Extracts of cistanthe sp. cell cultures cell cultures with skincare properties

Abstract

The invention relates to a composition with hydrating, antimicrobial, antioxidant and anti-inflammatory properties for skincare, which comprises aqueous, ethanol or hydroalcoholic extracts of Cistanthe sp. cells cultured in vitro, taken from one or more cell lines derived from the stems and leaves of the plant. The composition also includes a suitable carrier. The majority compounds present in the extract correspond to total polyphenols, with a concentration of 10-500 mg / ml equivalent to gallic acid, and betalains, with a concentration of 0.5-15 mg / L. The process for obtaining this composition and the use thereof allow the formulation of pharmaceutical or cosmetic skincare products, increasing hydration, antioxidants, UV radiation barriers and anti-inflammatory agents. This ingredient enhances the effect of a solar filter and protects the skin from oxidative stress caused by environmental factors such as UV radiation, blue light and air pollution.

Description

[0001] CISTANTHE SP CELL CULTURE EXTRACTS WITH SKIN CARE PROPERTIES

[0002] DESCRIPTIVE MEMORY

[0003] The present invention relates to a natural skin care composition containing, as an active ingredient, an extract of one or more than one cell line derived from stems and leaves of Cistanthe sp.

[0004] BACKGROUND OF THE INVENTION

[0005] There are approximately 400,000 plant species worldwide, and these organisms are known to synthesize various secondary metabolites that exhibit diverse physiological activities. These compounds, accumulated at specific sites within cells, are key to plant defense against the environment, microorganisms, and animals. Secondary metabolites include polyphenols, betalains, alkaloids, carotenoids, glycosides, and terpenoids, among others.

[0006] Cistanthe grandiflora is a plant endemic to Chile and belongs to the Caryophyllales order. It is a characteristic plant of the Atacama Desert, although it can be found in other places in the north-central region of our country. The Atacama Desert, located in northern Chile, is one of the driest places on the planet, with some areas without rain for centuries. It stretches for about 1,000 kilometers between the Andes mountain range and the Pacific coast. Surprisingly, after exceptional rainfall, the desert vegetation blooms spectacularly. Due to the extreme dryness of the region, seeds and plants remain dormant for long periods, but when rainfall is abundant, these seeds germinate quickly, covering the ground with a colorful variety of flowers.This phenomenon is relatively rare and occurs in specific years when climatic conditions are right, creating a stunning and fleeting landscape of vibrant colors amidst the desert's aridity. Cistanthe grandiflora emerges from the arid soil and covers the desert with red flowers, creating a striking contrast with the dry landscape. Its flowering is a clear example of how, despite the extreme dryness of the desert, life can flourish explosively when conditions permit. Cistanthe grandiflora produces secondary metabolites that allow it to defend itself and support its growth in an environment as hostile as the Atacama Desert. The characteristic reddish color of this plant is due to the production of betalains, nitrogenous secondary metabolites of plants that act as red and yellow pigments. These compounds are found, for example, in other plants such as beets, amaranth, and bougainvillea.Due to their strong dyeing properties, betalains are generally used as food colorants, but recently they have gained attention as they have demonstrated bioactive properties in vitro and in vivo, such as antioxidant, anti-inflammatory and antimicrobial activities in intestinal epithelium, but so far, no properties have been described in skin.

[0007] The present invention relates to the protection of an extract of Cistanthe sp. cells cultured in vitro, both in solid media (calluses) and in cell suspensions, where the extract is characterized by the presence of polyphenols and betalains. These extracts can be aqueous or ethanolic, and have moisturizing, sunscreen-enhancing, antioxidant, antimicrobial, and anti-inflammatory properties on the skin.

[0008] Prior art.

[0009] In the prior art, there are numerous compositions of plant extracts for skin care applications. The main goal is to develop extracts with anti-wrinkle, antioxidant, moisturizing, and anti-inflammatory properties that promote healthy aging. The signs of skin aging increase with age and are accelerated by external factors such as solar radiation, cigarette smoke, smog, and blue light from electronic screens, among others.

[0010] The same inventor has previously developed plant cell extracts for pharmaceutical or cosmetic use, for anti-aging. Patent application CL2018002863A1 presents the use of Fitzroya cupressoides cells cultured in vitro to obtain aqueous extracts with anti-aging and skin regeneration properties. Furthermore, the application and testing to analyze the cell regeneration and anti-aging potential of such extracts in dermatological applications are described.

[0011] Patent US9289375B2 of STEMTECH INTERNAT INC. presents cosmetic compositions made with combinations of natural ingredients for skin care that improve skin elasticity and hydration and act to reduce wrinkles, in addition to preventing aging due to age and environmental conditions. The main ingredient is a blue-green algae, in addition to aloe, cocoa, vanilla, maqui, among other components, where the plants or their extracts are used.

[0012] For its part, patent US8828458B2 by MORARIU MARIUS refers to a topical composition containing anthocyanins from maqui or a maqui extract, presenting maqui as a potent anti-aging ingredient, where the problem to be solved is providing an active compound that does not oxidize. The described composition uses the fruit alone or in combination with maqui leaves to prevent or treat skin damage. In addition to maqui, the composition could contain as additional ingredients collagen and elastin-enhancing agents, anti-glycation, myocontraction, anti-edema, antioxidants, etc.

[0013] There are also several plant extracts that have been shown to enhance sunscreens or offer sun protection properties. Some of these extracts contain antioxidant compounds and properties that help protect the skin from damage caused by ultraviolet (UV) radiation. Some examples of plant extracts with these effects are:

[0014] Green Tea Extract: Contains catechins, especially epigallocatechin gallate (EGCG), which are powerful antioxidants. Green tea helps reduce sun damage, protects against UV radiation, and has anti-inflammatory effects.

[0015] Red Raspberry Extract: Red raspberry seed oil is known for its high concentration of essential fatty acids and antioxidants such as polyphenols, which may offer natural protection against UV rays.

[0016] Brown seaweed extract (Fucus vesiculosus): This seaweed contains fucoidan, which has antioxidant properties and protects the skin against ultraviolet radiation, helping to improve sun protection.

[0017] Carrot extract: Rich in beta-carotene, a type of antioxidant that helps protect the skin from sun damage and promotes cell regeneration.

[0018] Calendula flower extract: With anti-inflammatory and antioxidant properties, this extract helps soothe skin damaged by sun exposure and improve skin repair.

[0019] Grape seed extract: Contains powerful antioxidants, such as flavonoids and tannins, which help protect the skin from UV damage. Although these extracts may have protective and complementary effects against solar radiation, they are not a substitute for conventional sunscreen with certified sunscreen filters. However, they are used in many cosmetic formulations to enhance sun protection or as an additional support for sunscreens.

[0020] The present invention provides an alternative for skin care based on composition extracts comprising a standardized extract obtained from Cistanthe sp. cell culture, which is not previously described. The extract and / or composition can be used as an active ingredient in the cosmetics industry due to its advantageous properties as a moisturizing agent, UV radiation protector, and oxidative stress protector, which helps maintain skin structure.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Visible light absorption spectrum for Cistanthe cell extracts in water and 50% ethanol solution. Both extracts show a peak at 538 nm, which is associated with the pigment betalain.

[0023] Figure 2. HPLC chromatograms of the aqueous extract of Cistanthe cells at different wavelengths: 254 nm Fig. 2-A, 280 nm Fig. 2-B, 325 nm Fig. 2-C, 365 nm Fig. 2-D, and 540 nm Fig. 2-E.

[0024] Figure 3. Toxicity analysis of Cistanthe cell extracts on human skin cells. The effect of aqueous and ethanolic extracts at different concentrations on HACAT keratinocytes was analyzed. Water (Ca) was added as a negative control, and the epidermal growth factor EGF (Cb) was used as a positive control.

[0025] Figure 4. In vitro antioxidant protection in human skin cells. Cell viability of HACAT keratinocytes treated with hydrogen peroxide (700uM H2O2 for 1 hour) is observed in cell cultures without pretreatment, pretreated with Vitamin C and pretreated with 1% aqueous and ethanolic extracts of Cistanthe. It is observed that the 3 pretreatments provide moderate protection against oxidation by H2O2.

[0026] Figure 5. UV light absorption. Figure 5-A, UV light absorption at different wavelengths between 250 and 400 nm, by Cistanthe cell extract according to the invention at 100%, 50%, 25%, 10% and 1% v / v of the Cistanthe cell extract. Water was used as a blank. Good absorption is observed at concentrations greater than or equal to 10%. Figure 5-B UV light absorption profile at wavelengths between 320 and 400 nm (UV-A) and Figure 5-C UV light absorption profile at wavelengths between 270 and 320 nm (UV-B), comparing the extract of the invention at 50% v / v with quillay and maqui extract at the same concentration of 50% v / v and which have the same total polyphenol content (parameter used for the standardization of the extracts). Better absorption of UV-A and UV-B light is observed by the Cistanthe extract of the invention.

[0027] Figure 6. Growth inhibition of Staphylococcus aureus in culture medium supplemented with concentrations of 50%, 5%, 2.5%, 1%, 0.5% and 0.25% v / v of a Cistanthe cell extract of the invention diluted to a total polyphenol concentration of 100 mg / l GAE. A 62% growth inhibition is observed with 50% extract, while concentrations of 5% and 2.5% reduce growth by 34% and 18% respectively. Figure 7. Expression of the aquaporin3 gene, the main water transporter in the skin, and of the proinflammatory gene interleukin 8 (IL8), in HACAT cells before and after treatment with 1% of the aqueous extract of the invention diluted to a total polyphenol concentration of 100 mg / l GAE. It is observed that the expression of aquaporin 3 increases, while the expression of IL 8 decreases significantly.

[0028] Figure 8. Figure 8-A, Toxicity evaluation of water extract of Cistanthe cells on FBH fibroblast culture (48h treatment). No decrease in cell viability is observed at any concentration of extract of the invention diluted to a total polyphenol concentration of 100mg / l GAE. Figure 8-B shows the effect of pretreatment of extract diluted to 100mg / l GAE of total polyphenols from Cistanthe cells at concentrations of 1%, 0.3% and 0.1% v / v on the production of interleukin 31 (IL-31) protein in human fibroblast cultures (FBH) after exposure to LPS. It is observed that IL-31 is lower in cells pretreated with the composition of the invention.Figure 8-C shows the effect of pretreatment of extract diluted to 100mg / IGAE of total polyphenols from Cistanthe cells at concentrations of 1%, 0.3% and 0.1% on the production of interleukin 13 (IL-13) in human monocyte cultures (THP-1) after exposure to LPS. It is observed that IL-13 protein is lower in cells pretreated with the composition of the invention at concentrations of 1% and 0.3%.

[0029] Figure 9. Figure 9-A. Fluorescent micrograph of the production of the Aquaporin 3 protein in cultured human skin fragments incubated with the product evaluated (extract diluted to 100 mg / l GAE of total polyphenols). Aquaporin 3 is marked in green mainly in the dermis (shiny area) and the blue mark represents the cell nucleus (opaque area). Figure 9-B, quantification of the presence of the AQUOPORIN3 protein in human skin explants treated or not with the extract of the invention.

[0030] Figure 10. Clinical trial of skin hydration at TO, T2H, T4H and T24H post-treatment, for the blank (no application, only hydration gel vehicle (Placebo) and hydration gel with 1 or 2% of the Cistanthe cell extract of the invention (extract diluted to 100 mg / l GAE of total polyphenols) DETAILED DESCRIPTION OF THE INVENTION

[0031] The invention relates to a composition comprising a plant extract of Cistanthe sp. cells with moisturizing, antimicrobial, UV radiation protection, oxidative stress protection, or antioxidant and anti-inflammatory properties for the skin. Wherein the extract is obtained from Cistanthe sp. cells cultured in vitro, preferably from one or more cell lines derived from the plant's stems and leaves. The extract is preferably aqueous, but could optionally be an ethanolic or hydroalcoholic extract. In one embodiment, the invention relates to a composition comprising the extract and a carrier, suitable for skin care. The composition of the invention protects the skin from oxidative stress induced by environmental stressors such as ultraviolet light, blue light, or air pollution, and has an anti-inflammatory effect.

[0032] Aqueous extracts of Cistanthe sp. are obtained from cells cultured in vitro. Specifically, they are obtained from Cistanthe grandiflora cells, where the cell culture is selected for the production of betalains, betaxanthin, which give the cells a yellow-orange color, and betacyanins, which give a red-purple color. Conveniently, in order to obtain an extract with stable composition, the extracts of the invention are obtained from in vitro cell cultures from one or more cell lines, which, as already indicated, are selected for their high production of betalain. It will be obvious to those skilled in the art that natural plant tissues contain differentiated cells where there is variability in the expression not only of betalain, but also of many metabolites.Therefore, it would not be possible to obtain the extract of the invention from natural plant tissue of Cistanthe plants, since not all of the plant's cells would have the same concentration of Betalain as can be obtained from a selected culture, such as those used in the invention. The plant culture can be developed using any method and culture medium suitable for plant cultivation and available in the art.

[0033] The plant extract of Cistanthe sp. cells comprises the following major compounds:

[0034] • total polyphenols of the order of 10-500 mg / L equivalent of gallic acid, preferably between 100 and 500,

[0035] • Betalains of the order of 0.5 to 15 mg / L, preferably between 3 to 10 mg / L.

[0036] This gives the extract of the invention an antioxidant capacity of the order of IC50 DPPH of between 0.5 and 1% v / v. This is the concentration of the extract necessary to reduce 50% of the DPPH radical. This composition is formulated for pharmaceutical or cosmetic use for skin care, combining the extract with at least one carrier suitable for skin care, conveniently the extract is standardized to a total polyphenol concentration of 100 mg / ml equivalent of gallic acid. This is done by diluting the extract with solvent (water and / or ethanol) since the extract in general has much higher concentrations of polyphenols, as indicated in the examples. If necessary, if the extract had a lower concentration of total polyphenols, it could be concentrated by evaporating the water or ethanol in a rotary evaporator.

[0037] Where the composition of the invention comprises formulation excipients and the extract of Cistanthe sp. cultivated in vitro in a final concentration of between 0.05% w / v and 50% w / v.

[0038] The invention also relates to the process for preparing the extract, which comprises the following steps: a) Collecting Cistanthe sp. cells with orange or red pigmentation from one or more cell lines derived from stems and leaves of the plant, cultured fresh in vitro and drying them at a temperature of between 30-40 9 C for 24 to 60 hours. For example, in a stove at 37 9 C for 48 hours, until the water is completely eliminated. b) Mix the dried in vitro cultured Cistanthe sp. cell powder with the solvent, either water or ethanol or a mixture of both, gradually until reaching a concentration between 0.05% w / v and 15% w / v; c) Incubate the mixture at a temperature between 10 and 50 °C. 9C with stirring at 100 to 250 rpm for a period of between 1 hour and 5 days; and d) Separate the liquid phase, which constitutes the extract e) Combine the extract with a carrier such as water, glycerin, sorbitol, propylene glycol or butylene glycol, or other appropriate excipient and mixtures thereof.

[0039] In a preferred embodiment of the invention, in step b), the concentration of the suspension is between 0.1% w / v and 5% w / v (dry weight with respect to the solvent) of water or ethanol or a mixture of both, for example, 50% ethanol.

[0040] In a preferred embodiment of the invention, the maceration or incubation of step c) is carried out between 1 and 3 days. More preferably, the incubation is carried out for 24 hours at 22 9 C with constant agitation and in darkness.

[0041] In a preferred embodiment of the invention, in step d), the separation can be carried out by centrifugation or filtration. By centrifugation, the samples are subjected to 10,000 to 20,000 rpm for 10 to 60 minutes, for example, at 14,000 rpm for 20 minutes. By filtration, gauze and paper filters are used, such as Whatman filter paper n 9 2.

[0042] Optionally, the extract is sterilized by filtration using, for example, 0.22um filters and stored cold at 4 9 C until further use.

[0043] The extract of the invention has demonstrated its safe use in dermatological applications, being harmless to skin cells.

[0044] The invention also relates to skin-appropriate preparations wherein the extract and a carrier are combined with dermatologically acceptable excipients and stabilizers, such as propanediol, sodium silicate carbomer, methylpropanediol, caprylyl glycol, phenylpropanol, among others. In one embodiment, the invention also relates to skin-appropriate formulations comprising the extract of the invention, carriers, customary formulation excipients, and other active ingredients, such as vitamins, peptides, antioxidants, sunscreens, or other plant extracts.

[0045] It will be obvious to those skilled in the art that a skin care formulation must have not only an aqueous carrier, but also an oily fraction that facilitates the absorption of the active compounds of the extract into the skin cells and that conveniently has preservatives and stabilizers to maintain the integrity or stability of the composition of the invention and its active compounds.

[0046] As shown in the examples, the extract of the invention and the compositions derived therefrom exhibit antioxidant activity, protecting the skin against stress and oxidative damage caused by the environment, including ultraviolet light, blue light, and air pollution. Additionally, the extract of the invention has an anti-inflammatory effect, decreasing the expression of IL-13 and IL-31, which helps prevent oxidative damage from inflammatory processes in the skin.

[0047] The extract of the invention also promotes skin hydration by stimulating the production of the main water transporter in the skin, the protein aquaporin 3. All these beneficial properties, which make the extract of the invention an ideal component for skin care compositions, will become clearer in light of the accompanying examples.

[0048] Example 1: Induction and proliferation of Cistanthe cells in vitro

[0049] Plant material from in vitro-grown plants was collected. To do this, Cistanthe grandiflora seeds were collected and germinated under sterile conditions. Leaves and stems were removed from plants 30 days post-germination and dissected into 1 mm long pieces.

[0050] The material was sterilized and cultured in Petri dishes with MS medium (Murashige & Skoog) supplemented with 30 g / l sucrose, 8 g / l agar, 0.5 mg / l BAP and 1 mg / l NAA to induce cell growth. The culture was maintained in the dark at a temperature of 10 ° C. 9 constant of 22 9 C

[0051] After cell induction, those cell lines that produced an orange or red pigment were selected. Under the microscope, the cells were observed to display intracellular coloration.

[0052] Selected pigmented Cistanthe cells grown on solid media were harvested, dried, and a 1% w / v extract was prepared with water and 50% ethanol. Each extract was macerated for 24 h at 22 °C. 9 C with stirring at 140 rpm. The supernatant was subsequently recovered, and the total polyphenols, betalain pigment, and antioxidant activity were quantified using the DPPH technique. The results are shown in Table 1.

[0053] Table 1

[0054] The ethanolic extract is observed to be richer in polyphenols, expressed as gallic acid equivalent (GAE), while the aqueous extract has a higher proportion of betalain and greater antioxidant capacity. The aqueous and ethanolic extracts of Cistanthe were analyzed in a visible light absorption spectrum between 400 nm and 700 nm. The results are shown in Figure 1, where a peak at 538 nm is associated with the presence of the betalain pigment. The peak at 538 nm is more abundant in the aqueous extract than in the ethanol extract, evidencing greater extraction of this pigment in water.

[0055] Example 2: Cistanthe cell suspensions and polyphenol profile

[0056] A liquid culture of Cistanthe cells from the cell lines chosen in Example 1 was established in flasks using the same culture medium but without agar. The liquid cultures were shaken at 120 rpm at 22 °C. 9 C. The nutrient medium was renewed every 15 days, increasing cell volume. After 3 months of culture, the cells were harvested and dried. Extracts in water and 50% v / v ethanol were prepared from this material, obtaining yields similar to those obtained with solid medium cultures. The yields obtained are shown in Table 2.

[0057] Table 2

[0058] A sample of the aqueous extract was then analyzed by HPLC to determine the profile of the polyphenols present. The method used is not a quantitative tool, but rather a qualitative way of identifying which polyphenol families are present in the extract, taking advantage of the fact that different polyphenol families have maximum absorbances at different wavelengths. The chromatograms were analyzed at wavelengths of 254 nm, 280 nm, 325 nm, 365 nm, and 540 nm. Figures 2. A; 2. B; 2. C; 2. D and 2. E show the polyphenol profiles obtained.

[0059] The chromatograms show that the highest intensities are observed at wavelengths of 254 nm and 280 nm. According to the literature, flavan-3-ols have UV absorption maxima around 280 nm, while several organic acids and lignans present maximum absorbances in the 200-300 nm range, so the extract compounds possibly belong to one of these groups. Additionally, the Cistanthe extract could contain low concentrations of anthocyanins.

[0060] In parallel, a quantitative HPLC analysis of some known polyphenols was performed for the aqueous and ethanolic extracts. The results are shown in Table 3 below.

[0061] Table 3

[0062] ND= not determined The identification and quantification of some polyphenols indicate that both extracts share more than 50% of the list of polyphenols analyzed, with the aqueous extract having a higher presence of known polyphenols, highlighting the presence of Vanillin and chlorogenic acid.

[0063] Example 3. Toxicity analysis of Cistanthe extracts

[0064] To evaluate the effect of applying a Cistanthe extract on skin cells, it is first necessary to assess whether the extracts are toxic to these cells, so HACAT toxicity tests were performed on keratinocytes.

[0065] Cistanthe water and ethanol extracts were diluted to a total polyphenol concentration of 100 mg / l GAE (extract 00). The toxicity of these extracts was evaluated on HACAT keratinocyte cell cultures.

[0066] A total of 6,000 HACAT cells were seeded in culture medium containing 15% FBS per well. After 24 hours, the medium was discarded, washed with PBS IX, and serum-free basal medium containing 2% water (Ca), medium containing EGF (Cb), and medium containing different concentrations of both extracts were added.

[0067] Concentrations ranging from 0.1% to 2% v / v were evaluated. The extract was diluted in the same culture medium as the HACAT cells. Cells were treated for 24 hours, and cell viability was measured using XTT.

[0068] After 24 hours, a photograph of each condition was taken, the medium was discarded, washed with PBS IX, and 50 pl of basal medium, 50 pl of XTT, and 1 pl of PMS were added per well. The sample was left to incubate for 2 hours, and the absorbance of each well was measured at 460 nm (reference 650 nm) using the Tecan Sunrise system. The data were processed in an Excel spreadsheet and graphed using GraphPad Prism 6.

[0069] The results are shown in Figure 3, where the results of concentrations between 0.1% v / v and 2% v / v of aqueous and ethanolic extract of Cistanthe are shown, which do not present toxicity in HACAT keratinocyte cell cultures, since there was no significant change in the viability of HACAT epidermis cells, compared to the control with water (Ca).

[0070] Example 4. In vitro antioxidant protection

[0071] We evaluated the in vitro antioxidant effect using HACAT keratinocyte cells treated with hydrogen peroxide. HACAT cells were treated for 24 hours with l% v / v of the extract lOO. (Extract diluted to a polyphenol concentration of 100 mg / l GAE). As a positive control for antioxidant protection, 500 uM of Vitamin C (ascorbic acid) was used for 24 hours. Subsequently, control cells were treated with 700 uM of H2O2 for 1 hour, after this time, cell viability was measured by XTT. The concentrations of hydrogen peroxide and vitamin C to be used in the antioxidant efficacy assays in HACAT cells were selected both with data taken from the literature and data obtained in our laboratory. As expected, cultures exposed only to 700 uM of H2O2, present a decrease of 38.93% (P <0.001) in cell viability.

[0072] In treatment with 500uM of Vitamin C and H2O2, cell viability is reduced by only 33.78% (5.15% protection, compared to 38.93% with H2O2 alone).

[0073] On the other hand, in the treatments with the aqueous extract of Cistanthe cells and H2O2 there was a decrease in cell viability of only 31.83% (P<0.001), that is, it provides greater protection than Vitamin C. While in the treatment with ethanolic extract, viability is reduced by only 34.12% (P<0.001), that is, a protection slightly lower than that provided by the positive control.

[0074] Treatments with vitamin C, aqueous extract, and ethanolic extract of Cistanthe significantly alleviated cell death caused by H2O2. There were no significant differences between vitamin C and the Cistanthe cell extracts.

[0075] Example 5. UV Absorption of Cistanthe Aqueous Extract

[0076] Cistanthe grandiflora grows in the Atacama Desert, the region with the highest solar radiation on the planet. Based on this information, we evaluated whether the aqueous extract of Cistanthe cells could serve as a sunscreen enhancer. To do so, we measured the UV absorption potential. Absorption scanning was performed from 250 nm to 400 nm, the wavelength range of UV radiation, with different concentrations of the concentrated Cistanthe extract. The 100% extract contains 330 mg / l GAE of polyphenols, which were used at 100%, 50%, 25%, 10%, and 1%. Water (solvent) was used as the blank. The results are shown in Figure 5-A.

[0077] UV absorption can be observed at concentrations greater than 10% of the concentrated Cistanthe extract, so extracts with a polyphenol content of 33 mg / l GAE are sufficient to absorb UV light and serve to enhance sunscreens. We evaluated in detail the UV-A and UV-B absorption profile of the 50% Cistanthe extract and compared it with the same concentration of extracts from other plants such as quillay and maqui prepared in the same way and containing the same total polyphenol content. It can be observed that, at the same concentration, the Cistanthe extract presents a higher absorption profile than the extracts of the other plants evaluated under both types of UV radiation (Figures 5-B and 5-C).

[0078] Example 6. Antimicrobial properties of Cistanthe aqueous extract

[0079] We evaluated whether Cistanthe cell extract has antimicrobial activity. To do so, we tested different concentrations of the extract on the growth of Staphylococcus aureus (one of the most abundant bacteria on the skin) for 24 hours. A concentration of 10 mg / ml of the purified molecule hinokitiol was used as a positive control.

[0080] The results are shown in Figure 6, where it is observed that 50% v / v of the extract 100 inhibits Staphylococcus aureus growth by 62%, while concentrations of 5% and 2.5% reduce growth by 34% and 18% respectively. Concentrations lower than 2.5% of the extract 100 do not inhibit bacterial growth.

[0081] Example 7. Evaluation of the effect of the extract of the invention on human keratinocyte cells by means of the expression of marker genes.

[0082] The expression of marker genes associated with improvements in skin cell health, which are intended to be enhanced for aesthetic purposes, was evaluated in HACAT human keratinocyte cells.

[0083] To this end, a culture of these cells was treated for 24 hours with a final concentration of 1% v / v of the aqueous extract diluted to a total polyphenol concentration of 100 mg / l GAE. After this time, RNA was extracted using the TRIZOL method, quantified using the QUBIT equipment, total RNA logs were taken, cDNA was synthesized using a standard method, and the expression level of different genes associated with cosmetic uses on the skin was measured by RT-qPCR, comparing the basal level of expression of these same genes without treatment with the extract.

[0084] Specifically, the expression of the following human genes was measured: Collagen synthesis (Metalloproteinase 1,2, 3, 7, 9; Metalloproteinase inhibitor 1; Collagen I alpha 1; Collagen III alpha 1; regenerating islet-derived protein 3-alpha and Thy-1); Antioxidant machinery (SOD1, SOD2, CATALASE, GSTP1); Anti-inflammation (ILL6, ILL8, CCL20); Skin epithelial barrier and water balance (Loricrin, filaggrin, Involucrin, Aquoporin 3, Aquoporin5, HPRT); Biorevitalizant products (hyaluronic acid synthase 1 (HAS1), HAS2, HAS3, hyaluronoglucosaminidase 1, elastin) and Senescence (P53, SIRT1, MITOL, Drpl, PGC-la). The primers used for each of these genes were selected from numerous publications reporting the expression of these genes in HACAT cells (e.g. Chen et al. 2019, Biomed Pharmacother 116:109011; Velazquez Pereda 2010, J Cosmet Dermatol 9(l):35-43; Yousefi et al. 2017 Iran J Allergy Asthma Immunol 16(3):228-234 and Jung 2019, Int J Mol Sci 20(17):4289)

[0085] Of these genes, we found that the aqueous extract of Cistanthe grandiflora induces the expression of the aquaporin3 gene, the main water transporter in the skin, and decreases the expression of IL8, a pro-inflammatory gene. These results are graphed in Figure 7. These results indicate that the application of the extract of the invention helps improve hydration, reduce inflammation, and therefore improve the appearance of skin cells. No significant changes in expression were found for the rest of the genes evaluated.

[0086] Example 8. In vitro anti-inflammatory assay

[0087] We evaluated the effect of Cistanthe extract on the production of Interleukin 13 (IL-13) and Interleukin 31 (IL-31) proteins in cultured human fibroblast cell lines (HFC) and human monocyte cell lines (THP-1) respectively.

[0088] Primary human fibroblasts (PHB) were treated with different concentrations between 0.001 and 10% of the evaluated product (extract OO) to determine non-cytotoxic concentrations through the MTT assay, with a 48 h treatment of different concentrations of the extract of the invention. The results are shown in Figure 8-A. It was observed that the extract OO of Cistanthe cells is not toxic to PHB cells at any of the concentrations evaluated. The same was observed for human monocytes (THP-1).

[0089] Next, human monocyte cultures (THP-1) and human fibroblast cultures (FBH) were pretreated with the three concentrations verified in the MTT assay (1%, 0.3% and 0.1% v / v) and subsequently exposed to 50pg / ml of bacterial lipopolysaccharide (LPS) to induce the inflammatory response. Finally, the interleukin 13 (IL-13) and interleukin 31 (IL-31) proteins were quantified by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figures 8-B and 8-C. Figure 8-B shows the effect of pretreatment of Cistanthe cell extracts at concentrations of 1%, 0.3% and 0.1% v / v on the production of IL-31 in human fibroblast cultures (FBH) after exposure to LPS. As expected, cultures exposed to only 50 pg / ml LPS, without pretreatment, showed a 74.92% increase (P<0.001) in IL-31 synthesis, contributing to the inflammatory state.While treatments with the aqueous extract of Cistanthe cells showed a significant decrease of 25.75% (P<0.001), 18.70% (P<0.01) and 18.00% (P<0.01) in the synthesis of IL-31, at concentrations of 1%, 0.3% and 0.1% v / v respectively.

[0090] Figure 8-C shows the effect of pretreatment with Cistanthe cell extract 100 at concentrations of 1%, 0.5% and 0.25% v / v on IL-13 production in human monocyte cultures (THP-1) after exposure to LPS. As expected, cultures exposed only to LPS 50 µg / ml, presented an increase of 73.09% (P<0.001) in the synthesis of IL-13, contributing to the inflammatory state. But treatments with the aqueous extract of Cistanthe cells showed a significant decrease of 25.63% (P<0.05) and 23.20% (P<0.05) in the synthesis of IL-13, at concentrations of 1% and 0.3% respectively. The concentration of 0.1% does not present a significant decrease in the expression of this interleukin.

[0091] These results demonstrate that the extracts of the invention contribute to reducing inflammation in skin cells, more completely at concentrations as low as 0.3% v / v, and there would be an effect, although smaller, at concentrations as low as 0.1% v / v.

[0092] Example 9. Ex-vivo aquaporin3 assay

[0093] Considering that in Example 7 it was established that the extract of the invention increased the expression of aquaporin3, we evaluated whether the aqueous extract of Cistanthe of the invention has properties to regulate water transport in the skin. To do so, we evaluated the presence of the AQUOPORIN3 protein in human skin explants. We prepared a formulation containing 100 mg / L of total polyphenols, which is composed of 50% aqueous extract of Cistanthe cells, 40% vegetable glycerin and 10% pentylene glycol as a way of applying the extract to these human skin explants.

[0094] Skin fragments were divided into pieces of approximately 1.5 cm2, placed in culture plates (Corning, USA) with specific culture medium and kept in an incubator at 37°C in the presence of 5% CO2 and the formulated extract with 25-30 mg / cm2 was evaluated. 2 maintaining the culture for 4 days.

[0095] After the treatment period, skin fragments were embedded in Tissue-Tek® OCT™ and serial 12-micron histological sections were then collected directly onto silanized slides using a cryostat (Leica, GER - CRYOCUT 1800). Sections were subsequently washed in phosphate buffer (PB) and incubated overnight with anti-aquaporin-3. Sections were then washed again and incubated for 1 hour with Alexa Flour 488 Secondary Antibody (1:1000). An additional incubation with DAPI (4'-6-diamidino-2-phenylindole; DNA marker) was performed followed by washes in phosphate buffer. The fluorescence intensity parameter emitted by labeling of the specific antibody was evaluated. After obtaining the images, the fluorescence intensity was quantified with the help of ImageJ software.

[0096] Figure 9-A shows the fluorescent micrograph evaluation of Aquaporin 3 production in cultured human skin fragments incubated with the product evaluated. Aquaporin 3 is marked in green mainly in the dermis (bright area) and the blue mark represents the cell nucleus (opaque area; DNA; DAPI). Figure 9-B graphs the quantification of the presence of the AQUOPORIN3 protein in human skin explants treated with the extract of the invention. As can be seen, treatment with the formulated extract promoted a 66.81% increase in Aquaporin 3 production, compared to the basal control (P<0.001).

[0097] Example 10. Clinical trial hydration

[0098] To evaluate the effect of hydration in vivo, a clinical trial was conducted with 20 volunteers using the following methodology:

[0099] Products to be evaluated: Placebo hydration gel, Hydration gel +1% Cistanthe cell extract, and Hydration gel +2% Cistanthe cell extract.

[0100] Trial type: This project is a single-center, blinded study. Volunteers are used as their own controls.

[0101] The specific inclusion criteria, defined in the protocol, were as follows:

[0102] Age: 18 to 70 years,

[0103] Gender: both, Skin type: dry skin (skin hydration < 40 AU).

[0104] The experimental area chosen was the forearm. The application and control areas were randomly selected by the researcher. The test product was applied by the researcher to one of the localized sites in a clockwise distribution, varying from subject to subject.

[0105] The researcher in charge of the test performed a light massage, with the help of a finger cover, until the complete penetration / homogenization of the product was achieved, in an area of ​​2.5 x 2.5 cm (6.25 cm 2 ) for each time point on the volar forearm, determined by randomization. The amount of product applied was 2 mg / cm2 and was measured using a micropipette with disposable tips. A blank control area was also measured to assess external influences. To ensure uniformity of testing procedures across subjects, the test product was applied by the same investigator. All measurements include a prior acclimatization period and are performed in a controlled environment (T=21°C ± 1°C, RH=50% ± 10%).

[0106] Hydration content is obtained using an electrometric system based on capacitance measurement, allowing the calculation of the dielectric constant of water. The measuring probe has an interdigital grid (I) of gold-coated electrodes. The interdigital electrode is coated with a vitrified, low-dielectric material. Therefore, there is no galvanic contact between the electrode and the skin surface. A constant application pressure is applied to the skin surface through a spring system. As a result of the probe's design, the system (electrode, superficial parts of the stratum corneum, and epidermis) behaves like a capacitor. Measurements are obtained in Arbitrary Units (AU) as a reference to a factory standard.

[0107] Measurements were performed using a CM825 Corneometer connected to an MPA 580®, MPA 6®, or MPA 2® Dual Corneometer (Courage & Khazaka, Germany). Each volar area of ​​the forearm (including the control group) was evaluated.

[0108] Measurements were taken at time = 0 h as a baseline level and at 2 h, 4 h and 24 h post-application. In each of the evaluation areas, measurements were taken in triplicate to completely sample the entire area, and the average was calculated. To obtain the T = 0 value, all time points in the evaluation area were sampled in triplicate, and the value was the average of all points. The results are shown in Figure 10, where it is observed that the compositions of the invention provide better hydration at all times evaluated, and that this is better with a higher proportion of extract of the invention (2% extract). Surprisingly, 24 hours after application, both compositions of the invention maintain skin hydration.

[0109] In conclusion, Cistanthe cell extract significantly increases skin hydration compared to the blank and placebo groups. The greatest results were observed at a 2% concentration, increasing by 20% at 2 hours, 10% at 4 hours, and maintaining a 5% increase 24 hours after application.

[0110] Example 11. Obtaining skin care composition.

[0111] The concentrated composition, for routine skin care, was obtained by mixing 5 g of Cistanthe cell extract with 5 g of glycerin, under gentle stirring, until a homogeneous mixture was obtained, which had 50% of the extract of the invention.

[0112] To obtain a composition for routine skin care, 1 g of Cistanthe cell extract was mixed with 9 g of glycerin, under gentle stirring, until a homogeneous mixture was obtained, which had 10% of the extract of the invention.

[0113] On the other hand, to obtain a hydration gel containing 1% w / v and 2% w / v of the active ingredient of the invention, two hydrating formulations were prepared with water and the excipients propanediol, sodium carbomer silica, methyl-propanediol, caprylyl glycol, phenylpropanol, and the Cistanthe grandiflora cell extract diluted to a total polyphenol concentration of 100 mg / l GAE (1% or 2% v / v). The quantities used are shown in Table 4 below.

[0114] Table 4

Claims

CLAIMS 1. A composition with moisturizing, antimicrobial, antioxidant and anti-inflammatory properties for the skin, CHARACTERIZED in that it comprises aqueous, ethanolic or hydroalcoholic extracts of Cistanthe sp cells cultured in vitro, of one or more cell lines derived from the stems and leaves of the plant; and a carrier, where the majority compounds comprising the extract correspond to total polyphenols in the range of 10 to 500 mg / ml equivalent of gallic acid and betalains in a concentration between 0.5 and 15 mg / L.

2. The composition according to claim 1, CHARACTERIZED in that the total polyphenols are in a concentration between 100 to 500 mg / ml equivalent of gallic acid and betalain in the order of 3 to 15 mg / L.

3. The composition according to claim 2, CHARACTERIZED in that the extract has an antioxidant capacity of between 0.5 to 1% v / v IC50 DPPH.

4. The composition according to claim 2, CHARACTERIZED in that the extract corresponds to an aqueous extract.

5. The composition according to claim 1, CHARACTERIZED in that the composition is formulated for pharmaceutical or cosmetic use for skin care, where the extract is standardized so that the total polyphenols are at a concentration of 100 mg / ml equivalent of gallic acid.

6. The composition according to claim 5, CHARACTERIZED in that the carrier is at least one excipient suitable for skin care, such as water, glycerin, sorbitol, propylene glycol or butylene glycol and mixtures thereof.

7. The composition according to claim 6, CHARACTERIZED in that it comprises formulation excipients and the Cistanthe sp. extract cultivated in vitro in a final concentration of between 0.05% w / v and 50% w / v.

8. Process for obtaining a composition comprising extracts of Cistanthe sp. cells cultured in vitro, according to claim 1, CHARACTERIZED in that it comprises the following steps: a) Collecting Cistanthe sp. cells with orange or red pigmentation from one or more cell lines derived from stems and leaves of the plant, cultivating them in vitro and drying them at a temperature between 30-40 9 C for 24 to 60 hours until the water is completely eliminated; b) Mix the dried Cistanthe sp. cell powder from step a) with a solvent, either water or ethanol or a mixture of both, little by little until reaching a concentration between 0.05% w / v and 15% w / v; c) Incubate the mixture at a temperature between 10 to 40 9 C with stirring at 100 to 250 rpm for a period of between 1 hour and 5 days; and d) Separate the liquid phase, which constitutes the extract, e) Combine the extract with a carrier such as water, glycerin, sorbitol, propylene glycol, butylene glycol, and mixtures thereof.

9. Process for obtaining according to claim 8, CHARACTERIZED in that in step a) the concentration of the suspension is between 0.1% w / v and 5% w / v.

10. Process for obtaining according to claim 8, CHARACTERIZED in that in step c) it is incubated between 1 to 3 days.

12. Process for obtaining according to claim 8, CHARACTERIZED in that in step d) the separation can be carried out by centrifugation or filtration.

13. Use of a composition according to any of claims 1 to 7, CHARACTERIZED in that it is used to prepare formulations for pharmaceutical or cosmetic use for skin care.

14. Use according to claim 13, CHARACTERIZED in that the formulation has a moisturizing, antioxidant, UV light protection and anti-inflammatory effect on the skin.

15. Use according to claim 14, CHARACTERIZED in that the formulation protects the skin from oxidative stress induced by environmental stressors such as ultraviolet light, blue light or air pollution.