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    • 专利摘要:
    Cotton cell cultures and their use in photoprotection. The invention relates to a cotton suspension cell culture (gossypium) which is useful as a skin photoprotective agent and for the preparation of medicaments. The invention also describes the process for obtaining this cell culture and cosmetic and pharmaceutical compositions comprising it. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2579385A1
    申请号:ES201530157
    申请日:2015-02-10
    公开日:2016-08-10
    发明作者:Òscar EXPÓSITO TARRÉS；Albert JANÉ FONT；Sara LAPLANA LASIERRA；Maria MAS DUARTE
    申请人:Phyture Biotech S L；Phyture Biotech SL；
    IPC主号:
    专利说明:

    DESCRIPTIONCotton cell cultures and their use in photoprotection.

    The invention relates to cotton cell cultures (Gossypium). It also refers to methods specially designed for obtaining them and medical and cosmetic uses of these crops.

    STATE OF THE TECHNIQUE
     10
    It is highly known that the skin is the first barrier of protection against external agents in most animals. It is also known that the skin must in turn protect itself from external agents in order not to compromise its integrity and thus not compromise its different functions. One of these external agents is precisely solar radiation (ultraviolet, visible and infrared spectrum), or any type of electromagnetic radiation that must be applied for other purposes; for example, UV ultraviolet radiation to achieve a controlled tanning or infrared IR radiation in case of physiotherapy treatments.

    Photoprotection of the skin using sunscreens or topical products for daily use 20 has basically focused on preventing acute damage, such as burns, or chronic skin damage, such as cancer or photoaging, caused by exposure to UV radiation , basically UVA and UVB.

    UVB radiation (280-315 nm) is responsible for most burns although UVA radiation (315-400 nm) also causes erythema to a lesser extent. Both types of radiation alter the immune system. UVB radiation is partially filtered by the ozone layer, but penetrates the superficial layers of the skin to the basal layer of the epidermis. It is the cause of the generation of reactive oxygen species (ROS), promoting inflammation and aging. UVB 30 radiation can also be absorbed by cellular DNA and cause mutagenic lesions. On the other hand, UVA radiation, less energetic than UVB but to a greater extent in the solar radiation spectrum (the latter, solar radiation of 200 nm at 1 mm wavelength) penetrates more than UVB radiation (UVA reaches basal levels of the dermis); and it is known that by generating ROS it can also alter DNA, 35
    proteins, and skin lipids, which leads to aging and wrinkle formation, and indirectly increases the risk of cancer.

    In recent years it has been found that other wavelengths of solar radiation, beyond UV, specifically the visible light spectrum (around 5% of the solar radiation spectrum) and IR radiation (more than 50 approximately% of the solar radiation spectrum) also cause damage to the skin and produce photoaging.

    As can be seen from the reference Dupont et al. "Review Article Beyond UV 10 radiation: A skin under challenge", International Journal of Cosmetic Science-2013, vol. No .: 35, pp.:224-232, visible light (from 400 to 700 nm) penetrates deeply into the dermis and 20% reaches the hypodermis (See for example Figure 6 of this reference).

    It also appears from the previous document that among the different types of IR radiation (IRA, IRB, IRC), IRA radiation (30% of the IR spectrum) reaches 65% of the dermis and 10% of hypodermis. IRA can induce an imbalance in the gene expression of extracellular matrix metalloproteinase 1 (MMP-1), altering the expression and functionality of collagen. With this, IRA is also involved in photoaging and can promote carcinogenesis. It is postulated that the IRA radiation can interact with elements of the electronic transport chain in the mitochondria and thus produce an imbalance favoring the appearance of ROS.

    While many compounds and compositions for the UV radiation filter are known, few are the compositions that also include active ingredients that can prevent damage caused by IR radiation or even visible radiation. However, there are references that indicate the effect of certain topical antioxidants as photoprotective agents of IRA radiation. An example of this is described by Grether-Beck et al., "Review Article Photoprotection of human skin beyond ultraviolet 30 radiation", Photodermatology, Photoimmunology & Photomedicine -2014, doi: 10.1111 / phpp.12111. Grether-Beck et al. indicate that a combination of vitamin C, ferulic acid and tocopherol came to inhibit the expression of messenger RNA of MMP-1 (extracellular matrix metalloproteinase 1) induced by ARF in fibroblasts and in human skin. The document also comments on the need for 35
    Locate antioxidants or compounds that are capable of filtering both UV and IR radiation.

    In order to provide as natural, effective and effective sunscreens that comprise only minimal amounts of synthetic assets, 5 different plant extracts have been developed that have been shown to be able to prevent damage from UV radiation. An example of this follows from the document Mishra et al., “Review Article. Herbal Cosmeceuticals for Photoprotection from Ultraviolet B Radiation: A review ”, Tropical Journal of Pharmaceutical Research-2011, vol. No. 10 (3), pp .: 351-360. This document lists extracts of plants and components isolated from them with the capacity to eliminate or prevent the effects of free radicals caused by UV radiation in particular. Among these plants are listed tomato (Solanum lycopersicum), garlic (Allium sativum), Aloe vera, saffron (Crocus sativus) or purslane (Portulaca oleracea), among others.
     fifteen
    It follows from all of this that, to date, new compositions are still necessary to prevent and / or treat the effects of solar radiation, and in particular of its various components, especially UV radiation and some components of the IR spectrum and even the visible light
     twenty
    EXPLANATION OF THE INVENTION

    The inventors propose a culture of suspension cells of cotton (genus Gossypium) that, surprisingly, acts as a filter for solar radiation, and in particular and very effectively for IR radiation. In addition, the culture is also highly effective against UV radiation. As will be detailed in the examples, the culture is a potent inhibitor of MMP-1 expression, thus preventing the degradation of collagen and maintaining the integrity of the skin. Its effect is comparable to that of quercetin (a flavonol used in cosmetic compositions as a radiation filter) even at lower doses. Cotton is a plant that grows in different parts of the planet. Some of its species are characterized, among other capacities, by growing in arid areas. The inventors have developed a reproducible cell culture that, surprisingly, is able to prevent the harmful effects of solar radiation with high effectiveness over other compounds.
     35
    Thus, in a first aspect the invention relates to a culture of cells in suspension of a plant of the genus Gossypium, characterized in that it comprises:
    - polyphenols at a concentration of 250 to 500 milligrams per liter of culture;
    - phytosterols at a concentration of 30 to 60 milligrams per liter of culture;
    - a mixture of sugars that includes glucose, fructose, sucrose, 5 furanosa, arabitol and myoinositol
    - lipids; Y
    - proteins

    The composition of the crop, comprising the amounts of polyphenols and 10 phytosterols, as well as the presence of lipids, sugars and proteins is a synergistic combination, so that it is the sum of its components and their proportions that gives rise to the advantageous effect on as for the prevention of the harmful effects of radiation. In addition, the crop supposes the advantage of avoiding the methodology for obtaining assets via traditional plant extracts and provides 15 many more active components than these extracts.

    In the sense of the invention it is understood as "culture of cells in suspension of cotton or of the genus Gossypium" to that group of dedifferentiated cells dispersed or in suspension in a liquid medium. The cell suspension may comprise cells at various stages of aggregation. Cell cultures in suspension, also called cell cultures or suspensions, are generally initiated from friable (disintegrable) corns in a liquid culture medium and under agitation. The callus is placed in a container (flask) with liquid culture medium and placed under stirring for several days at 25 ° C until free cells are obtained. The suspension is then filtered (250 µm) and added to another container with fresh culture medium and stirring is continued. Callus must be understood as that mass of dedifferentiated, amorphous cells that multiply in a disorganized manner. Calluses are usually induced in vitro from plant organs (usually also established in vitro) to suitable culture media comprising different plant growth regulators.

    The use of cell cultures in suspension of plants entails the advantage of having factories of compounds of interest in a controlled and reproducible manner once the system has been developed, which is not a routine task. So, 35
    Plant isolated cells undergo differentiation to obtain totipotent cells. These totipotent cells (also known as plant stem cells) can then be differentiated into the tissue of interest under the correct stimulation, or taken to a stage of production of compounds of interest. Suspended cultures are generally used for the production of 5 secondary metabolites of the plant in question and of special interest in cosmetics or pharmacy.

    In a particular embodiment of the culture, the polyphenols are in a concentration by weight of 200 to 400 mg / l of culture. 10

    Among the polyphenols present in the suspension cell culture of the invention are those present in cotton, such as phenols and flavonoids selected from gallic acid, catechins, gentisic acid, caffeic acid, ferulic acid, cumaric acid, salicylic acid, acid cinnamic, gosipetin, glycosylated gosipetin 15, kaempferol glycosides, herbacitin glycosides and mixtures thereof. Without being bound by any theory, the inventors propose that the crop comprises the total amount of polyphenols and within this total a particular composition of them that, together with the other components, confer the properties against solar radiation and in particular against IR radiation twenty

    Also, among the phytosterols present in the culture of cotton suspension cells are, in particular, lanosterol, stigmaesterol and β-sitosterol.
     25
    In another particular embodiment, the crop is characterized in that the plant of the genus Gossypium is selected from the group consisting of Gossypium herbaceum, Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium tomentosum, and mixtures thereof. In an even more particular embodiment it is from Gossypium herbaceum. 30

    The invention also has as a second aspect a method of obtaining a cell culture of a plant of the genus Gossypium in suspension, comprising the following steps:
     35
    (a) Cultivate dedifferentiated cells of the Gossypium genus plant in a growth culture medium comprising plant growth regulators, said regulators selected from the group consisting of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalenacetic acid (NAA) and cytokinins quinetin, and 6-Benzylaminopurine (BAP) and mixtures thereof 5 for a period of 5 to 10 days;
    (b) add to the cultivation of step (a), 2 to 4 times, an elicitor composition or combination, where this composition or combination is selected each time it is added from:
    - a composition or combination comprising cyclodextrins, and at least one jasmonate compound, wherein the jasmonate compound is selected from the group consisting of methyl jasmonate, ethyl jasmonate and propyl jasmonate; and where at the end of the 2 to 4 times of addition of the elicitor composition or combination, the cyclodextrins are at a final concentration in the culture of 18 to 72 g / l of culture and the jasmonate compound is at a final concentration in 100 µM to 500 µM culture; Y
    - a composition comprising at least one jasmonate compound, selected from methyl jasmonate, ethyl jasmonate and propyl jasmonate, where at the end of the 2 to 4 times of addition of the elicitor composition 20, the jasmonate compound is in a final concentration in the culture from 100 µM to 500 µM.

    In a particular embodiment, optionally in combination with one or more embodiments described above or below, the procedure is carried out in the dark.

    By "growth culture medium" means a culture medium adapted for the cultivation of cells in suspension of plants, so that they can carry out their entire cell cycle and divide until they reach a stationary stage, moment 30 when so many cells die as cells divide. These means comprise nutrients and mineral salts for the correct growth of cells, such as sugars and sodium, potassium or calcium salts. In addition, depending on what is intended with the cells in suspension, plant and vitamin growth regulators can be added. 35

    In another particular embodiment, the growth culture medium of step (a) consists of a basal culture medium of plant cells selected from the group formed by Murashige and Skoog (MS); LS Linsmaier and Skoog (LS) and Gamborg-B5, where to said basal medium are added to complement plant growth regulators (also known as phytohormones) and selected from the group consisting of auxins 2,4-dichlorophenoxyacetic acid (2, 4-D), naphthalenacetic acid (NAA) and cytokinins quinetin, and 6-Benzylaminopurine (BAP) and mixtures thereof. The expert knows these basal means and has the technique to complement them with nutrients and other components of interest, depending on the objective of the crop.

    In a particular embodiment, optionally in combination with any embodiment described above or below, the culture medium is the Murashige and Skoog (MS) medium which further comprises plant growth regulators selected from the group consisting of the acid auxins 2, 4-dichlorophenoxyacetic (2,4-D), naphthalenacetic acid (NAA) and cytokinins quinetin, and 6-Benzylaminopurine (BAP) and mixtures thereof.

    In another particular embodiment, also optionally in combination with one or more embodiments described above or below, the culture medium comprises auxins selected from those indicated above at a concentration of 1 to 4 mg per liter of culture (mg / l) of stage (a); and cytokinins selected from those indicated above at a concentration of 0.1 to 3 mg / liter of culture. In another more particular embodiment, the culture medium comprises auxin acid 2,4-25 dichlorophenoxyacetic acid (2,4-D), and cytokinin quinetin at a concentration of 0.5 to 4 mg / liter of culture and 0.1 to 3 mg / liter of cultivation, respectively. Still in a more particular embodiment, auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is at an initial concentration of 0.5 to 2 mg / l culture and cytokinin quinetin at a concentration of 0.1 to 2 mg / l. 30

    In an even more particular embodiment, the culture medium of step (a) comprises 0.5 mg / liter of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg / l of quinetin.
     35
    It is further understood that the concentrations of the members of the composition or elicitor combination of step (b) refer to the amount that is added in the culture such that, once added based on the number of times, the molar concentration it is the indicated one or the concentration in weight / volume is the indicated one. It will also be an elicitor composition (synthesis stimulator of another 5 compounds in the plant cell) if both components are added at the same time in the same matrix (for example in basal medium), and by elicitor combination if they are added simultaneously but separately or sequentially.

    In a particular embodiment of the process, the concentration of the 10 jasmonate compound is from 200 to 450 µM. More in particular from 300 to 450 µM. In another even more particular embodiment, the jasmonate compound is in a final concentration in the culture of 450 µM.

    Another particular embodiment of the process according to the invention, optionally in combination with the other embodiments of the process, comprises adding the cyclodextrins at a concentration in the culture of 25 to 50 g / l (grams per liter of culture), more particularly 30 at 40 g / l; and even more particularly from 35 to 40 g / l.

    In another particular embodiment of the process, this is characterized in that the step (b) is carried out with three additions of the elicitor composition or combination.

    In another even more particular embodiment, the process is characterized in that the addition of the elicitor composition or combination is carried out in accordance with the following addition pattern:
    (i) add a first dose of the elicitor composition or combination when the culture of step (a) has grown from 5 to 10 days;
    (ii) add a second dose 3 to 4 days after the first dose; Y
    (iii) add a third dose 2 to 3 days after the second dose.
     30
    In another even more particular embodiment, the addition of the elicitor composition or combination is carried out according to the following addition pattern:
     (i) adding a first dose of an elicitor composition or combination comprising cyclodextrins and a jasmonate compound, when the culture of step (a) has grown from 5 to 10 days; 35
    (ii) adding a second dose of a composition comprising a jasmonate compound, 3 to 4 days after the first dose; Y
    (iii) adding a third dose a composition comprising a jasmonate compound, 2 to 3 days after the second dose.
     5
    In an even more particular embodiment, the addition of the elicitor composition or combination is carried out according to the following addition pattern:
     (i) adding a first dose of an elicitor composition or combination comprising cyclodextrins and a jasmonate compound, when the culture of step (a) has grown from 5 to 10 days, where at the end of the addition the cyclodextrins are at a The final concentration in the culture of 18 to 72 g / l of culture and the concentration of jasmonate compound is 50 µM;
    (ii) adding a second dose of a composition comprising a jasmonate compound, 3 to 4 days after the first dose, where at the end of the addition the concentration of jasmonate compound is 50 µM; and 15
    (iii) adding a third dose a composition comprising a jasmonate compound, 2 to 3 days after the second dose, where at the end of the addition the concentration of jasmonate compound is 50 µM.

    Another particular embodiment of the process, optionally in combination with the rest of particular embodiments listed here, is characterized in that the elicitor composition comprises methyl jasmonate, preferably at a final dose or concentration of 300 to 450 µM once added 2 to 4 times .

    In another particular embodiment, also optionally in combination with the rest of 25 particular embodiments listed here, the elicitor composition comprising cyclodextrins comprises a cyclodextrin selected from α-cyclodextrin, β-cyclodextrin, Υ-cyclodextrin or a mixture of two or more of them. . Cyclodextrins as described in this invention also include modified or functionalized cyclodextrins with hydroxyl, alkylated (with C1-C4-linear or branched alkyls, including -CH3, -CH2-CH3,) or hydroxyalkylated, as are known in The state of the art.

    The method according to the invention is also characterized in that it also comprises the step (c) of lysing the cell culture obtained in step (b). 35

    In a particular embodiment of the process, the lysing step (c) is carried out by adding the culture obtained in step (b) to a liquid solution and homogenizing the mixture in a turrax at a stirring speed of 15,000 revolutions per minute ( rpm) at 25000 rpm. In an even more particular embodiment, the proportion by weight of culture in the liquid solution is from 10 to 40% by weight, more particularly from 25 to 40% by weight measured in grams of culture per grams of liquid solution. More particularly 30 to 35% by weight of culture of step (b) in the liquid solution.
     10
    In another particular embodiment, the liquid solution of step (c) comprises glycerin and, optionally preservatives and additives, preferably gluconolactones, benzoates (such as sodium benzoate) and gluconates.

    In another particular embodiment, the lysing stage (c) comprises the steps of:
    - freeze and lyophilize the culture of step (b);
    - adding a solid composition comprising a compound selected from maltodextrin, sorbitol, mannitol and mixtures of two or more of them; Y
    - homogenize the solid mixture.
     twenty
    The invention therefore also includes a culture of cells in suspension of a plant of the genus Gossypium, characterized in that it is obtainable by the method as described in any one of the previous variants. Specifically a suspension culture of cells of a plant of the genus Gossypium, preferably of the species Gossypium herbaceum, obtainable by the following 25 stages:

    (a) Cultivate dedifferentiated cells of the Gossypium genus plant in a growth culture medium comprising plant growth regulators, said regulators selected from the group consisting of auxins 30 2,4-dichlorophenoxyacetic acid (2,4-D) , naphthalenacetic acid (NAA) and cytokinins quinetin, and 6-Benzylaminopurine (BAP) and mixtures thereof for a period of 5 to 10 days;
    (b) add to the cultivation of step (a), 2 to 4 times, an elicitor composition or combination, where this composition or combination is selected each time it is added from:
    - a composition or combination comprising cyclodextrins, and at least one jasmonate compound, wherein the jasmonate compound is selected from the group consisting of methyl jasmonate, ethyl jasmonate and propyl jasmonate; and where at the end of the 2 to 4 times of addition of the elicitor composition or combination, the cyclodextrins are at a final concentration in the culture of 18 to 72 g / l of culture and the jasmonate compound is in a final concentration in the 100 µM to 500 µM culture; Y
    - a composition comprising at least one jasmonate compound, selected from methyl jasmonate, ethyl jasmonate and propyl jasmonate, where at the end of the 2 to 4 times of addition of the elicitor composition, the jasmonate compound is in a concentration final in the culture of 100 µM to 500 µM.

    The invention also has the appearance of a cell lysate of a suspension cell culture of a plant of the genus Gossypium, characterized in that it is obtainable by the method as described above, comprising the step (c) of lysis 20 of the culture.

    The expert will recognize how to proceed to the lysis of a cell culture, by disintegration or disruption of the cells mechanically or by ultrasound. With lysis it is possible to release the contents of the interior of the cells in the medium, which gives rise to a particular mixture comprising components of the cytoplasm, the cell membrane and the cell wall.

    Both the suspension cell culture of the invention, and a lysate thereof can be used in topical, cosmetic or pharmaceutical compositions, as well as active agents to prevent the harmful or unsightly effects caused by solar radiation, and in particular IR radiation. It can also be used in the form of a solid pharmaceutical composition, such as in capsules or tablets comprising the culture or its lysed form with pharmaceutically acceptable solid excipients. 35

    Thus, another aspect of the invention relates to topical pharmaceutical or cosmetic compositions, characterized in that they comprise an effective amount of a suspension cell culture of a plant of the genus Gossypium, as described above, or an effective amount of a lysate according to 5 is also defined above, together with one or more pharmaceutically or cosmetically acceptable excipients or vehicles.

    The term "effective amount of the cell culture or lysate" refers to the amount of this that provides a therapeutic or cosmetic effect after application without causing irritation and at the same time low enough to avoid side effects (at a benefit / risk ratio reasonable). The effective amount may vary with the age and physical conditions of the end user, either for a therapeutic effect and depending on the disease; or for a cosmetic effect.
     fifteen
    The term "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense that it must be compatible with the other ingredients of the pharmaceutical composition, while being suitable for use on the skin (or other tissues) without excessive toxicity or allergenic or immunogenic response. The term "cosmetically acceptable" or "dermatologically acceptable" refers to excipients or vehicles adapted for use in contact with human skin without excessive toxicity, incompatibility or allergenicity.

    It is accepted by the scientific community that the mechanisms triggered by the effect of UV radiation are different than those caused by IR radiation, given the different wavelength of radiation. This implies that compounds that efficiently filter or neutralize one type of radiation do not have to be effective with the other type of electromagnetic radiation. But in addition, it is added that the pathophysiology caused by one type of radiation is not identical to that caused by the other. All this means that, from the outset, the photoprotective effects of a composition against a type of radiation to another type with a different wavelength and resulting in different health consequences or aesthetic appearance are not extrapolated.
     35
    In a particular embodiment, the pharmaceutical composition or cosmetic composition is characterized in that it further comprises glycerin as an acceptable vehicle or excipient, regardless of being able to comprise other excipients and / or vehicles. In another particular embodiment, the composition comprises preservatives such as gluconolactone compounds and benzoate compounds (eg sodium benzoate) and additives such as calcium gluconate or other gluconate compounds.

    In another particular embodiment the concentration of polyphenols in these pharmaceutical or cosmetic compositions is at least 50 mg / l of composition, preferably 50 to 100 mg / l. 10

    The invention also has solid pharmaceutical or cosmetic compositions characterized in that they comprise an effective amount of a suspension cell culture of a plant of the genus Gossypium, as described above, or an effective amount of a lysate as also defined above. , together with one or more pharmaceutically or cosmetically acceptable excipients or solid carriers. Non-limiting examples of excipients or solid carriers refer to maltodextrins, sorbitol and mannitol.

    The invention also relates to nutraceutical compositions characterized in that they comprise an effective amount of a suspension cell culture of a plant of the genus Gossypium, as described above, or an effective amount of a lysate as also defined above, together with one or more acceptable excipients or solid vehicles.
     25
    The nutraceutical compositions refer to all those food, therefore mixtures of various edible ingredients, which are proclaimed as having a beneficial effect on the Health human Similarly, it can be said that the cultivation or lysate of the invention is as a product per se a nutraceutical agent, since administered in a food composition is one of the assets that exerts this beneficial effect on health, in particular the effect of avoid damage that could be caused by solar radiation or any of its spectrum members, in particular by IR radiation, as will be seen in the examples.

    Given the protective effects of the culture or lysates objects of the invention, it is also an aspect thereof the use of a suspension cell culture of a plant of the genus Gossypium, or of a cell lysate thereof as defined above, or of a topical pharmaceutical composition comprising them, for the preparation of a medicament for the prevention and / or treatment of a disease or condition 5 caused by radiation exposure, selected from the group consisting of UV radiation, IR radiation and a combination of both, being a combination of both, for example, solar radiation.

    In a particular embodiment the medicament is for the prevention and / or treatment of a disease or condition caused by exposure to infrared radiation.

    In another particular embodiment, optionally in combination with any previous or subsequent embodiment, the use is characterized in that the disease or condition caused by radiation exposure is selected from the group consisting of stinging 15 and / or itching of the skin and / or leather scalp, early skin aging, radiation skin burns, radiation dermal inflammation, atopic dermatitis and skin cancer.

    In another particular embodiment the medicament is for the prevention and / or treatment of a disease or condition caused by exposure to UV radiation. In another particular embodiment, the medicament is for the prevention and / or treatment of a disease or condition caused by exposure to IR radiation.

    The invention also relates to the use of a suspension cell culture of a plant of the genus Gossypium or of a cell lysate as defined above, or of a topical cosmetic composition as defined above, as a skin photoprotective agent and / o of the scalp. Skin photoprotection includes the improvement or prevention of at least one of these skin symptoms: wrinkles, itching, dryness, erythema, spots and aging. A photoprotective agent means that it is an entity that has the ability to protect the skin and / or scalp from adverse effects (pathological or aesthetic) that are associated with radiation, either solar with its full spectrum or relative to each of the radiation separately (UV, IR, visible).
     35
    The cosmetic effect of the culture, lysate or compositions that comprise them refers to the protection of the skin when it cannot be considered to be sick (or have any pathology), but rather that it is desired to preserve or improve its external appearance without There has been no damage or pathology. Cosmetic effects are derived from the photoprotection of cell culture, lysate or any composition that includes them, the comforting effect (also known as soothing), the regenerative anti-wrinkle effect, wrinkle prevention, prevention or improvement of erythema and / or eczema , dermal and epidermal photoregeneration, photoreparation, and the antioxidant effect in the sense that the appearance of free radicals (ROS) that worsen the appearance of the skin and / or scalp is prevented or cushioned. The cosmetic effect will be a combination of those mentioned above depending on the state of the skin and / or scalp as applied.

    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "understand" includes the case "consists of". For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

    BRIEF DESCRIPTION OF THE DRAWINGS
     25
    FIG. 1 corresponds to the production of MMP-1 (pg / ml) in a culture of human dermal fibroblasts (HDF) after exposure to IR radiation at an intensity of 150 J / cm2 (middle column) and 300 J / cm2 (right column in each type of sample tested), or without exposing them to radiation (left column in each type of sample tested). The lamp used was IR 30 Philips BR125 Infrared lamp hard glass Red (250 W, 230 V). The samples corresponded with cultures of untreated fibroblasts (NT), treated with Quercetin (Qu) 25 µM, with ascorbic acid (Asc) at 10 µM, or with different concentrations of a lysate of a cotton suspension cell culture from Arabia (Cott) at 23 µg / ml (mass of lysed cells by volume of glycerin), 45.6 µg / ml and 91 µg / ml. The 35
    MMP-1 amount in pg / ml was measured according to a specific ELISA.

    FIG. 2 shows the result of the same test as in FIG. 1 but the amount of MMP-1 detected in each type of sample is expressed as a percentage (%). The% is calculated considering as 100% that of untreated fibroblasts (NT) and not 5 exposed to radiation (first left column of NT). Abbreviations mean the same as in FIG. 1. The middle column of each sample corresponds to IR irradiation at 150 J / cm2 (middle column), while the right column in each type of sample tested at an intensity of 300 J / cm2 (column of the right). 10

    FIG. 3 corresponds to another test in a culture of human dermal fibroblasts (HDF). The MFI index (mean fluorescence intensity) in the fibroblasts is compared before (left column in each sample) and after irradiation with IR at 200 J / cm2 (right column in each sample). The different concentrations and products tested in the crops are shown on the abscissa axis. NT, means untreated; Qu (with quercetin); Asc with ascorbic acid (at 10 and 50 µM) and Cott with cotton suspension cell culture (23 µg / ml glycerin lysate, 45.6 µg / ml, and 91 µg / ml)
     twenty
    FIG. 4 shows the production of reactive oxygen species (ROS) (intracellular ROS production increase factor) in the fibroblast cultures of FIG. 3 after irradiation with IR (200 J / cm2).

    EXAMPLES 25

    Example 1. Obtaining cell culture

    To obtain the suspension cell culture, Gossypium herbaceum cultures were grown 7 days under constant agitation (90 to 110 rpm) in the dark and at room temperature. Before starting treatment with the elicitor composition, it was confirmed that there was at least 500 ± 50 ml of cells per liter final volume of culture. The basal culture medium was the MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (2 g / l), and quinetin (1 g / l).
     35
    Subsequently, elicitation was carried out with an elicitor composition comprising cyclodextrins (reference 121877 from Wacker) at a final concentration in the culture 50 g / l; and methyl jasmonate (reference 392707 from Sigma) at a final concentration in the culture of 50 µM. The cultures were left under stirring (from 90 to 110 rpm) and in the dark at an ambient temperature of 25 ° C. After 3-5 days from the first elicitation, a second elicitation was carried out with 50 µM methyl jasmonate. The conditions of agitation, darkness and temperature were also constant. Two days after the second elicitation, the scheme was repeated by eliciting again with 50 µM methyl jasmonate, so that the final jasmonate concentration after the first elicitation is 150 µM. He left 10 two more days in agitation and in the dark.

    Next, phenolics and flavonoids were analyzed by spectrophotometry (Agilent Technologies UV-visible spectrophotometer / Agilent model 8453 / serial number 0000338-02-01). For this, 10 ml of the sample was used. It was found that the sample for analysis had a cell concentration of 500 ± 50 ml of cells in 1 l of final culture volume. The same sampling was used to send a sample for microbiological analysis.

    The quantification of phenols and flavonoids was carried out by prior extraction of the same 20 from the culture and followed by its quantification. For this, the protocols described in the official European Pharmacopoeia Monograph (Ph Eur) of Herbal Drugs (European Pharmacopoeia 8.0) Markham, Andersen, pages 1205-1206; and in the methodology described in Marinova D et al., "TOTAL PHENOLICS AND TOTAL FLAVONOIDS IN BULGARIAN FRUITS AND VEGETABLES", Journal of University of 25 Chemical Technology and Metallurgy -2005, Vol. No. 40 (3), pp .: 255 -260.

    The results of the polyphenol analysis appear in Table 1.

    The product was formulated with glycerin (glycerinated) and lysed the cotton cells 30 with an Ultraturrax T-25 Basic device. Dispersion tool, model S 25 N - 25 G (Manufacturer IKA), lysate control was performed by observation with a BIOLUX-12 binocular microscope (KYOWA brand). For this, glycerin (69%), cell culture (30%) and Geogard ™ ultra preservative (1%) were added in a stainless steel container. Other preservatives that the expert will know are 35
    also appropriate. It was placed in an ice bath with the aim of not exceeding 30 ° during the process and dispersed until the preservative was completely dissolved and until an appropriate cell breakage was observed.

    The qualitative and quantitative composition of the crop, in terms of polyphenols and other 5 components, appears in Table 1. Table 2 details the composition in the glycerinated final product.

    Table 1.
     10
     Active  Analytical method Concentration range Identified assets
     Protein content  Bradford 700-1200 mg / L Total protein
     Lipid content  Thin layer chromatography Qualitative (visual) determination Phytosterols
     Triglycerides
     ac. free grasses

     Gas Chromatography and Mass Spectrometry (GC-MS) 1 Qualitative identification Stearic acid
     Monopalmitin
     Monostearin
     Phytosterial quantification  GC-MS1 30-60 mg / L Stigmasterol
     β-Sitosterol
     Lanosterol
     Sugar content  GC-MS1 Qualitative identification D-fructose
     2-keto-D-gluconic acid
     6-deoxy- β-L-manofuranose
     α-D-glucopiranose
     Arabitol
     Myoinositol
     Polyphenols  Colorimetry UV-visible spectrophotometer (method as indicated above) 250-500 mg / L Total polyphenols
    1 The GC-MS chromatographic conditions were as follows: Injection volume: 1µL; Column: Zebron ZB5MS 30m x 0.25mm x 0.25µm; Temperature program: Initial temperature of 40ºC maintained for 1 minute, temperature ramp of 10ºC / min, final temperature of 320ºC maintained for 15 minutes; Carrier gas: He at a constant linear speed of 36cm / sec; Split Ratio: 15
    1: 50; Injector temperature: 320 ° C; Interface temperature: 280ºC Detection: SCAN from m / z 41 to m / z 600. SCAN interval 0.5 seconds.


    Table 2. Composition and physical-chemical data of the product formulated in glycerin 5
     Test  Data Method
     Organoleptic exam
     Appearance  Viscose suspension of cell lysate
     Color  Brown-dark brown
     Physico-chemical parameters
     pH  4-5.5
     Density  1.10-1.30 g / ml pycnometer
     polyphenols  ≥ 50 mg / l
     Microbiological parameters
     Total aerobics  <100 cfu / g
     Fungi and yeasts  <10 cfu / g
     Pathogenic germs     European Pharmacopoeia 8.0 2.6.12. Microbial enumeration tests, pages 185 to 194.
     Staphylococcus aureus  Absence in 1 g
     Pseudomonas aeruginosa  Absence in 1 g
     Other components  Phytosterols (stigmasterol, β-sitosterol, lanosterol lipids (monoglycerides, triglycerides, free fatty acids), sugars (glucose, fructose, sucrose, furanosa, arabitol, myoinositol) Proteins (Bradford test) GC-MS (as detailed for Table 1)


    Example 2. Inhibitory effect of MMP-1 enzyme expression (responsible for the degradation of collagen in the extracellular matrix) of the cell lysate according to the invention.
     5
    In FIG. 1 the production of the MMP-1 enzyme (pg / ml) in a culture of human dermal fibroblasts (obtained from human skin samples-phimosis operations 0-3 years), cultivated and expanded in Dulbecco's 1g / culture medium is detailed. l glucose (Dulbeco's Modified Eagle's Medium 1g / L glucose, Lonza BioWhitaker) supplemented with 10% fetal bovine serum (FCS, Labclinics), 2 mM L-10 glutamine (Lonza) and antibiotics (10 µg / ml Penicillin 100 U / ml Streptomycin, Lonza). The fibroblasts were exposed to IR radiation with a Philips BR125 Infrared lamp hard glass Red IR lamp (250 W, 230 V). This lamp welcomes in its emission spectrum the entire IR region, with a pronounced peak at the wavelength of 1000 nm in the IRA region. fifteen

    The amount of MMP-1 was measured by a specific ELISA test (ELISA plate reader, Multiskan Ascent (Labsystems-Perseptive Biosystems)) from the supernatants of cultures of fibroblasts previously exposed to radiation (24 to 48 h exposure) and in the presence of different doses of lysate of 20 cotton cells or controls. The supernatants were collected 20 to 24 hours after irradiation.

    Fibroblast cultures were seeded in 24 wells until confluence. They were treated with the different assets (different concentrations of the glycerin cell lysate of the invention, in particular, 23 µg / ml, 45.6 µg / ml, 91 µg / ml; or with quercetin and ascorbic acid as positive controls. The target were untreated cells (NT))

    To obtain the different concentrations of cell lysate (also referred to herein as active biomass) in glycerin, dilutions of the lysate at 30% by weight of biomass in glycerin were made, as detailed below. The concentrations tested in fibroblast cultures are highlighted in bold. The dilution was made with glycerin.

     35

     Ratio of the original cell lysate sample of Gossypium herbaceum in glycerin (%)  30
     Sample density (g / ml)  1.21
     Actual concentration of biomass (lysate) in sample (g / ml):  0,363

     Sample dilution M06413 evaluated (times): Biomass concentration evaluated (%): Biomass concentration evaluated (mg / ml):
     1/500 dilution  500 0.06 0.73
     1/1000 dilution  1000 0.03 0.36
     1/2000 dilution  2000 0.015 0.18
     1/4000 dilution  4000 0.0075 0.09
     Dilution 1/8000  8000 0.0038 0.045
     Dilution 1/16000  16,000 0.0019 0.023


    From FIG. 1 it follows that the presence of the cell lysate at the indicated concentrations 5 and obtained as described in Example 1 in fibroblast cultures, is capable of reducing the expression levels of this enzyme responsible for the degradation of type 1 collagen. Although at higher intensity of IR radiation (300 J / cm2, right column in each type of sample tested) the expression was greater than at a lower intensity of irradiation (intensity of 150 J / cm2, middle column 10), fibroblasts to which different concentrations of the cell lysate of the invention had been added all showed a lower level of expression than in untreated cells (NT). In addition, the level of expression was similar to that of quercetin (25 µM), an antioxidant generally used as a control. Note that even at a much lower concentration of active (cell lysate), the effect is greater than that of the 15 reference assets quercetin and ascorbic acid.

    On the other hand, the same data in FIG. 1 are expressed as a percentage (%) of MMP-1 detected in each type of sample in FIG. 2. The% is calculated considering as 100% that of untreated fibroblasts (NT) and not exposed to 20
    radiation (first left column of NT). It is clearly seen that MMP-1 expression is similar to that of the group with quercetin and clearly significantly lower than in untreated fibroblasts.

    In another test the inhibitory activity of the cell lysate in glycerin was evaluated in the production of reactive oxygen substances (ROS), caused by the effect of IR radiation at 200 J / cm2, provided by the same lamp as in the previous test . The results are shown in FIGs. 3 and 4.

    For this, the fibroblast cultures were prepared in the same way (10-well 96-well format plate) and were subjected to radiation but without any added assets (NT untreated cells) or in the presence of glycerin cell lysate at concentrations before indicated. Also as a positive control, quercetin (25 µM) and ascorbic acid (concentrations 10 and 50 µM) were used. The cell lysate and the assets of the different controls were added 24 hours before irradiating. fifteen

    In this test, the production rate of reactive oxygen species was determined by the specific Carboxy-H2DCFDA (Molecular Probes) probe. This probe diffuses into the cells (fibroblasts) and remains in a non-fluorescent state. Under stress (irradiation) the acetate groups of this probe are eliminated by the action of intracellular esterases, giving rise to their fluorescent form, also granting them negative charges that prevent their exit from the cells. After irradiation, ROS levels were therefore measured indirectly by fluorimetry (Fluororoskan Ascent CF (Thermolabsystems)), since they are directly proportional to the fluorescence emitted by the probe. 25

    As indicated above, the data from this test are illustrated in FIGs. 3 and 4. In FIG. 3 average fluorescence intensity (MFI) values are shown at an excitation wavelength of 485 nm and an emission wavelength of 527 nm. Although in all the samples the IR radiation produced an elevation of 30 MFI with respect to the non-irradiated samples, the absolute value of MFI was always lower than in the untreated fibroblast (NT) sample. This shows the potential of the lysate according to the invention as a photoprotective agent of the skin in relation to IR radiation. FIG. 4, on the other hand, shows the analysis of the ROS detected in each sample. Samples treated with the cell lysate of 35
    Cotton showed a protective effect with significantly lower ROS levels than in untreated fibroblasts. Note again that at a lower concentration than the positive controls (quercetin and ascorbic acid), the cell culture (glycerin lysate) protected the same or even more than the latter. This proves that the composition of the lysate is such that its components act synergistically, and very low doses can be used to have a considerable protective effect.

    The data obtained with the tests with IR radiation demonstrate that with an ingredient of affordable and reproducible production, and at the same time being a natural product, the protective effect of radiation is comparable or even better than those 10 compounds currently used for it.

    Example 3. Effect of UV radiation on human fibroblasts treated with suspension cell cultures of the invention.
     fifteen
    In order to assess whether the cell lysate or the culture of cotton cells in suspension was also active against the physiological mechanisms that UV radiation causes in the skin, tests were also carried out regarding the protective capacity against oxidative stress produced by UV radiation in skin cells; tests relating to the protective capacity against degradation of UV-induced extracellular matrix 20 proteins (type I and III collagen and elastin); assays related to regenerative capacity by stimulating the synthesis of extracellular matrix proteins (type I and III collagen and elastin); and tests related to the modulation of the cytokine pattern involved in the processes of UV-induced skin inflammation. 25

    In the same way as for the IR tests, different concentrations of a cell lysate of cotton cells were used, obtained by cumulative dilution of a lysate composition initially at 30% by weight in glycerin matrix.
     30
    These samples were added in cultures of human dermal fibroblasts as in Example 2 above.

    On this occasion the irradiation was carried out with the SOL500 lamp (Dr. HÖNLE), Ref. 19671. Serial No. 0298 SOL 500 is a radiation system that simulates light 35
    natural sun. It consists of a high-pressure halogen lamp (Halogenide high-pressure SOL 500 S lamp) in a ballast unit, and a filter disk (H-filter) which partially blocks UVB radiation. The radiation emitted by the system is within the wavelength range of 300-2500 nm (UVB-UVA-VS-IR). This configuration allows the emission of a spectrum similar to that of the 5 sun light, which comprises UV light (mainly UVA, and 17.3% UVB light), visible and infrared light.

    3.1. Ability to inhibit the appearance of ROS
     10
    To assess the ability to inhibit the onset of ROS, the general carboxy-H2DCFDA oxidative stress indicator (Molecular Probes) was used as in example 2.

    Human dermal fibroblasts (HDF) were seeded in 96-well culture plates and kept growing (37 ° C, 5% CO2) until confluent. 15 Then, the cells were pre-treated with different concentrations of cell lysate of cotton cells in glycerin for 24 h. As a baseline production control of ROS, cells were maintained in culture medium during pretreatment. Quercetin-treated cells (25μM) were used as positive controls. Each experimental condition was evaluated in triplicate. After pretreatment, the test solutions were removed, and the cultures were washed twice with Hank's buffered salt solution (HBSS) for complete elimination. New HBSS solution was added to the wells before exposure to the sun. The cells were irradiated with the lamp at an intensity of 12J / cm2. In parallel, a control culture plate was not exposed to solar radiation. Then, the carboxy-H2DCFDA dye was applied to the cells and incubated at 37 ° C for 30 min. When finished, the fluorescence was measured and recorded in the fluorimeter as in Example 2. The relative mean fluorescence (MRFU) and standard deviation (SD) units were calculated for each experimental condition. MRFU values are directly proportional to the amount of intracellular ROS produced. Table 3 shows the 30 results (decrease in% of ROS compared to ROS in untreated cells):



     35

    Table 3:

     Comparative analysis vs untreated cells Comparative analysis vs irradiated cells

     S.D. Average percentage (%) S.D.
     Dye control  0.377 0.003 1.616 0.013
     Untreated cells  23,315 1,439 100,000 6,173
     Quercetin 25 µM  14,627 1,650 62,735 7,079
     % cell lysate in assay  1.910-3% (0.023 mg / ml) 16,332 1,400 70,049 6,006
     3.7510-3% (0.045 mg / ml)  15,894 1,186 68,170 5,085
     7.510-3% (0.091 mg / ml)  15,077 1,346 64,667 5,774

    As shown in Table 3, after 12J / cm2 of UV radiation, fibroblasts 5 increased intracellular ROS production (23,315 ± 1.44 increases) compared to baseline (non-UV) production. Quercetin, as a positive control in this assay, shows an important antioxidant action with a potent inhibitory effect of UV-induced intracellular ROS production in HDF (inhibition of 37.3 ± 7.08% with respect to baseline control). Antioxidant efficacy of 10 products was defined as the percentage reduction of ROS production in the pre-treatment of fibroblasts with respect to the production observed when fibroblasts were not treated.

    The fibroblasts (HDF) treated with the cell lysate according to the invention show a ROS production level of 30.21 ± 7.23%, 33.07 ± 8.18, and 35 052 ± 9.9%, lower than that observed in untreated HDF, at concentrations of 0.023, 0.045, and 0.091 mg / ml, respectively. These results demonstrate the powerful protective action of the lysate. Based on these results obtained with the lysate, its potent antioxidant action is demonstrated. Pretreatment with 0.023, 0.045 20 and 0.091 mg / ml of cotton cell cell lysate led to a reduction in ROS levels in HDF of 29,951 ± 6%, 31,830 ± 5.08, 35.33 ± 5.77% ,
    respectively. These values come out of subtracting 100% the values of Table 3 70.049, 68.170 and 64.667, respectively)

    3.2. Protective capacity of the lysate of the invention against degradation of extracellular matrix proteins (ECM) 5

    The protective capacity of the lysate of the invention against degradation of extracellular matrix proteins caused by solar radiation was evaluated on the basis of quantification of type I and type III collagen in HDF pretreated and exposed to solar radiation, by means of immunocytochemical analysis. For this, the fibroblasts were seeded in 96-well culture plates and kept growing (37 ° C, 5% CO2) until confluence (72-96h after cell seeding). Then, the cells were pre-treated with different concentrations of Arabian cotton (Gossypium herbaceum) for 24 h. Untreated cells and cells treated with 25 μM quercetin were used as a baseline control and positive control, respectively. After pretreatment, the test solutions were removed and the cells washed twice with Hank buffered saline (HBSS). For the irradiation process, new HBSS solution was added in the cell culture wells and the cells were exposed to a radiation dose of 40 J / cm2, with the SOL 500 lamp. In parallel HDF culture plates were evaluated in the same 20 experimental conditions and treatments and controls without being exposed to solar radiation.

    Immediately after irradiation, the HBSS solution was removed from the cultures and new culture medium with low concentration of FCS (1%) was added. The 25 cultures were kept in incubation for an additional 24 h. After this period, the cells were washed and fixed with para-formaldehyde for quantification of type I and type III collagen present in the treated extracellular matrix and HDF was irradiated by immunocytochemical analysis. The detection of these matrix proteins was performed by the immunocytochemistry technique by ELISA. For this analysis, the specific primary human antibodies against both proteins were used (Sigma human type I anti-collagen monoclonal antibody, MP Biomedicals human type III anti-collagen monoclonal antibody and Sigma human anti-elastin monoclonal antibody) , followed by the horseradish peroxidase conjugated secondary antibody (HRP) and incubation with its
    substrate, o-phenylenediamine, in urea buffer H2O2. At the end of the HRP reaction, the absorbance was measured in a spectrophotometer (492 nm). Cultures that were incubated with the secondary antibody were only used as controls for non-specific antibody binding. The values obtained in these control wells were subtracted from the test wells. The absorbance values are proportional to the amount of protein present in the extracellular matrix of the HDF cultures evaluated.

    Although the data is not shown, a significant reduction in the amount of extracellular matrix (ECM) proteins was detected in UV-induced fibroblasts compared to non-irradiated. The reduction of type I collagen, type III collagen and elastin was 58.97 ± 8.14%, 47.91 ± 8.11% and 62.47 ± 4.42%, respectively, in HDF exposed to 40 J / cm2 of UV with respect to the non-irradiated. This reduction in ECM protein levels was slightly lower in quercetin-treated fibroblasts after UV-exposure (reduction of 15 46.78 ± 6.91%, 25.03 ± 8.08% and 49.15 ± 10, 17% in type I collagen, type III collagen and elastin, respectively). These results support the protective effect of quercetin against photo-induced degradation of ECM proteins.

    The protective effect of the lysate of the invention was greater than that shown by quercetin. The reduction values of type I collagen in HDF pre-treated with lysate compared to those not irradiated was 36.59 ± 7.72%, 37.81 ± 4.62% and 32.95 ± 7.25% for 0.091, 0.182 and 0.363 mg / ml lysate concentration, respectively. Consequently, HDF treated with 0.091, 0.182 and 0.363 mg / ml of lysate had 54.55 ± 4.18%, 51.58 ± 11.26% and 63.41 ± 17.67% more of type 25 collagen I in its extracellular matrix, which untreated fibroblasts.

    The lysate also showed a protective effect of type III collagen and elastin. Fibroblasts treated with 0.091, 0.182 and 0.363 mg / ml lysate had 44.46 ± 23.43%, 53.13 ± 22.2% and 61.02 ± 17.8% more type III and 59 collagen , 18 ± 30 11,045%, 58.58 ± 1.12% and 43.46 ± 29.01% more elastin in extracellular matrix than untreated fibroblasts.

    3.3. Production of extracellular matrix proteins (ECM)
     35
    ECM protein production was evaluated in order to measure skin firmness, elasticity and anti-aging effects of lysate. Fibrillar collagen (type I and type III collagen) and elastin production were analyzed by the previous immunocytochemical method. For this, HDF cells were seeded in 96-well culture plates and kept under growth conditions (growth medium, 5 37 ° C, 5% CO2) up to 75-80% confluence. Then, the cells were treated with different concentrations of lysate for 72 h (time required for the synthesis, processing and insertion of the selected proteins in the extracellular matrix). Untreated cells were used as a negative control. As a positive control, the cells were incubated with 10 ng / ml of transforming growth factor beta-1 (TGF-β1), a known inducing agent for the synthesis of ECM proteins in HDF. Each experimental condition was evaluated in triplicate. At the end of the cell treatment, the cultures were washed with HBSS and fixed with paraformaldehyde for the analysis of type I and type III collagen and the elastin content in the extracellular matrix of the treated cells. The absorbance of the test cultures was measured in a spectrophotometer at 492 nm. Absorbance values obtained are directly proportional to the amount of the protein detected in the ECM of the HDF culture. Cultures that were incubated with the secondary antibody were only used as controls for the binding of nonspecific antibodies and their absorbance values were subtracted from the test wells. The results obtained in the 20 cells treated with the lysate were compared with those of the untreated control, to determine the inducing effect of ECM protein synthesis.

    The transforming growth factor beta-1 (TGF-β1) induced the production of type I collagen, type III collagen and elastin by 98.71 ± 9.31%, 109.97 ± 6.73% and 25 46, 74 ± 18.59%, respectively, after HDF treatment for 72 h compared to untreated fibroblasts. The samples treated with the lysates showed (data not shown) a slightly stimulating effect on the production of type I collagen in a dose-dependent manner at the concentrations evaluated. In particular, an increase in the production of type I collagen of 31.63 30 ± 8.53%, 37.24 ± 7.98%, 58.22 ± 8.6 and 51 ± 16.63% in HDF treated with 0.091, 0.182, 0.363 and 0.726 mg / ml of lysate with respect to untreated fibroblasts. Fibroblasts treated with 0.363 and 0.726 mg / ml glycerin lysate according to Example 1 for 72 h showed an increase in the synthesis of type III collagen of 50.29 ± 10.23% and 78.97 ± 26.16%, respectively , compared to untreated HDF. 35
    In addition, HDF treated with cotton cell cell lysate also induced elastin production by 68.86 ± 14.44% at the highest concentration evaluated (0.726 mg / ml).

    These ECM protein production data demonstrate that the lysate of the invention has a regenerative effect (regenerative activity) due to its ability to promote the synthesis of these proteins.

    3.4. Modulation of the inflammatory response
     10
    Although the data is not shown, the concentrations of different pro-inflammatory cytokines were also determined in the fibroblast cultures. The cell lysate of cotton cells reduced the production of the cytokines responsible for the onset of skin inflammation IL-1α, IL-1β and TNF-α. It also reduced the production of most cytokines involved in the inflammation amplification phase of the immunomediated skin, such as interleukins IL-4, IL-6, IL-8, IL-10, IL-13, γ interferon ( INF-γ) and monozyme-1 chemotactic protein (MCP-1).

    3.5. Efficacy of lysate against UV radiation in in vivo assay in humans
     twenty
    In vivo assays were performed with the cell lysate (30% in glycerin) according to the invention in 16 volunteers. The lysate was applied together with a commercial basal skin cream at a concentration of glycerinated lysate in the cream of 1% by weight. The volunteers included several phototypes (I-III) according to Fitzpatrick scale
     25
    One test was to evaluate the potential of the cell lysate according to the invention, as a photoprotective agent.

    The sunscreen effect was evaluated by determining the minimum dose of erythema (MED) before and after 4 days of treatment. The MED was calculated by inducing an erythema on the skin with a solar simulator. The lysate effect was compared with placebo (commercial cream without cell lysate). For the induction of erythema, an artificial light source was used with the Multiport® filtering system (Sunlight Co light): xenon arc (300 W) and Schott WG 320 and UG 11 (1 mm) filters. The emission spectrum of the simulator ranges between 290 and 400 nm. 35

    The irradiation areas were chosen by the responsible technician, taking into account the appearance of the skin (without markings and having uniform color), and that the area had not been exposed to the sun's irradiation 1 month before the start of the study, avoiding friction areas (clothing), bone projections and extreme areas of curvature. The areas were limited with a special surgical pen (Surface: 35 cm2). The exposures were made before the application of the product and after 4 applications.

    MED values were read when the erythema response was optimal (i.e. 10 20 ± 4, between 16 to 24 hours) after exposure to UV rays. The determination of the MED was done visually. The MED was defined as the quality radiant energy (the lowest UV dose) necessary to produce the first noticeable and unambiguous redness, with defined edges in most of the area of UV exposure and read 16 to 24 hours later of the exhibition. fifteen
    The MED was then calculated according to:
    MED = (Dose (in MED / min) x exposure time (in seconds)) / 60

    The following tables 4 to 6 show the results obtained:
     twenty
    Table 4. Results with 1% lysate cream

     MED Minimum dose of erythema
     Before treatment (Day 1) After treatment (day 8)% variation (before-after)
     Half  1.43 1.54 11.50%
     S.D.  0.32 0.39 28.40%

    Table 5. Results with placebo cream

     MED Minimum dose of erythema
     Before treatment (Day 1) After treatment (day 8)% variation (before-after)
     Half  1.43 1.42 0.80%
     S.D.  0.32 0.33 18.10%

    Table 6. Control results (untreated area)

     MED Minimum dose of erythema
     Before treatment (Day 1) After treatment (day 8)% of variation (before-after)
     Half  1.43 1.36 -2.90%
     S.D.  0.32 0.34 20.00%

    The results show an improvement of the MED (11.5%) in the area treated with the lysate of cotton cells. On the other hand, no significant variation was observed in the MED 5 for the control zone and the placebo treated area.

    It can then be concluded that, the experimental conditions adopted and taking into account the evolution of the decisive parameter considered, the product (lysate) of the invention has a sun protection effect after 4 applications. 10

    On the other hand, the repair effect (soothing effect; after-sun action) provided by the 1% lysate in the same commercial basal cream was also evaluated in another trial.
     fifteen
    For this, areas of the skin with the same selection criteria as for the photoprotection test were irradiated with the same lamp in the same 16 volunteers. UV radiation was applied by 6 optical fibers (8 mm in diameter) that define on the skin 6 subsites of 0.5 cm².
     twenty
    The calming effect of the product was evaluated, objectively and quantitatively, by colorimetric measurements, with the Mexameter® MX18 (Courage & Khazaka), which has a measuring probe with a measuring surface diameter of 5 mm. The measurement was performed in the area of the skin treated with the test product and the placebo product and the untreated area, after exposure to radiation. 25

    The surface of the exposure sub-sites (irradiation) was 0.5 cm² and the minimum distance between the edges of two adjacent sub-exposure sites was at least 0.8 cm.

    The energy produced by each fiber of the simulator was regulated by the observation screens of increasing values according to a geometric progression 1,25.

    The UV doses were measured by a previously calibrated solar light dosimeter and expressed in MED / min. (scale up to approximately 3.00 MED / min). All irradiance measurements were made using the same dosimeter.

    The exposure time of the area (established by the estimated MED, per 10 colorimetry) was 15 to 60 seconds and varied according to the subjects (phototypes). The results were expressed in mean values of parameter E or the level of erythema.

    The difference between the value of parameter E obtained in the irradiated area and that obtained in the adjacent area (∆E) was calculated. fifteen

    Erythema level measurements were made in D2 / T0H (6h after irradiation of the skin and before application of the test product and the placebo product) and in D2 / T1h and D2 / T2H (1h and 2h after the application of the test product and the placebo product). twenty

    The percentage reduction of erythema observed in each volunteer for the cream with the cotton cell cell lysate (1% by weight), for the placebo and for the control is detailed in the following Table 7:
     25
    Table 7. Percentage reduction of erythema after irradiation and application of cell lysate, placebo or in untreated areas
     Treatment with cotton cell cell lysate
     Subject  Reduction percentage D2 / T1h-D2 / T0h (%) Reduction percentage D2 / T2h-D2 / T0h (%)
     one  -10% -25%
     2  -15% -27%
     3  -26% -4%
     4  -16% -31%
     5  -44% -34%
     6  -11% -13%
     7  -9% -6%
     8  -28% -52%
     9  -31% -33%
     10  -eleven%
     eleven  -12% -26%
     12  -9% -10%
     13  -20% -8%
     14  -13% -8%
     fifteen  -twenty-one%
     16  -8% -18%
     Half  -15.9% -18.5%
     Standard Deviation (SD)  11.3% 14.6%
     Placebo
     Subject  Reduction percentage D2 / T1h-D2 / T0h (%) Reduction percentage D2 / T2h-D2 / T0h (%)
     one  2% -2%
     2  1% 12%
     3  -7% -55%
     4  -3% 1%
     5  -12% 6%
     6  6% 8%
     7  1% 11%
     8  -10% -1%
     9  9% -9%
     10  -44% 5%
     eleven  17% 20%
     12  4% 3%
     13  -14% 4%
     14  -4% -17%
     fifteen  2% -8%
     16  3% -1%
     Half  -3.1% -1.4%
     Standard Deviation (SD)  13.4% 16.8%
     Control
     Subject  Reduction percentage D2 / T1h-D2 / T0h (%) Reduction percentage D2 / T2h-D2 / T0h (%)
     one  3% 25%
     2  -8% -23%
     3  -10% -12%
     4  -19% -11%
     5  -38% 14%
     6  11% 16%
     7  0% 10%
     8  151% 195%
     9  -28% 5%
     10  20% 33%
     eleven  46% 52%
     12  -4% -7%
     13  -5% 4%
     14  -5% -16%
     fifteen  7% 7%
     16  -12% -17%
     Half  6.8% 17.3%
     Standard Deviation (SD)  42.9% 51.5%

    These results show that the cell lysate of cotton cells as described in Example 1 (applied at 1% by weight in a basal cream), show that the product reduces the level of erythema by 16%, 1 hour after its application and in 19%, 2 hours after its application. In the same way, the percentage of reduction of the level of erythema detected in the "reactive subjects" is 16.5% and 23.3%, 1h and 2h after the application of the product, respectively.
    Under the experimental conditions adopted and taking into account the evolution of the fundamental parameters of analysis, the cell lysate of the invention demonstrates a statistically significant calming effect. This effect was detected in 81% of 10 volunteers tested.

    Example 4. Topical composition with cell lysate of a suspension cotton cell culture.

     Component  Percentage (%)
     Glycerin  69
     Gossypium herbaceum cell culture lysate (cotton)  30
     Preservatives
     Gluconolactone  0.74
     Sodium benzoate  0.25
     Additives
     Calcium gluconate  0.01
     5
    This composition may be an ingredient added to other compositions or galenics for topical use, such as creams, gels, ointments, sprays, makeup bases, or scalp shampoos.

    REFERENCES CITED 10

    - Grether-Beck et al., "Review Article Photoprotection of human skin beyond ultraviolet radiation", Photodermatology, Photoimmunology & Photomedicine -2014, doi: 10.1111 / phpp.12111.
    - European Pharmacopoeia 8.0 Markham, Andersen, pages 1205-1206. fifteen
    - Marinova D et al., "TOTAL PHENOLICS AND TOTAL FLAVONOIDS IN BULGARIAN FRUITS AND VEGETABLES", Journal of University of Chemical Technology and Metallurgy -2005, Vol. No. 40 (3), pp .: 255-260.
    权利要求:
    Claims (15)
    [1]

    1.- Cultivation of plant cells of the genus Gossypium in suspension, characterized in that it comprises: 5
    - polyphenols at a concentration of 250 to 500 milligrams per liter of culture;
    - phytosterols at a concentration of 30 to 60 milligrams per liter of culture;
    - a mixture of sugars, which in turn comprises glucose, fructose, sucrose, furanosa, arabitol and myoinositol;
    - lipids; and 10
    - proteins

    [2]
    2. Culture according to claim 1, characterized in that the polyphenols are in a concentration of 200 to 400 mg / l of culture.
     fifteen
    [3]
    3. Cultivation according to any one of the preceding claims 1 to 2, characterized in that the plant of the genus Gossypium is selected from the group consisting of Gossypium herbaceum, Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium tomentosum, and mixture thereof.
     twenty
    [4]
    4.- Procedure for obtaining a culture of cells of a plant of the genus Gossypium in suspension comprising the following stages:
    (a) Cultivate dedifferentiated cells of the Gossypium genus plant in a growth culture medium comprising growth regulators of 25 plants, said regulators selected from the group consisting of auxins 2,4-dichlorophenoxyacetic acid (2,4-D) , naphthalenacetic acid (NAA) and cytokinins quinetin, and 6-Benzylaminopurine (BAP) and mixtures thereof for a period of 5 to 10 days;
    (b) add to the culture of step (a), 2 to 4 times, a composition or elicitor combination, where this composition or combination is selected each time it is added of:
    - a composition or combination comprising cyclodextrins, and at least one jasmonate compound, wherein the jasmonate compound is selected from the group consisting of methyl jasmonate, jasmonate
    ethyl and propyl jasmonate; and where at the end of the 2 to 4 times of addition of the elicitor composition or combination, the cyclodextrins are at a final concentration in the culture of 18 to 72 g / l of culture and the jasmonate compound is in a final concentration in the 100 µM to 500 µM culture; and 5
    - a composition comprising at least one jasmonate compound, selected from methyl jasmonate, ethyl jasmonate and propyl jasmonate, where at the end of the 2 to 4 times of addition of the elicitor composition, the jasmonate compound is in a concentration final in the culture of 100 µM to 500 µM. 10

    [5]
    5. Method according to claim 4, characterized in that step (b) is carried out with three additions of the elicitor composition or combination.

    [6]
    6. Method according to claim 5, characterized in that the addition of the elicitor composition or combination is carried out according to the following addition pattern:
    (i) add a first dose of the elicitor composition or combination when the culture of step (a) has grown from 5 to 10 days;
    (ii) add a second dose 3 to 4 days after the first dose; and 20
    (iii) add a third dose 2 to 3 days after the second dose.

    [7]
    7. Method according to any one of claims 5 to 6, characterized in that the addition of the elicitor composition or combination is carried out in accordance with the following addition pattern:
     (i) adding a first dose of an elicitor composition or combination comprising cyclodextrins and a jasmonate compound, when the culture of step (a) has grown from 5 to 10 days;
    (ii) adding a second dose of a composition comprising a jasmonate compound, 3 to 4 days after the first dose; and 30
    (iii) add a third dose of a composition comprising a jasmonate compound, 2 to 3 days after the second dose.

    [8]
    8. Method according to any one of claims 4 to 7, characterized in that the composition or elicitor combination comprises methyl jasmonate. 35

    [9]
    9. Method according to any one of claims 4 to 8, characterized in that it further comprises step (c) of lysing the cell culture obtained in step (b).
     5
    [10]
    10. Cell lysate of a suspension culture of cells of a plant of the genus Gossypium, characterized in that it is obtainable by the method according to claim 9.

    [11]
    11. Topical pharmaceutical or cosmetic composition, characterized in that it comprises an effective amount of a suspension culture of cells of a plant of the genus Gossypium as described in any one of claims 1 to 3, or of a lysate as defined in claim 10, together with one or more pharmaceutically or cosmetically acceptable excipients or vehicles.
     fifteen
    [12]
    12. Composition according to claim 11, characterized in that it further comprises glycerin as an excipient.
    13. Use of a suspension culture of cells of a plant of the genus Gossypium as defined in claims 1 to 3, or of a cell lysate as defined in claim 10, or of a topical pharmaceutical composition as defined in any one of claims 11 to 12, for the preparation of a medicament for the prevention and / or treatment of a disease or condition caused by exposure to radiation selected from the group consisting of ultraviolet (UV), infrared (IR) radiation and A combination of both. 25

    [14]
    14. Use according to claim 13, characterized in that the medicament is for the prevention and / or treatment of a disease or condition caused by exposure to infrared radiation.
     30
    [15]
    15. Use according to any one of claims 13 to 14, characterized in that the disease or condition caused by exposure to radiation is selected from the group consisting of stinging and / or itching of the skin and / or scalp, early aging of skin, radiation skin burns, radiation dermal inflammation, atopic dermatitis and skin cancer. 35

    [16]
    16. Use of a suspension culture of cells of a plant of the genus Gossypium as defined in claims 1 to 3, or of a cell lysate as defined in claim 10, or of a topical cosmetic composition as defined in any one of claims 11 to 12, as a photoprotective agent of the skin and scalp.
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    同族专利:
    公开号 | 公开日
    ES2579385B1|2017-05-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP3808335A1|2019-10-15|2021-04-21|Faes Farma, S.A.|Compositions for topical administration|
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    优先权:
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    ES201530157A|ES2579385B1|2015-02-10|2015-02-10|COTTON CELL CULTIVATIONS AND ITS USE IN PHOTOPROTECTION|ES201530157A| ES2579385B1|2015-02-10|2015-02-10|COTTON CELL CULTIVATIONS AND ITS USE IN PHOTOPROTECTION|
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