Pseudostellaria heterophylla root extract and application thereof

Abstract

The present invention relates to a root extract of the plant Pseudostellaria heterophylla, to a composition comprising the root extract of Pseudostellaria heterophylla, to a method for the preparation thereof, and to the use thereof. The invention also relates to the cosmetic use of a composition comprising a root extract of the plant Pseudostellaria heterophylla, for combating the signs of aging, for improving the overall appearance of the skin. In addition, the invention also relates to the cosmetic application of the pseudostellaria heterophylla cyclic peptide B, which can be obtained from the root extract of the plant pseudostellaria heterophylla, as an anti-aging agent, especially as an anti-wrinkle agent.

Description

Technical Field

[0001] The present invention relates to a root extract of the plant Pseudostellaria heterophylla, a composition comprising the root extract of Pseudostellaria heterophylla, a method for preparing the same and its use. The present invention also relates to the cosmetic use of the composition comprising the root extract of the plant Pseudostellaria heterophylla for combating signs of aging and for improving the overall appearance of the skin. In addition, the present invention also relates to the cosmetic use of Pseudostellaria heterophylla cyclotide B obtainable from the root extract of the plant Pseudostellaria heterophylla as an anti-aging agent, especially as an anti-wrinkle agent. Background Art

[0002] The skin is the first barrier of the human body, protecting organs from attacks from the external environment. However, excessive chemical and physical stimulation impairs the normal function of the skin and causes its aging.

[0003] Skin aging is a multifactorial process driven by intrinsic factors (e.g., time, genetic factors, hormones) and extrinsic factors (e.g., UV exposure, pollution). Characteristics of intrinsic or time-dependent aging include visible signs such as thin and dry skin, fine wrinkles, reduced elasticity, abnormal pigmentation. Among the causes of epidermal thinning, a reduction in the proliferation and renewal capacity of basal keratinocytes has been reported. During the aging process, the extracellular matrix undergoes structural changes and degradation, leading to increased wrinkles and loss of skin elasticity.

[0004] Retinoids are well-known active agents used for cosmetic purposes in preventing aging, treating acne and psoriasis, and other skin disorders.

[0005] Vitamin A and its derivatives, especially retinol, are the most effective substances in slowing down the aging process. In keratinocytes, retinol stimulates cell activity, promotes their proliferation, strengthens the protective function of the epidermis, and reduces transepidermal water loss.

[0006] Retinoids, such as retinol, retinal and retinyl palmitate, are known to have many beneficial effects, such as improving skin elasticity, inhibiting matrix metalloproteinases and enhancing the synthesis of tissue inhibitors of metalloproteinases, reducing skin pigmentation and shrinking stimulated melanocytes. In addition, skin damage caused by ultraviolet radiation can be partially reversed by the application of retinoic acid.

[0007] Several clinical studies have demonstrated that retinol preparations lead to significant improvement in fine lines in combination with in vitro assays of matrix metalloproteinases, collagenases secreted by fibroblasts.

[0008] Although retinol is one of the most commonly used skin anti-aging agents in the cosmetic field, it is also known to cause irritation, erythema, itching, scaling, stinging or burning sensations and sensitivity on the skin, all of which constitute drawbacks. Therefore, in order to avoid or limit these drawbacks, retinol is usually stabilized and / or encapsulated in a delivery system, such as liposomes.

[0009] Pure bakuchiol is the first active molecule to replace retinoids such as retinol. Bakuchiol is a meroterpene found primarily in the seeds of Psoralea corylifolia that has been shown to possess anti-inflammatory, antioxidant, anti-aging, and anti-acne activities. Mechanistically, bakuchiol reportedly targets several cellular pathways similar to those targeted by retinoids, including upregulation of many retinoid-responsive genes and collagen and extracellular matrix synthesis enzymes, while having no effect on genes encoding retinoic acid receptors (RAR-B and RAR-G). All of these modulations result in noticeable effects on the skin, namely improvements in wrinkles, pigmentation, and firmness.

[0010] Pseudostellaria heterophylla, also known as Kid Ginseng, Child Ginseng, Crown Prince Ginseng or False Starwort, is an adaptogenic plant belonging to the Caryophyllaceae family, which is used in traditional Chinese medicine and herbalism.

[0011] The roots of Pseudostemon heterophylla contain two types of cyclic peptides (also called cyclic peptides or ringpeptides): pseudostemon heterophylla and heterophylla. The roots of Pseudostemon heterophylla additionally contain polyphenols, sugars, and polysaccharides, the latter being considered to be one of the main bioactive components of the entire extract. Both the nature and the amount of the aforementioned components are significantly affected by many factors, such as the region where the plant grows, its origin and variety, and the extraction process using the roots. Therefore, the active components and their amounts may vary even between the same plant species.

[0012] The root extract of Pseudostemon heterophylla contains Pseudostemon heterophylla cyclotide A, B, D, J and Pseudostemon heterophylla cyclotide A to H (Hu et al., Chinese Medicine, 14:21, 2019). The alcohol root extract of Pseudostemon heterophylla contains Pseudostemon heterophylla cyclotide A, B, D and Pseudostemon heterophylla cyclotide A to F and G (Hu et al., Molecules, 21, 1538, 2016). It is reported that the quantity and quality of active components in wild and cultivated Pseudostemon heterophylla show significant differences depending on the ecological environment. In fact, the methanol root extract of Pseudostemon heterophylla contains cyclotides A to E and G (Chao Han et al., Journal of Chromatography B, 862, 125-131, 2008), while the methanol root extract of the same plant from another region contains Pseudostemon heterophylla cyclotides A to J and Pseudostemon heterophylla cyclotides A to H (Pomilio et al., Current Organic Chemistry, 10, 2075-2121, 2006). The roots of Pseudostemon heterophylla extracted with ethyl acetate contain Pseudostemon heterophylla cyclotides A, B, D, J and Pseudostemon heterophylla cyclotides A to G, fatty acids and amino acids (Lu et al., Frontiers in Pharmacology, 11, 850, 2020).

[0013] The use of extracts from the roots of Chickweed is known in the cosmetic field.

[0014] For example, patents CN-B 107929127 and CN-B 109528804 disclose that the fibrous roots of Pseudostemon heterophylla extracted with polar solvents contain cyclic peptides. The fibrous root extracts obtained can be used for skin treatment. CN-A 104138342 discloses an ethanol extract of the fibrous roots of Pseudostemon heterophylla containing cyclic peptides that can be used to protect DNA, retain moisture due to polysaccharides, and act as anti-inflammatory agents. CN-A105106280 discloses a water-methanol extract of the roots of Pseudostemon heterophylla titrated in cyclotide B for inhibiting tyrosinase activity. JPH-A 07324095 discloses cyclotides A to G obtained from the root extract of Pseudostemon heterophylla after solid resin fractionation, which are used to inhibit tyrosinase activity. More generally, purified tropanopeptides A to E and G are known to inhibit tyrosinase activity and melanin formation (Morita et al., Tetrahedron, 50, 33, 1994).

[0015] The extracts of the above-mentioned Schizonepeta heterophylla have several technical drawbacks. Since the roots of Schizonepeta heterophylla contain large amounts of both polyphenols and polysaccharides, the relevant biological effects are mainly related to these components. Polyphenols may be chemically unstable over time and often result in a dark color of the final extract, which may be undesirable in a formulated cosmetic product.

[0016] As cosmetic consumers are always looking for effective products to combat skin aging, it is highly desirable to have available natural products like retinols, which are most acceptable to the end consumer, meaning products with equivalent efficacy to retinol, but without the side effects and which are easy to formulate at a low cost.

[0017] Therefore, it is desirable to treat the root extract of Pseudostemon heterophylla with a lower content of both polysaccharides and polyphenols, but rich in cyclic peptides, before processing, which is later considered to be more stable and expected to be biologically more effective even at a lower content.

[0018] It would also be desirable to provide a cyclic peptide based root extract of Chickweed heterophylla which has different and / or improved biological effects on the skin than those known in the art to cause any irritation on the skin and which can be used, for example, in skin care products.

[0019] There remains an unmet need to provide a root extract of Chickweed heterophylla having a conventionally high cyclic peptide content that is guaranteed to be stable over time and independent of the seasons and geographic origin of the plant.

[0020] In general, peptides are known to show beneficial effects even at low concentrations and can be used in many cosmetic applications. Of all forms of peptides, the cyclic form appears to be advantageously more effective compared to linear peptides, probably due to its chemical stability over time and its three-dimensional structure that is not easily accessible to enzymes, resulting in weak degradation. In addition, cyclic peptides can pass through the dermis more easily than linear peptides. Therefore, it would be very desirable for cosmetic purposes to have a composition essentially based on cyclic peptides, from natural sources, showing efficacy equivalent to that of retinol without the side effects. Summary of the invention

[0021] A first aspect of the present invention relates to a root extract of Pseudostemon heterophylla comprising:

[0022] - one or more phenolic components,

[0023] - one or more cyclotides of ginseng,

[0024] - one or more Pseudostellariae cyclotide, including Pseudostellariae cyclotide B,

[0025] Among them, relative to the total weight of the root extract, the content of Pseudostellaria heterophylla cyclotide B is equivalent to at least 10 mg / kg.

[0026] Another aspect of the present invention relates to a method for preparing a root extract of Pseudostemon heterophylla, comprising the following steps:

[0027] a. by contacting the optionally dried roots of Pseudostemon heterophylla with a polar solvent, performing a solid / liquid extraction of the roots to obtain a solution,

[0028] b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution to obtain a powder,

[0029] c. Perform the following classification steps:

[0030] i. the concentrated solution obtained in step b); or

[0031] ii. the powder obtained in step b) dissolved in a polar solution;

[0032] d. recovering the cyclic peptide-containing fraction obtained in step c).

[0033] Another aspect of the present invention relates to a cosmetic or pharmaceutical composition comprising:

[0034] - the root extract of Pseudostemon heterophylla as active ingredient,

[0035] - at least one additional cosmetically and / or pharmaceutically acceptable ingredient other than the root extract of Schefflera heterophylla,

[0036] Wherein, the composition is a composition for topical use selected from the group consisting of: solutions, suspensions, emulsions, creams, pastes, gels, lotions, powders, soaps, water containing surfactants, oils, lotions and sprays; and / or wherein the composition is a health food composition for oral administration.

[0037] The invention also relates to the cosmetic use of a composition comprising as active ingredient an extract of the roots of Pseudostemon heterophyllus or a composition as defined above, for combating and / or preventing signs of aging of the skin and hair follicles, for improving the overall appearance of the skin, in particular for improving the firmness, elasticity, radiance, brightness, hydration of the skin, for reducing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

[0038] Another aspect of the present invention relates to a non-therapeutic method for preventing and / or for delaying the signs of aging in the skin of a subject by applying to the skin in need thereof an effective amount of a composition comprising as active ingredient a root extract of Pseudomonas heterophylla or a composition as defined above.

[0039] Yet another aspect of the invention relates to the cosmetic use of Pseudostellaria cyclotide B obtainable (or obtained) from a root extract of Pseudostemon heterophylla for preventing and / or reducing the appearance of fine lines or wrinkles, in particular for promoting collagen synthesis, for reducing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] exist Figure 1 In the present invention, the content of type I procollagen released by normal human dermal fibroblasts (NHDF) treated or not treated with the root extract of Pseudostemon heterophylla of the present invention or with retinol or with bakuchiol for 3 days was measured by ELISA test. The results are expressed as the percentage change compared with the control condition.

[0041] exist Figure 2 In the present invention, the content of IL-6 released by normal human skin fibroblasts (NHDF) treated or not treated with the root extract of Pseudostemon heterophylla of the present invention or with retinol or with bakuchiol for 5 days was measured by ELISA test. The results are expressed as the percentage change compared with the control condition.

[0042] exist Figure 3 The extracellular melanin content was measured in B16-F1 cells treated or untreated with kojic acid (positive control) or with the root extract of Pseudostemon heterophyllum of the present invention at different concentrations (0.3%, 0.15% and 0.05%). The released melanin was measured by optical density (OD). The results are expressed as a percentage change compared to the untreated condition.

[0043] FIG. 4 shows that the root extract of Pseudostemon heterophylla of the present invention improves the lightness (measured as parameter L*, Figure 4A ) and reduced redness (measured by parameter a*, Figure 4B ) and the ability to improve skin complexion. For both parameters (L*, a*), the proportion of volunteers with improvement is shown below the corresponding graphs for the placebo and the root extract of Pseudochia heterophylla of the invention.

[0044] Figure 5 shows the improvement in the mechanical properties of the skin. Figure 5A The ability of the root extract of Scutellaria heterophylla of the present invention to reduce skin sagging after topical application for 28 days was demonstrated. Figure 5B Shown is an improvement in skin firmness measured by reduction of the R0 cutometer parameter after topical application for 28 and 56 days, which increases over time. For both parameters (sagging and firmness), the proportion of volunteers with improvement is shown for the placebo and the root extract of Pseudo-Schlumbergera heterophylla of the invention below the corresponding graph.

[0045] FIG. 6 shows the comparison between the root extract of Pseudostemon heterophylla of the present invention and Bakuchiol after topical application for 28 days with respect to brightness ( Fig. 6A ), sagging after 28 days of topical application ( Figure 6B ) and firmness after 28 and 56 days of topical application ( Figure 6C For all parameters, the proportion of volunteers with improvement is shown below the corresponding graphs for the root extract of Pseudochia heterophylla of the present invention and Bakuchiol. DETAILED DESCRIPTION

[0046] A first aspect of the present invention relates to a root extract of Pseudostemon heterophylla comprising:

[0047] - one or more phenolic components,

[0048] - one or more cyclotides of ginseng,

[0049] - one or more Pseudostellariae Radix Cyclopeptides, including Pseudostellariae Radix Cyclopeptide B,

[0050] Among them, relative to the total weight of the root extract, the content of Pseudostellaria heterophylla cyclotide B is equivalent to at least 10 mg / kg.

[0051] As used throughout this disclosure, the root extract of Pseudostemon heterophyllum of the present invention is also designated as "(the) extract" or "root extract" or by its abbreviation "PHE" to represent the Pseudostemon heterophyllum extract.

[0052] As used herein, the term "extract" may be any substance made by extracting the roots of Pseudostemon heterophylla. According to the present disclosure, the extract is a solvent-based extract obtained by solvent extraction of the plant. As used herein, the extraction is achieved by using at least one solvent, preferably a polar solvent, followed by a fractionation process. Thus, the extract is obtained by performing a solid / liquid solvent extraction process followed by a fractionation step, wherein a chromatographic solid column may be used to obtain the final extract.

[0053] As used herein, the singular or plural terms "cyclic peptide" or "cyclic peptide" can be understood interchangeably in the broadest sense in the art. It means a peptide having at least one ring, which is produced by a non-cyclic peptide by forming an amide or ester bond, by a disulfide connection, or by a new carbon-carbon, carbon-nitrogen, nitrogen-oxygen or carbon-sulfur bond. In particular, the cyclic peptide of the present invention can be selected from the group consisting of Pseudostellariae Pseudostellariae cyclic peptide and Pseudostellariae Pseudostellariae cyclic peptide.

[0054] In one embodiment, one or more cyclotides may be selected from the group consisting of cyclotides A to E and G. In a preferred embodiment, the cyclotides may be selected from at least two or more of the group consisting of cyclotides A to E and G. More preferably, the cyclotides may be selected from at least three or more of the group consisting of cyclotides A to E and G. In particular, the cyclotides may be a combination of cyclotides A to E and G.

[0055] In one embodiment, one or more Pseudostellariae cyclic peptides may be selected from the group consisting of Pseudostellariae cyclic peptides A, B, C and J, preferably two or more thereof, especially a combination thereof.

[0056] In a preferred embodiment, relative to the total weight of the extract, the amount of Pseudostellariae Cyclotide B by weight may be equivalent to at least 20 mg / kg, more preferably at least 25 mg / kg, even more preferably at least 35 mg / kg, especially at least 45 mg / kg.

[0057] In another preferred embodiment, relative to the total weight of the extract, the content of Pseudostigma tuberculin B may be equivalent to between 20 and 500 mg / kg, preferably between 25 and 450 mg / kg, more preferably between 35 and 430 mg / kg, and especially between 45 and 400 mg / kg.

[0058] In a preferred embodiment, the amount of one or more phenolic components (including polyphenols) relative to the total weight of the extract is less than 900 mg / kg. Advantageously, the one or more phenolic components (including polyphenols) may be in the range of 100 to 300 mg / kg, preferably 150 to 250 mg / kg, by weight, relative to the total weight of the extract. Advantageously, the one or more phenolic components may be, for example, non-glycosylated polyphenols. It should be noted that the root extract of Pseudo-Schlumbergera heterophylla extracted with a polar solvent such as water or hydroethanol contains between 900 and 3000 mg / kg of polyphenols by weight, relative to the total weight of the extract. Therefore, such a high content of phenolic components, mainly polyphenols, is at least partly responsible for the dark color of the final extract.

[0059] In a preferred embodiment, the total cyclic peptide content may be in the range of 70 to 900 mg / kg, preferably 100 to 850 mg / kg, more preferably 150 to 800 mg / kg, especially 200 to 750 mg / kg, relative to the total weight of the extract.

[0060] According to the present invention, relative to the weight of the total cyclic peptides, the Pseudostellariae cyclotide B may be approximately equivalent to between 2 and 55% by weight.

[0061] In a preferred embodiment, the extract may further comprise less than 0.1%, preferably less than 0.01%, more preferably less than 0.001%, about 0% by weight of polysaccharides relative to the total weight of the extract.

[0062] In one embodiment, the dry content of the extract may correspond to 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.

[0063] The extract according to the present invention can be in liquid, pasty or solid form under standard conditions (room temperature of about 20° C., atmospheric pressure of about 1013 hPa). In this context, the terms “liquid / liquid state” and “fluid” can be understood interchangeably. Preferably, the extract can first be obtained in liquid form. Then, one or more solvents can be optionally removed to obtain a solid form of the extract. The liquid extract can also be concentrated under reduced pressure, using but not limited to a vacuum concentrator or a rotary vacuum evaporator. In addition, after concentration, the extract can be dried. Then, preferably a portion of one or more solvents is removed. Optionally, a liquid or pasty extract can be obtained. Optionally, the extract can also be diluted again with one or more solvents to obtain a solvent-based extract. Optionally, the extract can also be purified by any means (e.g., crystallization, chromatographic means, etc.).

[0064] The extract may optionally be referred to as a "tincture" or "absolute". The solvent used to prepare the extract may be an aqueous solvent (water or buffer), or it may be a combination of an aqueous solvent and an alcohol. The extract of the present invention is a solvent-based extract, wherein the diluting solvent may be selected from water, aqueous buffer, alcohol, glycol, ethyl lactate, isopropyl myristate, triglyceride, triethyl citrate, dioctyl ether, glyceryl isostearate, glyceryl stearate, ethyl acetate or a mixture thereof. The alcohol may preferably be selected from methanol, ethanol and isopropanol. The glycol may preferably be selected from pentanediol and glycerol.

[0065] Thus, the ready-to-use root extract of Pseudostemon heterophylla according to the invention may be dried, or frozen or freeze-dried, or fresh, or dissolved in a glycerol:water mixture in a volume ratio of 95 / 5 to 5 / 95, preferably 80 / 20 to 20 / 80.

[0066] As used herein, the term "dry extract" can be understood in the broadest sense as the dry matter form, i.e. the extract according to the invention without solvent. The dry extract may optionally be physically present in the form of dry matter. However, it will be directly understood that in the calculations involving the percentage of dry matter, it is not necessarily the dry matter that is physically present, 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.

[0067] Dry matter (physical form) can be obtained by a suitable extraction method, followed by the step of drying the extract obtained. Drying can be performed by any method known in the art suitable for the purpose. Drying means removing one or more solvents used for extraction. One or more solvents can be removed by any means. For example, the extract can be subjected to a hot dry atmosphere and / or the extract can be placed on a heated plate to evaporate the solvent. For example, drying can be achieved by evaporation in a vacuum and / or at an elevated temperature (e.g., in a rotary (vacuum) evaporator) or by crystallization of the solid material of interest. Alternatively or additionally, drying can be achieved by atomization or by freeze drying.

[0068] Another aspect of the present invention relates to a method for preparing a root extract of Pseudostemon heterophylla, comprising the following steps:

[0069] a. by contacting the optionally dried roots of Pseudostemon heterophylla with a polar solvent, performing a solid / liquid extraction of the roots to obtain a solution,

[0070] b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution to obtain a powder,

[0071] c. Perform the following classification steps:

[0072] i. the concentrated solution obtained in step b); or

[0073] ii. the powder obtained in step b) dissolved in a polar solution;

[0074] d. recovering the cyclic peptide-containing fraction obtained in step c).

[0075] Advantageously, the method of the invention can be carried out starting from a dry extract of the roots of Pseudostemon heterophylla obtained after carrying out steps a) and b), preferably in powder form. Such a dry extract may optionally be commercially available, or may be prepared partly or entirely from the roots of Pseudostemon heterophylla. In such a case, the method according to the invention further comprises steps c) and d), wherein the powder in step ii) may be dissolved in a polar solvent selected from water or a mixture of water and an alcohol, the proportion of alcohol in the polar solvent being between 1 and 20% (v / v), preferably between 1.5 and 15% (v / v), in particular between 2 and 10% (v / v). In a preferred embodiment, the polar solvent used in step ii) is water.

[0076] Advantageously, the root of Pseudo-Schwansonii in powder form can be dissolved in a polar solvent at a ratio of between 5 and 20%, preferably between 10 and 40%, in particular between 15 and 30% (weight or powder / volume solvent). In other words, a 5% dissolution means putting 5 g of powder in 1 L of polar solvent. After the dissolution step, the liquid can be filtered.

[0077] As used herein, the term "polar solvent" may be understood in the broadest sense as generally understood in the art. Preferably, it means a solvent used in a known solid / liquid extraction method. Thus, the solid / liquid extraction is carried out at ambient pressure (i.e. 980 to 1200 hPa) and at ambient temperature (i.e. 18 to 25° C.). Preferably, the polar solvent may be selected from water, alcohol or a mixture thereof in any proportion.

[0078] In one embodiment, the alcohol may be selected from ethanol, methanol, propanol, butanol, pentanol, phenol, glycerol, 1,3-butanediol, propylene glycol or a mixture of two or more thereof. Preferably, the alcohol may be selected from methanol, ethanol, propanol and glycerol.

[0079] In a preferred embodiment, the polar solvent used in step a) can 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. Thus, a preferred polar solvent can be a water / ethanol mixture with an ethanol ratio of 30 / 70% (v / v) or an ethanol ratio of 40 / 60% (v / v).

[0080] Advantageously, step a) may also comprise a step of separating solid matter from the solution, which may be a crude solution.

[0081] According to the present invention, the fractionation steps are known in the art. In one embodiment, fractionation, also referred to as purification, can be performed, for example, with the aid of a chromatography resin column, using a convenient elution solvent, or by liquid / liquid separation.

[0082] As used herein, "chromatographic resin" refers to a chromatographic medium present in a chromatographic column and responsible for separating the starting components present in the extract. Thus, the chromatographic resin may be packed in a packed bed or an expanded bed. Different types of chromatographic resins may be used, such as chromatographic resins made of porous polymer resins. Typically, the chromatographic column may be washed with water before subjecting the solution containing the extract to a fractionation step.

[0083] An example of a chromatography column that can be used to perform the fractionation step is the Diaion TM HP20 resin.

[0084] In one embodiment, the eluent solvent can be identical with the polar solvent as described above. In a preferred embodiment, the eluent solvent can be selected from water or a mixture of water / alcohol, wherein the alcohol is equivalent to 60 to 98% (v / v) of the mixture, preferably 70 to 95% (v / v), more preferably 80 to 92% (v / v). More preferably, the eluent solvent can be selected from water or a mixture of water / ethanol, wherein the ethanol can be equivalent to between 10 to 90 to 90 to 10% (v / v).

[0085] In one embodiment, the fractionation step can be performed with the aid of a chromatography resin column, wherein the eluent solvent can be a water / alcohol mixture. Advantageously, the water / alcohol eluent can contain an alcohol equivalent to between 5 and 95% (v / v). Preferably, the alcohol can be ethanol or methanol.

[0086] According to one embodiment, step c) can be carried out by: c.1) eluting with a water / alcohol mixture, wherein the alcohol may correspond to 5 to 20% (v / v), and additionally c.2) eluting with a water / alcohol mixture, wherein the alcohol may correspond to 80 to 95% (v / v).

[0087] According to a preferred embodiment, the elution in c.1) can be performed with an ethanol / water mixture containing 5% (v / v) ethanol, preferably 10% (v / v) ethanol, more preferably 20% (v / v) ethanol.

[0088] According to one embodiment, the elution in c.2) can be performed with an ethanol / water mixture containing 80 to 95% (v / v) ethanol, preferably 85 to 90% (v / v) ethanol.

[0089] Advantageously, each elution in c.1) and c.2) can be performed within 30 to 120 min, preferably 45 to 90 min. The collected eluate constitutes the cyclic peptide-based fraction of the extract, which can be resuspended in a hydroalcoholic solvent, preferably in a mixture of glycerol / water in any proportion.

[0090] Another aspect of the present invention relates to a cosmetic or pharmaceutical composition comprising:

[0091] - as active ingredient an extract of the root of Pseudostemon heterophyllum as described above,

[0092] - at least one additional cosmetically and / or pharmaceutically acceptable ingredient other than the root extract of Chickweed heterophylla,

[0093] Wherein, the composition is a composition for topical use selected from the group consisting of: solutions, suspensions, emulsions, creams, pastes, gels, lotions, powders, soaps, water containing surfactants, oils, lotions and sprays; and / or wherein the composition is a health food composition for oral administration.

[0094] Another aspect of the invention relates to the cosmetic use of a composition comprising or consisting of a root extract of Pseudostemon heterophyllus as described above, for combating and / or preventing signs of aging of the skin and hair follicles, for improving the overall appearance of the skin, in particular for improving the firmness, radiance, brightness, hydration of the skin, for reducing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

[0095] As used herein, the term "signs of skin aging" means any changes in the appearance of the skin caused by aging over time, such as wrinkles and fine lines, wrinkles, eye bags, dark circles, sagging, loss of elasticity and / or skin tone (lightness), dullness or lack of radiance, lack of pigmentation, but also includes any internal changes in the skin that are not systematically manifested in the form of an altered external appearance, such as a reduction and loss of dermal density, thickening of the stratum corneum. The skin may also thicken and become rough. Signs of aging also include any degeneration of the skin after exposure to ultraviolet rays, such as premature fine lines around the eyes and mouth, expression lines on the face, pigmentation in different areas, such as freckles and solar lentigo, irregular skin color, general loss of skin tone, loss of color and thinning of the lips.

[0096] Notably, the experimental studies provided in the present disclosure demonstrate that the composition comprising (or even consisting of) the root extract of Pseudostemon heterophyllum of the present invention can be used as an active ingredient for cosmetic and / or pharmaceutical purposes, in particular for skin care and hair care.

[0097] In a preferred embodiment, the root extract of Pseudostemon heterophylla according to the present invention can be used to obtain the following effects:

[0098] a. Improve skin complexion,

[0099] b. Strengthen and / or restore epidermal barrier function,

[0100] c. Increase collagen synthesis and improve extracellular matrix components,

[0101] d. Increase retinoic acid receptor γ transcription,

[0102] e. Improve skin cell regeneration and epidermal differentiation,

[0103] f. Regulate oxidative stress in skin cells,

[0104] g. Improve skin defense,

[0105] h. Strengthen skin homeostasis,

[0106] i. Improve skin soothing,

[0107] j. Counteracts hyperpigmentation on the skin,

[0108] k. Improve skin microbiome,

[0109] l. A combination of two or more of these.

[0110] Here, the extract according to the invention may optionally be considered as (or optionally even as the only) active ingredient. As used herein, the term "active ingredient" may be understood in the broadest sense as a component that may exhibit the desired and expected activity alone or together with one or more other ingredients, such as one or more carriers that are inactive by themselves or other active ingredients that may optionally act synergistically. Preferably, the active ingredient of the invention is a cosmetic active ingredient.

[0111] The cosmetic or pharmaceutical composition can be used for any purpose and in any form. In a preferred embodiment, the composition is a composition for topical use selected from the group consisting of: solutions, suspensions, emulsions, creams, pastes, gels, lotions, powders, soaps, water containing surfactants, oils, lotions and sprays; or wherein the composition is a health food composition for oral administration.

[0112] The composition may also be or may be contained in a product selected from the group consisting of a cream, a gel, an ointment, a tonic, a liquid soap, a bar soap, a bath oil, a shower oil, a massage oil, a makeup, a scalp care, an aftershave, a shaving product, a deodorant, a shower gel, a shampoo, and a combination of two or more thereof.

[0113] Optionally, the cosmetic or pharmaceutical composition can also be a health food composition that can be orally administered to a subject. Then, the cosmetic or pharmaceutical composition can be optionally included in food, such as a food supplement. Then, the composition can typically have a systemic effect. The health food composition according to the present invention can be formulated into any form suitable for oral administration with an acceptable carrier, such as a tablet, capsule, granule, powder, solution, emulsion or suspension.

[0114] The cosmetic or pharmaceutical composition may contain any amount of the extract of the present invention. In a preferred embodiment, the composition contains less than 20% by weight of the extract according to the present invention, based on the total weight of the composition. In a preferred embodiment, the composition contains 0.0001 to 15% by weight, more preferably 0.001 to 10% by weight, even more preferably 0.005 to 8% by weight, especially 0.01 to 5% by weight of the extract according to the present invention, based on the total weight of the composition.

[0115] At least one additional cosmetic and / or pharmaceutically acceptable ingredient different from the extract of the present invention can be any cosmetic and / or pharmaceutically acceptable ingredient. In a preferred embodiment, the cosmetic and / or pharmaceutically acceptable ingredient is or includes at least one cosmetic and / or pharmaceutically acceptable carrier.

[0116] 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 pharmaceutical acceptability or usability of the composition containing the root extract of Pseudostellaria heterophylla according to the present invention. Preferably, neither the extract of Pseudostellaria heterophylla or the composition containing at least one such extract according to the present invention is toxic when applied to tissue.

[0117] The composition ready for use may preferably be a liquid preparation, especially a composition suitable for topical and oral administration. The storage form of the composition may also be a liquid, but may also be a dried form (e.g., a powder, such as a powder containing or consisting of a root extract of Pseudostellaria heterophylla), or may be a paste or syrup, etc. Optionally, the dried form, paste or syrup may be dissolved or emulsified before being applied to the skin of interest.

[0118] The cosmetic and / or pharmaceutically acceptable carrier can be exemplarily selected from the list consisting of: aqueous buffer, saline, water, alcohol, vegetable oil, mineral oil, polymer or a combination of two or more thereof. Optionally, the cosmetic and / or pharmaceutically acceptable carrier can contain one or more cosmetic and / or pharmaceutically acceptable additives.

[0119] In a preferred embodiment, such cosmetically and / or pharmaceutically acceptable additives may be selected from the group consisting of flavors / essences, dyes, pigments, emulsifiers, lubricants, chelating agents, acidity regulators, antimicrobial agents, preservatives, antioxidants, and combinations of two or more thereof.

[0120] Furthermore, in addition to the root extract of Pseudostemon heterophylla, the composition of the present invention may also contain one or more other known active ingredients that act synergistically and / or complementarily with the extract of Pseudostemon heterophylla without affecting its activity or its intended use as described above.

[0121] Composition according to the present invention can be a cosmetic product or can be included in a cosmetic product. The composition can be used for any purpose. Any route of administration that can reach the desired purpose of protection is suitable. Application can be local or systemic administration. Preferably, application is topical application. Application can be topical application, transdermal application, oral administration, by means of injection (for example, intravenous (iv), intraarterial (ia), intraperitoneal (ip), intramuscular (im) and subcutaneous (sc) injection) application or nasal administration. For example, the composition according to the present invention and an acceptable carrier can be formulated into any suitable form for topical, oral, rectal, mucosal, nasal, intestinal, enteral and parenteral administration.

[0122] In a preferred embodiment, the composition is a composition for topical use. In a preferred embodiment, the composition is a composition for topical use that is topically applied to a subject. In a preferred embodiment, the composition is a composition for topical use that is topically applied to a portion of the skin, such as the face, scalp, or any part of the body. The skin can be all skin types, including damaged skin, dry skin, healthy skin, normal skin, and oily skin.

[0123] As mentioned above, the root extract or composition of Pseudostemon heterophylla of the present invention can be used for any purpose. Preferably, it is used for cosmetic and / or pharmaceutical purposes.

[0124] The extract of the invention or the composition comprising it may have a local and / or systemic effect. In a preferred embodiment, when applied locally (locally and / or topically), it has (predominantly or exclusively) a local effect.

[0125] Subjects having tissues of interest, particularly skin, scalp and hair, may suffer from or may be at risk of developing the above-mentioned diseases and / or injuries.

[0126] As used in the context of the present invention, the term "subject" may be understood in the broadest sense as any organism. The subject is preferably a human or an animal, more preferably a human or a mammal, in particular a human.

[0127] Another aspect of the invention relates to a composition comprising (or consisting of) a root extract of Pseudostellaria heterophylla as described above, for use in combating and / or preventing signs of aging of the skin and hair follicles, for improving the overall appearance of the skin, in particular for improving the firmness, elasticity, radiance, brightness, hydration of the skin, for reducing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for use in two or more methods thereof.

[0128] Another aspect of the present invention relates to the cosmetic use of Pseudostellariae Cyclic Peptide B for combating and / or preventing signs of aging of the skin and hair follicles, in particular for reducing wrinkles and fine lines, for improving the firmness and / or elasticity of the skin, for counteracting irregular hyperpigmentation in the skin, for relieving skin inflammation, or for two or more thereof.

[0129] Advantageously, the Pseudostellariae Cyclotide B of the present invention can be purified from the root extract of Pseudostemon heterophylla or can be purchased commercially. In such a case, Pseudostellariae Cyclotide B can be obtained synthetically.

[0130] Another aspect of the present invention is directed to a cosmetic composition comprising Pseudostellariae Cyclic Peptide B for the use defined above, wherein, relative to the total weight of the composition, by weight, Pseudostellariae Cyclic Peptide B may be equivalent to between 0.00001 and 2%, preferably 0.00005 to 1.5%, more preferably between 0.0001 and 1%, and wherein the composition may further comprise a cosmetically acceptable carrier.

[0131] Throughout this specification and claims, unless the context requires otherwise, open-ended terms such as "comprise", "contain", "include", etc., and variations such as "comprises / comprising", "contains / containing", "includes / including", etc., may be understood to imply the inclusion of stated components, members, integers or steps, or groups of components, members, integers or steps, but not the exclusion of any other components.

[0132] The terms "one" and "a kind of" and "the / said" and similar references used in the context of describing the present invention (particularly in the context of the claims) should be understood to cover both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. The description of the value range herein is intended only as a shorthand method for quoting each individual value falling within the range individually. Unless otherwise specified herein, each individual value is incorporated into the specification as it is described individually and in combination with each other herein. Unless otherwise specified herein or clearly contradictory to the context, all methods and procedural steps, including the method-defined product steps described herein, can be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., "such as", "for example") provided herein is intended only to better illustrate the present invention, and is not intended to limit the scope of the present invention otherwise protected. Any language in the specification should not be interpreted as representing any unclaimed element that is essential to the practice of the present invention.

[0133] More generally, it should be understood that these aspects and embodiments can be combined in any manner and any number into further embodiments. Unless otherwise indicated by the context, any arrangement and combination of all described elements in this application should be considered to be disclosed by the description of this application.

[0134] Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by those skilled in the art. Although the methods and materials described in this article are preferred, other methods and materials similar or equivalent to the methods and materials described herein can also be used in the practice or testing of the present invention.

[0135] All documents cited or referenced herein are hereby incorporated by reference and may be employed in the practice of the present invention. More specifically, all cited documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0136] The following examples and figures are intended to illustrate further embodiments of the invention without limiting its scope.

[0137] Example

[0138] 1. Preparation of Root Extract of Pseudostemon heterophylla

[0139] a. Solid / liquid extraction:

[0140] The roots of Pseudostemon heterophylla were collected, washed, dried and ground. The crude powder was extracted 7 times in a water-ethanol solvent containing 60% ethanol (v / v) under reflux for 2 hours. The obtained solution was diluted 6 times, concentrated under reduced pressure at 50°C, and then spray-dried to obtain a dried powder. 200 g of the dried powder was dissolved in 1 L of demineralized water, stirred at 500 rpm for 1 h at room temperature, and centrifuged. The supernatant was filtered to remove insoluble matter, and filtered to obtain a supernatant.

[0141] b. Grading steps:

[0142] The chromatographic resin column ( HP20, Thermo Fischer Scientific) was previously washed with ethanol and water. Then, the supernatant was loaded into the resin column at a flow rate of 6mL / min (6.4 bed volumes / min). The column resin was eluted by a water / ethanol mixture of 80 / 20 (v / v) of 190mL of ethanol at a flow rate of 3mL / min (3.2 bed volumes / min). The relevant fractions were collected.

[0143] The resin column was further eluted with 380 mL of a water / ethanol mixture having 90% (v / v) ethanol at a flow rate of 3 mL / min (3.2 bed volumes / mn) to obtain a cyclic peptide-rich root fraction of Pseudostemon heterophylla containing:

[0144] - polyphenols,

[0145] -641mg / L cyclic peptides: Pseudostellariae cyclic peptides A to E and G, among which Pseudostellariae cyclic peptide B is equivalent to 291mg / L solvent.

[0146] -Pseudostellariae Cyclotide A, C and J

[0147] The obtained cyclotide-rich root fraction of Pseudo-Schlumbergera heterophylla was mixed with 50 mL of demineralized water and then concentrated under reduced pressure at 50° C. on a rotary evaporator until the final mass was a maximum of 50 g. Glycerol and water were added in a ratio of 80 / 20 (v / v), respectively. The hydroglycerolated extract (hereinafter referred to as “PHE”) was further clarified by filtration at 1 μm.

[0148] 2. In vitro experiments and results

[0149] Transcriptome analysis was performed one day after skin explants were treated with a formulation containing PHE. The results are presented as relative expression ratios between the control and treatment groups.

[0150] All data are expressed as mean ± standard deviation. Since all data were normally distributed, the statistical significance of the data was determined by Student's t test using Minitab software. The significance is indicated as follows: ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: p ≥ 0.05.

[0151] a. PHE improves epidermal barrier function and extracellular matrix integrity

[0152] method: After receiving an abdominal biopsy, adipose tissue was removed and skin explants were cut into pieces (2 cm x 2 cm). Test compounds (PHE and positive control) were then topically applied (5 mg / cm 2 ) or not applied (control) to the surface of skin explants, and then cultured in a humidified incubator at 37°C and 5% CO2 for 24 hours.

[0153] All experimental conditions were performed with n=3.

[0154] At the end of incubation, skin explants were washed in phosphate-buffered saline (PBS) solution and placed in RNAlater at 4 °C for 24 h and then frozen at -20 °C until RNA extraction. The samples were mechanically homogenized using an Evolution homogenizer (Bertin Instrument) and purified using TriPure Isolation according to the supplier's instructions. Total RNA was extracted from each sample.

[0155] The quantity and quality of RNA were assessed using capillary electrophoresis (Bioanalyzer 2100, Agilent technologies).

[0156] According to the supplier's instructions, use PCR (polymerase chain reaction) was performed using the Roche Molecular System Inc.

[0157] result:

[0158] Table 1

[0159]

[0160] Table 1 shows the significant upregulation of transcription of some genes related to the retinoid signaling pathway.

[0161] P450 cytochromes are heme proteins responsible for the oxidative metabolism of a variety of endogenous molecules. They are able to detoxify and eliminate xenobiotic chemicals, preventing the accumulation of potentially toxic substances in the body.

[0162] 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 of certain retinoid species that are not used by the cell into polar metabolites.

[0163] The latest generation of retinoids is designed to specifically target RAR-B or RAR-G to avoid the side effects of retinoids, primarily skin inflammation. Due to its selectivity for RAR-G, trefarotene (the latest generation of retinoids) has shown strong efficacy in improving retinoid-regulated pathways involved in epidermal differentiation, acne-prone skin regression. In addition, targeting of RAR-G has demonstrated anti-inflammatory properties and depigmentation activity.

[0164] The results showed that after PHE treatment, RAR-G transcription was significantly increased, which is a key player in the signaling of retinoic acid's effects on acne, pigmentation, and inflammation.

[0165] Table 2

[0166]

[0167] Table 2 shows a significant increase in the transcription of genes involved in mitochondrial biology, such as mitochondrial fusion protein-1 (MFN1), involved in the regulation of oxidative stress through ROS production due to the superoxide dismutase 2 gene (SOD2), and involved in the proliferation capacity of cells through the positive regulation of PCNA (proliferating cell nuclear antigen).

[0168] These results highlight the beneficial role of PHE in regulating proliferation and generation of oxygen reactive species. With aging, the epidermis thins, in part due to a lack of cell turnover and excessive production of ROS, which exacerbate skin aging and inflammation.

[0169] Table 3

[0170]

[0171] Table 3 shows the positive regulation of several genes involved in skin barrier function and epidermal differentiation processes. Keratin 1 (KRT1) and its heterodimeric partner keratin 10 (KRT10) are major components of the intermediate filament cytoskeleton in the suprabasal epidermis. Their upregulation precedes the expression of filaggrin and cornified envelope protein. This has led to the hypothesis that a subset of keratins and the related protein filaggrin are essential for barrier function.

[0172] In the outermost layer of the stratum granulosum, loricrin is involved in the formation of the cornified envelope beneath the plasma membrane and in its mechanical resistance. An increase in CALML5 (calmodulin-like 5), which is also involved in the terminal differentiation of keratinocytes, as well as desmoglein 1 (DSC1) and desmoglein (DSG1), which constitute adhesion proteins of desmosomal cell-cell junctions and are required for cell adhesion and desmosome formation, was demonstrated.

[0173] Kallikrein (KLK) is an essential protein involved in skin desquamation by degrading the stratum corneum, a junctional structure that allows corneocytes to adhere. Corneodesmosomes consist of an extracellular protein, corneodesmosin (CDSN), and two transmembrane proteins, desmoglein 1 (DSG1) and desmoglein 1 (DSC1). Degradation of these proteins by KLK allows proper skin desquamation and is required for skin homeostasis.

[0174] The interior of corneocytes is mainly composed of keratin filaments aggregated by filaggrin (FLG). FLG degradation products partially explain the water-holding capacity of the stratum corneum and the maintenance of acidic pH, both of which are crucial for epidermal barrier homeostasis by regulating the activity of multiple enzymes that control desquamation, lipid synthesis, and inflammation.

[0175] PHE has shown its ability to improve skin barrier function by increasing keratin and adhesion proteins such as DSC1 and DSG1. PHE has shown its ability to strengthen skin homeostasis by increasing kallikrein, which is useful for skin desquamation and epidermal renewal.

[0176] Table 4

[0177]

[0178] Table 4 shows that PHE significantly increased the transcription of genes involved in extracellular matrix composition and dermal remodeling.

[0179] PHE was observed to upregulate genes encoding collagen subunits (COL1A1, COL3A1, COL4A2), which are known to be retinoid-responsive genes. In addition, upregulation of the elastin gene (ELN) was demonstrated.

[0180] TIMP1 encodes a natural inhibitor of matrix metalloproteinases (MMPs), a group of peptidases involved in the degradation of the extracellular matrix. In addition to its inhibitory effects on most known MMPs, the encoded protein is able to promote cell proliferation in a variety of cell types and may also have anti-apoptotic functions.

[0181] In short, PHE regulates genes involved in epidermal differentiation, cell turnover, regulation of dermal extracellular matrix composition, and dermal-epidermal junction composition similarly to retinol and bakuchiol.

[0182] b. PHE improves the synthesis of type I collagen

[0183] like Figure 1 As shown in , PHE has the potential to synthesize type I collagen by human fibroblasts.

[0184] Type I collagen is a very important structural protein that provides strength and elasticity to the skin. Aging (aging over time and photoaging) is associated with a decrease in collagen production and an increase in its degradation. Therefore, type I collagen synthesis is therefore a good marker for evaluating the efficacy of anti-aging actives. It is also known that retinol and bakuchiol promote collagen formation and application to the skin has been shown to reduce wrinkles and fine lines.

[0185] method:

[0186] Primary human fibroblasts (NHDF) from 3 different donors (35, 47 and 50 years old) were processed for 3 days by 10ppm of retinol, 10ppm of bakuchiol, PHE containing 0.10ppm of Pseudo-P ...

[0187] result:

[0188] In NHDF, 10 ppm retinol was a potent promoter of type I procollagen synthesis, and 10 ppm bakuchiol was less effective (+57% vs. control, +150% for retinol). PHE with 0.10 ppm HB was demonstrated to significantly increase type I procollagen release by fibroblasts to the same level as retinol (+164%). Interestingly, treatment of cells with HB alone highlighted that the effect of PHE was primarily supported by its Pseudomonas aeruginosa content.

[0189] In short, the results confirm the potential of PHE as a natural alternative to retinol to become an effective skin anti-aging agent.

[0190] c. PHE reduces the synthesis of pro-inflammatory cytokines

[0191] like Figure 2 As shown in , both PHE and HB can reduce the secretion of interleukin-6 (IL-6).

[0192] Retinoids reduce several signs of skin aging, but they also have many side effects, such as skin redness, itching, peeling, stinging or burning sensations, and sensitivity.

[0193] method:

[0194] NHDF from a 47-year-old donor was treated with PHE or 0.10ppm Pseudo-P ...

[0195] result:

[0196] The results confirmed that after 5 days of treatment, both PHE and HB significantly reduced the secretion of IL-6 by 60% and 63%, respectively. IL-6 is synthesized at the local lesion during the initial stages of inflammation. Interestingly, it was shown that neither retinol nor bakuchiol significantly reduced the synthesis of IL-6 by NHDF.

[0197] Thus, the potent anti-inflammatory effects of both PHE and HB were demonstrated, strengthening their use as natural alternatives to retinol.

[0198] d. PHE reduces melanin secretion in melanocytes

[0199] like Figure 3 As shown in , different concentrations of PHE and HB can reduce the melanin content in melanocytes.

[0200] Retinoids are also known to affect the function of melanocytes, providing a regular arrangement of melanin in the epidermis. They also block the transport of melanin to epidermal cells and reduce the activity of stimulated melanocytes.

[0201] method:

[0202] B16-F1 cells were treated with 0.3% kojic acid or three different concentrations of PHE for 4 days, corresponding to HB contents of 0.21ppm, 0.10ppm or 0.035ppm. At the end of the treatment, the dose of secreted melanin was completed by optical density measurement at 475nm. Protein standardization was applied to all conditions after Bradford analysis and 595nm plate reading.

[0203] result:

[0204] The results showed that PHE was able to reduce melanin release from B16-F1 melanocytes in a HB content dose-dependent manner (up to 60%, 54% and 48% reductions for 0.21 ppm PHE, 0.10 ppm PHE and 0.035 ppm HB, respectively).

[0205] The potent anti-pigmentation activity of PHE and its use to counteract the irregular hyperpigmentation observed in skin aging is thus demonstrated.

[0206] 3. Clinical evaluation

[0207] 3.1. Formulated PHE compared to placebo formulation

[0208] a. Materials and methods

[0209] A randomized, double-blind, placebo-controlled clinical trial was performed to evaluate the effects of PHE on the skin. This study was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines for non-pharmacological research.

[0210] All data are presented as mean ± standard deviation. Since all data were normally distributed, the statistical significance of the data was determined by Student's t-test using Minitab software. The significance is indicated as follows: ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: p ≥ 0.05.

[0211] 21 Caucasian women were selected, with an age range of 45 to 65 years (mean 55 ± 2 years). The volunteers showed a lack of firmness and redness on the face, and dull skin tone. They were asked to apply a cream formulation containing 0.3% by weight of PHE and a placebo on each half of the face, in a random distribution, twice a day for 56 days.

[0212] Table 5: Cream formulations containing 0.3 wt% PHE and placebo

[0213] Composition of cream Placebo (%) Creams with PHE (%) Ammonium Acryloyldimethyltaurate / VP Copolymer 0.80 0.80 Dioctyl ether 1.00 1.00 Buffers (citric acid and sodium citrate) 1.00 1.00 Phenoxyethanol / Methylparaben / Ethylparaben 1.10 1.10 spices 0.003 0.003 Pseudochia heterophylla root extract 0.00 0.30 water 96.10 95.80

[0214] Biometric measurements were performed on day 0, after day 28 and after day 56 of applying the cream. To evaluate whether the formulation improves skin complexion, the lightness and redness of the skin were evaluated by measuring the L* parameter and a* parameter using a spectrophotometer on day 0 and day 28, respectively. Redness is a component of skin color, but it also reflects the degree of irritation of the skin. To calculate the mechanical properties of the skin, the evaluation focused on parameters related to firmness and sagging. The effect on sagging was evaluated by ptosis scores on day 0 and day 28, using a validated clinical ptosis scale from the Skin Aging Atlas, Volume 1, Caucasian type, independently by 3 trained experts. On day 0, day 28 and day 56, the firmness analysis was completed by R0 parameter measurement using a cutometer, and an additional time point was added to highlight the effect over time.

[0215] b. result

[0216] Figure 4A A significant improvement in skin brightness was shown, achieved by a 1.9% increase in the L* parameter from day 0 to day 28 with PHE, while the change with placebo was not significant. Moreover, the effect of PHE was significantly different from that of placebo, being 84% higher. In addition, a higher proportion of volunteers with an improvement in the L* parameter were observed in the PHE group (90%) compared to the placebo group (57%).

[0217] Figure 4B It was demonstrated that skin redness was significantly reduced from day 0 to day 28 with PHE (a*: -10.3%) compared to placebo (a*: -4.1%). Moreover, the effect of PHE was significantly different from that obtained with placebo, being 60% higher. In addition, the proportion of volunteers with an improvement in the a* parameter was higher in the PHE group (81%) than in the placebo group (62%).

[0218] Since the a* parameter is associated with redness, which itself is closely related to skin irritation, the above results confirm that the effects on inflammatory mediators (IL-6) observed during the in vitro experiments lead to improved skin soothing and prevention of skin irritation. Therefore, these results confirm the ability of PHE to be a non-irritating alternative to retinol. The above results confirm that PHE is not irritating and even has a soothing effect on the skin.

[0219] Therefore, PHE has been clinically proven to improve skin complexion by measuring L* and a* parameters.

[0220] It is known that aging, and in particular photoaging, reduces the mechanical properties of the skin. In fact, the skin loses its firmness, ultimately leading to sagging.

[0221] Three trained experts independently assessed skin sagging. Figure 5A It was shown that the change in skin sagging from day 0 to day 28 was significantly reduced by PHE treatment (-10.7%), while the change in placebo treatment was not significant. In addition, the effect of PHE was significantly different from that obtained with placebo, which was 84% ​​higher. In addition, the proportion of volunteers with improved sagging in the PHE group (95%) was much higher than that in the placebo group (38%).

[0222] Skin firmness is measured by the R0 cutometer parameter, which decreases if the skin is tighter. From this decrease, taking the absolute value of the percentage change, a positive increase in firmness can be assessed. Figure 5B The ability of PHE to improve skin firmness was shown, which increased over time: from 12.6% from day 0 to day 28, increasing to 19% at day 56. In contrast, the placebo did not show any significant improvement in firmness at any time point. Moreover, the effect of PHE was significantly different from that obtained with placebo at both measured time points, being 95% and 84% higher at day 28 and day 56, respectively. Likewise, for the firmness parameter, the proportion of volunteers with improvement was higher in the PHE group (100% at day 28 and day 56) than in the placebo group (60% at day 28 and 65% at day 56).

[0223] Finally, the clinical study confirmed the efficacy of PHE in improving skin complexion at day 28, as measured by brightness and redness. The reduction in redness was likely related to the soothing effect, which was completed by the improvement in sagging at day 28 and the improvement in firmness at day 28, which was further enhanced at day 56.

[0224] In conclusion, these results demonstrate the efficiency of the root extract of Pseudostemon heterophyllus of the present invention in combating and overall improving the signs of skin aging.

[0225] 3.2. Formulated PHE vs. formulated bakuchiol

[0226] a. Methods

[0227] A randomized double-blind clinical trial was conducted to compare the effects of PHE and Bakuchiol. The test dose of Bakuchiol was 0.5% as this is the recommended dose. The clinical study was conducted according to the principles of the Declaration of Helsinki and the guidelines of the above-mentioned international conferences, showing the same time points and measurements as in the above-mentioned clinical study (see point 3.1) (L* by spectrophotometry, sagging by scoring at day 0 and day 28, R0 by cutometry at day 0, day 28 and day 56).

[0228] Twelve Caucasian women were selected, with an age range of 45 to 65 years (mean 55 ± 3 years). The volunteers showed a lack of firmness and redness on the face, and a dull complexion. They were asked to apply a formulation containing 0.3% PHE (Table 6) or 0.5% bakuchiol on each half of the face, in a random distribution, twice a day for 56 days.

[0229] Table 6: Cream formulations containing 0.3 wt% PHE and 0.5% Bakuchiol

[0230]

[0231] Fig. 6A A clear improvement in skin brightness was shown, which was achieved by an increase of 4.3% in the L* parameter from the baseline value for PHE and 3.5% for Bakuchiol. PHE and Bakuchiol can be considered as effective as each other as there was no significant difference in their effects. In addition, both preparations were effective in 100% of the volunteers at day 28.

[0232] Bakuchiol is known to improve the firmness of the skin. Figure 6B It was shown that both PHE and Bakuchiol enhanced skin firmness significantly over time. Moreover, these changes were highly significant for both PHE and Bakuchiol compared to day 0. Bakuchiol increased firmness by about 8.8% at day 28 and then by 17.0% at day 56, and PHE increased firmness by about 7.9% at day 28 and then by 14.9% at day 56. The efficacy of PHE and Bakuchiol was equivalent, as the difference between the two products was not significant at both time points. In addition, both formulations were effective for 100% of the volunteers at day 28 and day 56.

[0233] Finally, skin sagging of PHE and bakuchiol was measured on day 28. Skin sagging is one of the most difficult consequences of skin aging to address. Figure 6C It was shown that from day 0 to day 28, Bakuchiol was effective in 67% of the volunteers, with a significant decrease of 8.9%. Interestingly, PHE showed a better performance, as the decrease was greater and more significant: -12.8%. In addition, the proportion of volunteers with improvement was also higher than Bakuchiol, as it led to improvement in 100% of the volunteers. PHE's ability to improve sagging of the skin was significantly better than Bakuchiol.

[0234] The results obtained from all the above experiments (in vitro and clinical) confirm the effectiveness of PHE and Pseudostellariae Cyclotide B in preventing signs of skin aging and improving the overall appearance of the skin. As a previous study (Dhaliwal et al., Br Journal of Dermatology, 2019; 180(2): 289-296) demonstrated that bakuchiol is as effective as retinol for hyperpigmentation after 12 weeks of use, it can be assumed that the root extract of Pseudostemon heterophylla of the present invention is also as effective as retinol in these parameters. In addition, the root extract of Pseudostemon heterophylla of the present invention performs better than bakuchiol in combating skin sagging.

Claims

1. A root extract of Pseudochilophora heterophylla, comprising - one or more phenolic components, - one or more cyclotides of ginseng, - one or more Pseudostellariae cyclotide, including Pseudostellariae cyclotide B, wherein, Relative to the total weight of the root extract, the Pseudostellariae Cyclotide B is equivalent to at least 10 mg / kg.

2. The root extract according to claim 1, wherein The cyclotide is selected from the group consisting of cyclotides A to E and G, preferably at least two thereof, more preferably at least three or more, especially a combination thereof.

3. The root extract according to any one of claims 1 or 2, wherein The Pseudostellariae cyclic peptide is selected from the group consisting of Pseudostellariae cyclic peptide A, B, C and J, preferably two or more thereof, especially a combination thereof.

4. The root extract according to any one of claims 1 to 3, wherein Relative to the total weight of the root extract, Pseudostellariae Cyclotide B is equivalent to at least 20 mg / kg, preferably at least 40 mg / kg, more preferably at least 50 mg / kg, especially at least 60 mg / kg.

5. The root extract according to any one of claims 1 to 4, wherein The total cyclic peptide content corresponds to 70 to 900 mg / kg, preferably 100 to 850 mg / kg, more preferably 150 to 800 mg / kg, especially 200 to 750 mg / kg by weight relative to the total weight of the root extract.

6. The root extract according to any one of claims 1 to 5, wherein The phenolic component ranges between 100 and 300 mg / kg, preferably between 150 and 250 mg / kg by weight relative to the total weight of the root extract.

7. The root extract according to any one of claims 1 to 6, wherein The root extract is a hydroalcoholic extract, preferably a hydroglycolic extract.

8. A method for preparing a root extract of Pseudostemon heterophylla, comprising the following steps: a. by contacting the optionally dried roots of Pseudostemon heterophylla with a polar solvent, performing a solid / liquid extraction of the roots to obtain a solution, b. optionally, concentrating the solution obtained in step a), and preferably drying the concentrated solution to obtain a powder, c. Perform the following classification steps: i. the concentrated solution obtained in step b); or ii. the powder obtained in step b) dissolved in water; d. recovering the cyclic peptide-containing fraction obtained in step c).

9. The method according to claim 8, wherein: Step c) is carried out in a chromatography resin column by elution with water / alcohol, wherein the alcohol corresponds to 5 to 95% (v / v).

10. The method according to any one of claims 8 or 9, wherein: Step c) is performed by: c.1) eluting with a water / alcohol mixture, wherein the alcohol corresponds to 5 to 20% (v / v); and c.2) Elution with a water / alcohol mixture, wherein the alcohol corresponds to 80 to 95% (v / v).

11. The method according to any one of claims 8 to 10, wherein: The elution in c.1) is carried out with an ethanol / water mixture containing 5% (v / v) ethanol, preferably 10% (v / v) ethanol, more preferably 20% (v / v) ethanol, and wherein the elution in c.2) is carried out with an ethanol / water mixture containing 80 to 95% (v / v) ethanol, preferably 85 to 90% (v / v) ethanol.

12. A cosmetic or pharmaceutical composition comprising: - a root extract of Pseudostemon heterophylla according to any one of claims 1 to 7 as an active ingredient; and - at least one cosmetically and / or pharmaceutically acceptable ingredient different from said extract of the roots of Pseudostemon heterophyllus, in, The composition is a composition for topical use selected from the group consisting of: solutions, suspensions, emulsions, creams, pastes, gels, lotions, powders, soaps, water containing surfactants, oils, lotions and sprays; and / or wherein the composition is a health food composition for oral administration.

13. Cosmetic use of a root extract of Pseudostemon heterophyllus according to any one of claims 1 to 7 or a composition comprising a root extract according to claim 8 for combating and / or preventing signs of aging of the skin and hair follicles, for improving the overall appearance of the skin, in particular for improving the firmness, elasticity, radiance, brightness, hydration of the skin, for reducing or preventing skin inflammation, for preventing and / or alleviating acne-prone skin, or for two or more thereof.

14. The cosmetic use according to claim 13, wherein the root extract is used for: a. Improve skin complexion, b. Strengthen and / or restore epidermal barrier function, c. Increase collagen synthesis and improve extracellular matrix components, d. Increase retinoic acid receptor γ transcription, e. Improve skin cell regeneration and epidermal differentiation, f. Regulate oxidative stress in skin cells, g. Improve skin soothing, h. Improve skin defense, i. Strengthen skin homeostasis, j. Counteracts hyperpigmentation on the skin, k. Improve skin microbiome, l. A combination of two or more of these.

15. Cosmetic use of Pseudostellariae Cyclic Peptide B for combating and / or preventing signs of aging of the skin and hair follicles, especially for reducing wrinkles and fine lines, for improving skin firmness and / or elasticity, for counteracting irregular hyperpigmentation in the skin, for relieving skin inflammation, or for two or more thereof.

16. A cosmetic composition comprising Pseudostellariae Cyclotide B for use according to claim 15, wherein: Relative to the total weight of the composition, by weight, Pseudostigmine B is equivalent to between 0.00001 and 2%, preferably 0.00005 to 1.5%, more preferably between 0.0001 and 1%, and wherein the composition further comprises a cosmetically acceptable carrier.

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