also have an anti-apoptotic function.

[0141] In a nutshell, PHE shows a similar regulation of gene involved in the epidermal differentiation, the cellular renewal, the dermal extracellular matrix component regulation and dermal epidermal junction composition than retinol and bakuchiol. b. PHE improves the synthesis of collagen type I As shown in Figure 1 , PHE is capable to potentialize the synthesis of collagen I by human fibroblasts.

[0142] Collagen type I is a very important structural protein which provide to the skin its strength and elasticity. Aging - chronological and photoaging - is associated to a decrease of collagen production and an increase of its degradation. Collagen type I synthesis is thus a good marker to assess the efficacy of an anti-aging active. Moreover, it is well known that both retinol and bakuchiol promote the collagen formation and application to the skin has been shown to reduce wrinkles and fine lines.

[0143] Methods:

[0144] Human primary fibroblasts (NHDF) from 3 different donors (35 years old, 47 years old and 50 years old) were treated 3 days either by retinol at 10 ppm, bakuchiol at 10 ppm, PHE containing 0.10 ppm of heterophyllin B (HB), and pure HB at 0.15 ppm. The amount of pro-collagen I released in supernatants was dosed by ELISA test according to manufacturer’s instruction and compared to non-treated controls. All values were expressed in percentage of control.

[0145] Results:

[0146] In NHDF, retinol at 10 ppm is a powerful agent to potentialize the synthesis of procollagen I, and that bakuchiol at 10 ppm is less effective (+57% compared to control and +150% for retinol). It was demonstrated that PHE - containing 0.10 ppm of HB - significantly increases the pro-collagen I release by fibroblasts at the same level than retinol (+164%). Interestingly, the treatment of cells by HB alone highlights that the effect of PHE was mainly supported by its heterophyllin B content.

[0147] In a nutshell, this result demonstrates the potential of PHE, as a natural alternative of retinol, to be an efficient skin anti-aging agent. c. PHE decreases the synthesis of pro-inflammatory cytokine

[0148] As shown in Figure 2, both PHE and HB are capable to reduce the secretion of interleukin-6 (IL-6).

[0149] Retinoids reduce several signs of skin aging, but they also have many side effects such as cutaneous erythema, pruritus, peeling, stinging or burning, and sensitivity.

[0150] Methods: NHDF from a 47-year-old donor were treated 5 days either by PHE - containing 0.10 ppm of HB - or heterophyllin B at 0.10 ppm. The amount of IL-6 released in supernatants was dosed by ELISA test according to the instructions of manufacturer and compared to respective non treated controls. All values were expressed in the percentage of control.

[0151] Results:

[0152] It was demonstrated that both PHE and HB, significantly decrease by 60% and 63% respectively, the secretion of IL-6 after 5 days of treatment. IL-6 is synthetized at the local lesion in the initial stage of inflammation. Interestingly, it was shown that neither retinol nor bakuchiol significantly reduce the IL-6 synthesis by NHDF.

[0153] Hence, it was demonstrated the potent anti-inflammatory effect of both PHE, and HB reinforcing their application as natural alternatives to retinol. d. PHE decreases the secretion of melanin by melanocytes

[0154] As shown in Figure 3, both PHE at different concentrations, and HB content, is capable to reduce the melanin content in melanocytes.

[0155] Retinoids are also known to influence the function of melanocytes, providing regular arrangement of melanin in the epidermis. They also block transport of melanin to epidermal cells and diminish the activity of stimulated melanocytes.

[0156] Methods:

[0157] B16-F1 cells were treated 4 days with either kojic acid 0.3% or PHE at three different concentrations corresponding to the content of HB of 0.21 ppm, 0.10 ppm or 0.035 ppm. At the end of treatment, the dosage of secreted melanin was done by optical density measure at 475 nm. Protein normalization was applied for all conditions after a Bradford analysis and plate read at 595 nm.

[0158] Results: It was shown that PHE is able to decrease in a HB content dose-dependent manner the release of melanin by B16-F1 melanocytes (by up to 60%, 54% and 48% for 0.21 ppm of PHE, 0.10 ppm of PHE and 0.035 ppm of HB respectively).

[0159] Hence, it was demonstrated the potent anti-pigmentation activity of PHE and its use to counteract the irregular hyperpigmentation observed in skin aging.

[0160] 3. Clinical evaluation

[0161] 3.1 . Formulated PHE compared to placebo formula a. Material and methods

[0162] A randomized double-blind placebo controlled clinical trial was conducted to evaluate the effects of the PHE on the skin. The study was conducted in accordance with the principle of the Declaration of Helsinki and the guidelines of the International Conference on Harmonization Good Clinical Practice, as applicable to a non-drug study.

[0163] All data were expressed as mean ± standard deviations. The statistical significance of the data was determined by Student t-tests with Minitab software as all data were normally distributed. Significance is expressed as following: ***: p<0.001 ; **: p<0.01 ; *: p<0.05; ns: p>0.05.

[0164] 21 Caucasian females, with an age range of 45-65 years old (average 55 ± 2), were selected. The volunteers presented a lack of firmness and redness on the face and a dull complexion. They were asked to apply a cream formulation containing PHE at 0.3% by weight and a placebo, on each half of their face, randomly distributed, twice a day for 56 days.

[0165] Table 5: Cream formulation containing PHE at 0.3% by weight and placebo

[0166] Biometrological measurements were done at day 0, after 28 days and after 56 days of application of the cream. To assess whether the formulations improve the skin complexion, it was evaluated the brightness and the redness of the skin respectively thanks to the measurements of L* parameter and a* parameter using a spectrophotometer at day 0 and day 28. The redness is a component of skin color, but it also reflects the level of irritation of the skin. To calculate the mechanical properties of the skin, the evaluation was focused on parameters related to firmness and sagging. The effect on sagging was assessed through the scoring of ptosis at day 0 and at day 28 independently by 3 trained experts, using the validated clinical scale Ptosis from the “Skin Aging Atlas, Volume 1 , Caucasian type”. This data was completed by the firmness analysis through RO parameter measurements using a cutometer at day 0, day 28 and day 56, with an additional timepoint to highlight the effect over time. b. Results

[0167] Figure 4A shows a significant improvement of skin brightness materialized by an increase variation of the L* parameter by 1 .9% from day 0 to day 28 with PHE whereas the variation with the placebo is non-significant. Moreover, the effect of PHE was significantly different from the one obtained with the placebo, and it was 84% higher. Additionally, the part of volunteers presenting an improvement of the L* parameter was higher in the PHE group (90%) than in the placebo group (57%).

[0168] Figure 4B demonstrates a significant decrease of skin redness (a*: -10.3%) from day 0 to day 28 with PHE compared to the placebo (a*: -4.1 %). Furthermore, the effect of PHE was significantly different from the one obtained with the placebo, and it was 60% higher. Additionally, the part of volunteers presenting an improvement of the a* parameter was higher in the PHE group (81 %) than in the placebo group (62%).

[0169] As the a* parameter is linked to redness, itself closely correlated to the skin irritation, the above results confirm that the effects observed during the in vitro experiments on inflammatory mediators (IL-6) lead to the improvement of skin soothing and the prevention of skin irritation. Therefore, these results corroborate the ability of PHE as being a non-irritant alternative of retinol. The above results demonstrate that PHE is not irritant, it even exhibits a soothing effect on the skin.

[0170] Hence, it was clinically demonstrated by the measurement of L* and a* parameters, PHE is capable to improve the complexion of the skin.

[0171] Aging, particularly photoaging, is known to decrease the mechanical properties of the skin. Indeed, skin loses firmness ultimately leading to sagging.

[0172] Sagging of skin was evaluated independently by 3 trained experts and figure 5A displays a significant decrease of skin sagging variation (-10.7%) from day 0 to day 28 treated by PHE against a non-significant variation for the placebo treatment. Moreover, the effect of PHE was significantly different from the one obtained with the placebo, and it was 84% higher. Additionally, the part of volunteers presenting an improvement of sagging was much higher in the PHE group (95%) than in the placebo group (38%).

[0173] Skin firmness was measured by the RO cutometer parameter which decreases if the skin is firmer. From this decrease, taking the absolute value of the percentage of variation, a positive increase of firmness can be evaluated. Figure 5B shows the ability of PHE to improve the skin firmness which was enhanced over time: 12.6% from day 0 to day 28 and was increased to 19% to day 56. On the contrary, the placebo did not present any significant improvement of the firmness in any timepoint. Moreover, the effect of PHE was significantly different from the one obtained with the placebo at both measured timepoints, and it was 95% and 84% higher, respectively at day 28 and day 56. For the firmness parameter also, the part of volunteers presenting an improvement is higher in the PHE group (100% at day 28 and 56) than in the placebo group (60% at day 28 and 65% at day 56). Finally, the clinical studies demonstrated the efficiency of PHE in improving the skin complexion at day 28 according to the measurements of both brightness and redness. The redness decrease could be linked to a soothing effect, which was completed by an improvement of sagging at day 28, and firmness at day 28 which was further enhanced at day 56.

[0174] Taken altogether, these results demonstrate the efficiency of the root extract of Pseudostellaria heterophylla of the present invention in combating and improving globally the signs of skin aging.

[0175] 3.2. Formulated PHE compared to formulated bakuchiol a. Methods

[0176] A randomized double-blind clinical trial was conducted to compare the effects of PHE and bakuchiol. The tested dose of bakuchiol was of 0.5% as it is the recommended dose. The clinical study was conducted in accordance with the principle of the Declaration of Helsinki and the guidelines of the International Conference on shows The same timepoints and measurements as in the above clinical study (Cf. point 3.1 ) were performed (L* by spectrophotometry, sagging by scoring at Day 0 and 28, RO by cutometry at day 0, 28 and 56).

[0177] 12 Caucasian females, with an age range of 45-65 years old (average 55 ± 3), were selected. The volunteers presented lack of firmness and redness on the face and a dull complexion. They were asked to apply the formulations containing PHE at 0.3% (table 6) or bakuchiol at 0.5% on each half of their face, randomly distributed, twice a day for 56 days.

[0178] Table 6: Cream formulation containing PHE at 0.3% by weight and bakuchiol at 0.5%

[0179] Figure 6A shows a significant improvement of skin brightness materialised by the increase of L* parameter by 4.3% for PHE and by 3.5% for bakuchiol from basal values. PHE and bakuchiol can be considered as efficient as each other as their effects are non-significantly different. Moreover, both formulations had an effect on 100% of volunteers at day 28.

[0180] Bakuchiol is known to improve the firmness of the skin. Figure 6B indicates a significant increase of skin firmness which is enhanced over time for both PHE and bakuchiol. Additionally, the variations have a high significance for both PHE and bakuchiol compared to day 0. Bakuchiol increased the firmness about 8.8% at day 28 then 17.0% at day 56 and PHE about 7.9% at day 28 then 14.9% at day 56. The efficiency of both PHE and bakuchiol is equivalent as the difference between the 2 products are nonsignificant at both timepoints. Additionally, both formulations had an effect on 100% of volunteers at day 28 and day 56.

[0181] Ultimately, the sagging of the skin for both PHE and bakuchiol at day 28 was measured. Sagging of skin is one of the most difficult consequences of skin aging to address. Figure 6C displays a significant decrease by 8.9% from day 0 to day 28 with bakuchiol with an efficiency of 67% on the volunteers. Interestingly, PHE showed a better performance as the decrease was higher combined with a higher significance: - 12.8%. Additionally, the part of volunteers with improvement was also higher than bakuchiol as it led to an improvement on 100% of the volunteers. The performance ability of PHE in improving sagging aspect of the skin was significantly superior compared with bakuchiol. Results obtained according to all the above experiments - in vitro and clinical - confirmed the efficiency of PHE as well as heterophyllin B in preventing the signs of skin aging, for improving the global appearance of the skin. Bakuchiol being as efficient as retinol on hyperpigmentation after 12 weeks of use as demonstrated in previous studies (Dhaliwal et al., Br Journal of Dermatology, 2019;180(2):289-296), it can be postulated that the root extract of Pseudostellaria heterophylla of the present invention is efficient as retinol also on these parameters. Moreover, the root extract of Pseudostellaria heterophylla of the present invention performed better on skin sagging counteraction than bakuchiol.

Claims

Claims1 . A root extract of Pseudostellaria heterophylla comprising:- one or more phenolic components,- one or more pseudostellarins,- one or more heterophyllins, including heterophyllin B, wherein the heterophyllin B represents at least 0.5% by weight relative to the total weight of the dry extract.

2. A root extract according to the claim 1 , wherein pseudostellarins are selected from the group consisting of pseudostellarins A to E and G, preferably at least two thereof, more preferably at least three or more, in particular a combination thereof.

3. A root extract according to any of the claim 1 or 2, wherein heterophyllins are selected from the group consisting of heterophyllins A, C and J, preferably two or more thereof, in particular a combination thereof.

4. A root extract according to any of the claims 1 to 3, wherein the heterophyllin B represent at least 1 % by weight, more preferably at least 1 .5% by weight, even more preferably at least 2% by weight, in particular at least 2.5% by weight relative to the total weight of the dry extract.

5. A root extract according to any of the claims 1 to 4, wherein the heterophyllin B represents approximatively between 2 to 55% by weight relative to the weight of the total cyclopeptides.

6. A root extract according to any of the claims 1 to 5, wherein the total cyclopeptides content represent between 3.5% to 45%, preferably from 5% to 42.5%, more preferably from 7.5% to 40%, in particular from 10% to 37.5% relative to the total weight of the dry extract.

7. A root extract according to any of the claims 1 to 6, wherein the phenolic components range between 5% to 15%, preferably between 7.5% to 12.5% by weight relative to the total weight of the dry extract.

8. A root extract according to any of the claims 1 to 7, wherein the root extract is an hydroalcoholic extract, preferably a hydroglycolic extract.

9. A cosmetic active ingredient comprising the root extract of Pseudostellaria heterophylla according to any of the claims 1 to 8 in a cosmetically acceptable vehicle selected from the group consisting of water, aqueous buffer, alcohols, glycols, ethyl lactate, isopropyl myristate, triglycerides, tri-ethyl citrate, dicaprylyl ether, glyceryl isostearate, glyceryl stearate, ethyl acetate or a mixture thereof.

10. A method for preparing a root extract of Pseudostellaria heterophylla comprising the steps of: a. performing a solid / liquid extraction of a root, optionally dried, of Pseudostellaria heterophylla, by contacting the root with a polar solvent for obtaining a solution, b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution for obtaining a powder, c. performing a fractionation step on: i. the concentrated solution obtained in step b); or ii. the powder solubilized in water obtained in step b); d. recovering the cyclopeptides containing fraction obtained in step c).

11. The method according to the claim 10, wherein step c) is performed in a chromatographic resin column by eluting with water / alcohol, wherein alcohol represents between 5 to 95% (v / v).

12. The method according to any of the claims 10 or 11 , wherein step c) is conducted by c.1 ) eluting with a mixture of water / alcohol wherein alcohol represents 5 to 20% (v / v); and c.2) eluting with a mixture of water / alcohol wherein alcohol represents 80 to 95% (v / v).

13. The method according to any of the claims 10 to 12, wherein eluting in c.1 ) is conducted with a mixture of ethanol / water containing 5% (v / v) of ethanol, preferably 10% (v / v) of ethanol, more preferably 20% (v / v) of ethanol, and wherein eluting in c.2)is conducted with a mixture of ethanol / water containing 80 to 95% (v / v) of ethanol, preferably 85 to 90% (v / v) of ethanol.

14. A cosmetic or pharmaceutic composition comprising:- a root extract of Pseudostellaria heterophylla, according to any of the claims 1 to 8, as an active ingredient; and- at least one further cosmetically and / or pharmaceutically acceptable ingredient other than the root extract of Pseudostellaria heterophylla, wherein the composition is a composition for topic use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampooing, and a spray; and / or wherein the composition is a nutraceutical composition which is administered orally.

15. Cosmetic use of a root extract of Pseudostellaria heterophylla according to any of the claims 1 to 8, or a cosmetic active ingredient according to the claim 9, or a composition comprising a root extract according to claim 14, for combating and / or preventing the signs of aging on the skin and the hair follicles, for improving the global appearance of the skin, in particular for improving the firmness, the radiance, the brightness, the hydration of the skin, for decreasing and / or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

16. The cosmetic use according to the claim 15, wherein the root extract is for: a. improving the complexion of the skin, b. reinforcing and / or restoring the epidermal barrier function, c. increasing the synthesis of collagen and improving the extracellular matrix components, d. increasing retinoic acid receptor gamma transcription, e. improving skin cell regeneration and epidermal differentiation, f. regulating oxidative stress in skin cells, g. improving skin soothing, h. improving skin defense, i. reinforcing the homeostasis of the skin,j. counteracting the hyperpigmentation on the skin, k. improving the skin microbiota l. combination of two or more thereof.

17. Cosmetic use of heterophyllin B for combating and / or preventing the signs of aging on the skin and the hair follicles, in particular for reducing wrinkles and fine lines, for improving the firmness and / or the elasticity of the skin, for counteracting the irregular hyperpigmentation in skin, for alleviating skin inflammation, or for two or more thereof.

18. Cosmetic composition comprising heterophyllin B for use according to the claim 17, wherein heterophyllin B represents between 0,00001 to 2%, preferably 0,00005 to 1 ,5%, more preferably between 0,0001 to 1 % by weight relative to the total weight of the composition, and wherein the composition further comprises cosmetically acceptable carrier.

19. A non-therapeutic method for combating and / or preventing the signs of aging on the skin by topically applying an effective amount of heterophyllin B onto the skin on need, as an active ingredient, wherein preventing signs of aging on the skin is for reducing wrinkles and fine lines, for improving the firmness and / or the elasticity of the skin, for counteracting the irregular hyperpigmentation in skin, for alleviating skin inflammation, or for two or more thereof.

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