Procedure to identify properties of a grape extract (Machine-translation by Google Translate, not le

专利摘要:
The invention involves a series of processes that establish the solubility, non-cytotoxic and cytotoxic concentrations of grape extract, subsequently establishing its antioxidant and antidegenerative capacity and its efficacy using human retinal pigment epithelial cell cultures and various assay strategies that combine different oxidative treatments. Additionally, a composition for treating or ameliorating an ophthalmological disorder is described, which comprises a grape extract. (Machine-translation by Google Translate, not legally binding)
公开号:ES2779984A1
申请号:ES201930142
申请日:2019-02-20
公开日:2020-08-21
发明作者:Girish Kumar Srivastava；Rodriguez Alberto Guadarrama；González Luis Carlos Moro；Laguna Victor David Vendrell；Jimeno José Carlos Salvador Pastor
申请人:Bodega Matarromera Sl；Universidad de Valladolid；
IPC主号:

专利说明:

[0002] Procedure to identify properties of a grape extract
[0004] BACKGROUND
[0006] Grapes are natural antioxidants and can act as a natural chemotherapy to alleviate some of the symptoms of eye diseases such as retinitis pigmentosa, age-related macular degeneration, glaucoma, dry eye syndrome, conjunctivachalasia, keratoconus, cataracts, diabetic retinopathy and some uveitis among others, and other pathologies other than ocular such as cardiovascular edema or impaired capillary integrity, or atherosclerosis and also hematological pathologies such as excessive spontaneous platelet aggregation among others. In the process of obtaining it, as in this case "the extract", its formulation together with other components to make a product, its benefits in human use and the level of effectiveness of these antioxidant characteristics at the cellular and / or tissue level are It can alter, in addition to depending on the grape varieties. On the other hand, the evaluation of the antidegenerative characteristics is crucial because oxidative damage can cause cellular and / or tissue degeneration, processes that can also be acute or chronic. Based on the less potent antioxidant capacity, it may be that it does not rescue cells and / or tissues that are in degeneration (that is, it does not have antidegenerative capacity). Therefore, it is necessary to evaluate these characteristics -antioxidant and antidegenerative- including an assessment of its solubility, its cytotoxicity and its comparison with other products already on the market, to achieve new, more powerful and effective products for human use.
[0008] The risk of oxidative damage occurs in almost all tissues of the body, but is especially relevant in some such as the skin, neuronal tissue, the heart, and the eye among others. Taking the eyes as an example, it can be stated that they present a special risk of suffering the effects of oxidative damage due to their high exposure to oxygen, their large amount of oxidizable fatty acids and also their high exposure to light, environmental pollutants and very especially to ultraviolet radiation. Much of the cells and tissues of the eye are continually exposed to oxidative damage as a result of their constant exposure to light and atmospheric oxygen, increasing the risk of eye diseases. There are numerous studies that suggest that some of these diseases (such as Age-Related Macular Degeneration, AMD) have their origin in the loss or serious alterations of the cells of the retinal pigment epithelium (RPE) (cellular degeneration), which they are essential for the survival of photoreceptors and other retinal cells (tissue degeneration), and consequently, for the maintenance of vision. And there is evidence that this loss of RPE cells may be a consequence of the oxidative stress to which this cell layer is constantly subjected, so the search for new treatments or products based on antioxidant and antidegenerative compounds that are more powerful and effective for preventing, delaying or stopping it, than those currently available, has become a highly relevant issue in research on these diseases. There is also evidence that this oxidative damage can lead to functional and morphological deficiencies in photoreceptors, endothelial cells, and ganglion cells of the retina, among others. Therefore, it is necessary to study procedures that can act against the environment that produces oxidative damage in cells and / or tissues. The presence of antioxidant compounds has been proven both in wine and in grape extracts and, in the case of polyphenols such as catechins, flavonoids and resveratrol, their effectiveness against cellular oxidative damage has been shown. It is also worth noting the presence of other antioxidants such as carotenoids (lutein, zeaxanthin) and small concentrations of Cu and Zn. However, the procedures for obtaining wine or an extract from grapes depend on variable protocols and the technologies involved that are not uniform. Furthermore, it is necessary to find out the effect of these procedures on certain characteristics such as the solubility of the products, their cytotoxicity and the antioxidant and antidegenerative capacities.
[0010] The first indicator on whether or not it is acceptable to use a product for human use is the evaluation of its cytotoxicity on cells and / or tissues (that is to say, establishing an essential part of its bio-compatibility). In this way, cytotoxicity studies establish a very important aspect of the bio-compatibility, and therefore, the level of safety of the product, before initiating experimental studies with animals. Current advances in science and technology propose several types of cytotoxicity tests, but a good cytotoxicity test is one that follows standard protocols, has high sensitivity and produces quantitative and comparable data quickly to be evaluated. The guidelines of the ISO standard (UNE-EN ISO 10993-5: 2009 Biological evaluation of products Part 5: In Vitro Cytotoxicity Assays ) recommend standard protocols for direct and indirect methods for cytotoxicity studies. The direct contact method allows to detect weaker levels of cytotoxicity due to its high sensitivity.
[0012] Oxidative stress is defined as an imbalance between oxidants and antioxidants in favor of oxidants that promote damage to biological macromolecules (lipids, proteins, carbohydrates and nucleic acids) and alter cellular processes (functionality of membranes, production of enzymes, cellular respiration and gene induction among others). Oxygen is essential for life because almost everything in nature (fats, carbohydrates among others) is oxidized by the action of oxygen (oxide-reduction reaction) and during this process the so-called reactive oxygen species (ROS) are generated. . ROS are produced during metabolic reactions, while the body's cells transform food into energy, especially in situations of hyperoxia, intense exercise and ischemia, and also by exposure to certain external agents such as ionizing radiation and ultraviolet light, environmental pollution, tobacco smoke, among others. There is sufficient evidence that ROS increase oxidative stress in cells and / or tissues that cause the development and evolution of a wide variety of degenerative processes and diseases. The term "reactive species" refers to two types of molecules: free radicals and non-radicals. This series of molecules is formed as a result of cellular metabolism and is represented within biological systems by ROS. Within ROS The most important inorganic ones are molecular oxygen O2, the radical-superoxide anion (O2-), the hydroxyl radical (HO-) and its immediate precursor, hydrogen peroxide (H2O2). The secondary or organic ROS are the peroxyl radical (ROO -), organic hydroperoxide (ROOH), and peroxidized lipids. They are generally very small molecules highly reactive due to the presence of an unpaired valence electron shell. These species are naturally formed as a by-product of normal oxygen metabolism and play an important role in cell signaling, initially causing mitochondrial depolarization and then triggering a positive feedback loop of the release of ROS induced by the ROS themselves.
[0014] Cell culture systems are very useful for studying the effects of oxidative stress, both in terms of cyto-toxicity and cellular adaptive responses. There are several models of oxidative cell damage produced by indexing or blocking of cellular signaling mechanisms. The model systems for oxidative stress most used in the studies are: extracellular sources of O2- and H2O2 and normobaric hyperoxia (high levels of ambient oxygen).
[0016] In humans, diseases can be caused by acute damage or by chronic damage. Therefore, it is necessary to understand the mechanisms involved in both damages and reproduce models based on these mechanisms. There is sufficient evidence to show that incubating cells with H2O2 under certain conditions such as exposure time, concentration or cell growth among others, generate acute damage and when cultures are exposed to an enzyme called glucose oxidase, chronic damage occurs .
[0018] ROS have a fairly short half-life and for this reason it is difficult to directly measure their amounts in vivo in cells and / or tissues. The "electron spin resonance" is a technique that allows the measurement of ROS. However, there are other indirect techniques that can help to estimate their concentrations. These techniques allow to see the damage caused by ROS and the activity of certain parameters within the oxidative stress and include protein evaluation (Lowry method and Bradford method), chemiluminescence, thiobarbituric acid reactive substances (TBARS), SOD activity, catalase determination, glutathione peroxidase enzyme determination, glutathione S-transferase measurement , measurement of glutathione and determination of nitrites and nitrates. In the ISO / TS 19006: 2016 standard it is mentioned in the test with CM-H2DCFDA. In this study to determine the antioxidant effect on cells, intracellular oxidant species are determined by use of 2 ', 7'-Dichlorodihydrofluorescein diacetate (DCFH-DA). Its mode of action consists of diffusion into the cells of l DCFH-DA (non-fluorescent). Subsequently, DCFH-DA will be de-acetylated by intracellular esterases to 2 ', 7'-Dichlorodihydrofluorescein (DCFH), (non-fluorescent), and rapidly oxidized to fluorescent 2', 7'-Dichlorodihydrofluorescein (DCF) by reactive oxidizing species . The intensity of the fluorescence will be proportional to the concentration of reactive oxidizing species in the cytosol.
[0020] The same ISO standards recommend cell lines CCL 1 (NCTC clone 929; mouse fibroblasts), CCL 163 (Balb / 3T3 clone A31; mouse fibroblasts), CCL 171 (MRC-5; human fibroblasts) , CCL 75 (WI-38; human fibroblasts), CCL 81 (Vero; non-human primate epithelial cells), CCL 10 [BHK-21 (C-13); hamster fibroblasts] and V-79379A (hamster fibroblasts) from the "American Type Culture Collection" (ISO 10993-5: 2009). However, primary cell and / or tissue cultures obtained directly from living tissues, also are recommended for analysis requiring specific sensitivity, provided that the reproducibility and accuracy of the cellular and / or tissue response can be demonstrated against the samples studied. The choice of cell and / or tissue cultures depends on the This is the subject of the study and may vary depending on the standards of the certifying authorities in different countries. In vitro studies commonly use mammalian cell cultures; more specifically, established cell lines obtained from recognized repositories have been used. The cells of an ARPE-19 cell line are pigmented epithelial cells of human origin located in the retina, characterized by Dunn (Dunn et al. Exp Eye Res.
[0021] 1996; 62: 155-69), and can be easily purchased from the "American Type Culture Collection." They are easily accessible, widely used in research, and require less effort to grow and maintain under laboratory conditions. from other cells of the retina, such as ganglion cells, are not well established and primary cultures of other cells of the retina are difficult to grow and maintain. Thus, a good alternative is RPE cells and within them those of the line ARPE-19 cells that show advantages over other types of retinal cells in terms of availability, growth, maintenance and their application in research.
[0023] Therefore, taking into account all these reasons, it is necessary to design appropriate strategies to evaluate in a sensitive and reliable way the solubility, cytotoxicity, antioxidant and antidegenerative capacity, and the efficacy of grape extracts to develop the products of human use that may have a beneficial effect on human pathologies. Specifically, the detection of the antioxidant and antidegenerative capacity and its potencies and effectiveness of said extract will allow its use in other fields such as nutritional, therapeutic, gastronomic or cosmetic, among others.
[0025] It is therefore the object of the present invention to evaluate the solubility, cytotoxicity, antioxidant and antidegenerative capacity, and its potential efficacy of the aforementioned grape extract that will provide the scientific bases for the development of beneficial products in different fields such as nutritional, therapeutic, gastronomic or cosmetic, among others.
[0026] The present invention refers to procedures that make it possible to establish the solubility, non-cyto-toxic and cyto-toxic concentrations of said grape extract, subsequently its antioxidant and antidegenerative capacity, its potency and efficacy are established by applying the epithelium cell cultures retinal pigmentation of human origin and different assay strategies.
[0028] There are several grape extracts worldwide, but each one of them has characteristics that must be identified by applying different test strategies to propose them to different nutritional, therapeutic, gastronomic or cosmetic industries, among others, as a basis for the development of products for use. such as the treatment of ocular pathologies.
[0030] This object is achieved by means of the present invention as claimed in the independent claim. Other advantageous embodiments are described in the dependent claims.
[0032] The object of the present invention is also achieved by means of a method. Each individual step of the method is described in greater detail in the following paragraphs.
[0034] The problem of the present invention is solved by a process, which comprises the following steps:
[0035] a) Provide a grape extract obtained by procedures involved;
[0036] b) Determine the solubility of the extract in one or more solvents;
[0037] c) Determine the non-cytotoxic and cytotoxic concentration of said extract for the cells to be tested;
[0038] d) Determine the antioxidant capacity of said extract based on a nocytotoxic concentration determined in step a) for the cells to be tested.
[0040] Also, additional steps not explicitly specified can be part of the method. In particular:
[0041] e) Determine the antioxidant capacity of said extract in a cellular model of acute oxidative damage.
[0042] f) To determine the antioxidant capacity of said extract in a cellular model of chronic oxidative damage.
[0043] j) Determine that said grape extract rescues previously damaged cells (antidegenerative effect).
[0044] k) To determine the antidegenerative capacity of said extract in a cellular model of acute damage.
[0045] l) To determine the antidegenerative capacity of said extract in a cellular model of chronic damage.
[0046] m) Determine that said grape extract rescues damaged cells to a greater extent than other compounds also obtained from grapes, such as Resveratrol and that to some extent it can be considered as the reference.
[0048] The solubility of the extract is preferably determined for an aqueous medium and dimethylsulfoxide (DMSO), more preferably for the culture medium of the cells to be tested and dimethylsulfoxide (DMSO). It may be necessary to filter the solution before use, preferably using a 0.2 µm filter.
[0050] The non-cyto-toxic and cyto-toxic concentration of the extract is preferably measured by exposing the extract directly in contact with cells and / or tissues. Preferably, the concentration measurement is carried out using the Alamar Blue test (alamarBlue®) or the MTT® test.
[0052] For the subsequent steps a concentration is used, which has been established as non-cyto-toxic (viability of at least> 90%) in at least one test, and preferably in all tests.
[0054] In the next stage, the antioxidant capacity of said extract is determined. Preferably this determination comprises the treatment of the cells with the enzyme glucose oxidase (GOx) and / or with H2O2. For both treatments, the determination of intracellular oxidant species is then carried out, preferably using 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA).
[0056] In the next stage, the antidegenerative capacity of said extract is determined. Preferably this determination comprises the treatment of the cells with the enzyme glucose oxidase (GOx) and / or with H2O2. For both treatments, the determination of the metabolic activity of the cells is then carried out, preferably using the MTT® test.
[0058] In another embodiment of the present invention, comparative tests are carried out using Resveratrol.
[0060] In another embodiment of the present invention, the antioxidant and antidegenerative capacities of the extract are measured in at least one of the following treatment strategies:
[0061] - Previous treatments (PRE): the cells are exposed to the grape extract and subsequently the oxidative intracellular species and the metabolic activity are measured with and without oxidative treatment, using glucose oxidase after treatment with the extract.
[0062] - Simultaneous treatments (SIMU): intracellular oxidant species and metabolic activity are measured during treatment with the extract with and without oxidative treatment, using glucose oxidase.
[0063] - Combined treatments (COMB = PRE + SIMU): the cells are exposed to the grape extract and subsequently the intracellular oxidant species and the metabolic activities are measured with and without oxidative treatment, using glucose oxidase, during a subsequent treatment with the extract.
[0065] The duration of each treatment can vary from 1 minute to 48 hours, preferably from 1 minute to 24 hours.
[0067] In a preferred embodiment, the antioxidant and antidegenerative treatment in each of the strategies is a treatment with glucose oxidase for 1 to 40 hours. The duration depends on the concentration of the extract used and the type of cells.
[0069] In a preferred embodiment cell viability is measured after each strategy.
[0071] In another preferred embodiment, the measurement of intracellular oxidizing species is performed by measuring the fluorescence of 2 ', 7'-dichlorodihydrofluorescein diacetate.
[0072] In another embodiment of the present invention, the oxidative treatment comprises a treatment of the cell culture with H2O2. The treatment is preferably carried out for at least 30 minutes to 4 hours, preferably for 1 to 2 hours. The H2O2 concentration is between 0.05 M and 2 mM, preferably 0.25 mM.
[0074] Hereinafter, the expression used to refer to this process will be "the process of the present invention".
[0076] Another object of the invention is a composition for treating or ameliorating an ophthalmological disorder comprising a grape extract.
[0078] These ophthalmological disorders are eye diseases such as retinitis pigmentosa, age-related macular degeneration, glaucoma, dry eye syndrome, conjunctivochalasia, keratoconus, cataracts, diabetic retinopathy and some uveitis, preferably macular degeneration, plus preferably macular degeneration, with or without, reduced or impaired vision.
[0080] Such a grape extract is preferably identified using the method of the invention. Preferably, such a grape extract shows a better performance than 5 to 20 µM of resveratrol in the process of the invention, more preferably the grape extract shows a better performance than 10 µM of resveratrol.
[0082] In another embodiment, the grape extract in a concentration of 1 to 5 mg / l, preferably in a concentration of 2 mg / l, has a better performance than 10 µM of resveratrol in the process of the invention.
[0084] By behavior is preferably meant a high antioxidant capacity and / or a low cytotoxicity, preferably a high antioxidant capacity for identical treatment of retinal pigment epithelium cell cultures, preferably antioxidant treatment against damage caused by H ² O ² or glucose oxidase. Better performance is preferably an improvement of at least 5%, 10% or 15%, when compared to a control having the same conditions, but without anti-oxidative treatment. Preferably, the enhancement is measured as viability and / or DCF fluorescence, preferably as DCF fluorescence.
[0086] The grape extract is preferably obtained from grape pomace.
[0088] The composition comprises as active ingredients a grape extract comprising as active ingredients the following components:
[0090] a) one or more concentrations of polyphenols in a concentration between 0.5 and 100 mg / g of the composition;
[0091] b) dietary fibers, wherein the dietary fibers comprise one or more sugars or derivatives thereof at a concentration of 50 to 600 mg / g based on the composition.
[0093] Polyphenols are chemical compounds or substances found in a large amount of plant-derived foods. The term encompasses a wide variety of molecules with a common structure, characterized by having several hydroxyl groups attached to aromatic rings. However, polyphenols also include molecules with a phenol ring, as in the case of phenolic acids or phenolic alcohols.
[0095] Polyphenol Classification:
[0097] Polyphenols are classified based on the number of phenol rings they contain, as well as the structural elements that link these rings. As such, the main groups of polyphenols are:
[0099] Flavonoids: which have a diphenylpropane carbon skeleton and two benzene rings joined together by a linear chain of 3 carbon atoms. To date, more than 6,000 flavonoids have been identified in plants, and the list continues to grow. Flavonoids can be divided into 6 subclasses, based on the oxidation state of the central pyran ring. Flavonoids can be divided into Flavonols, Flavones, Flavanones, Lysoflavones, Anthocyanins, and Flavanols.
[0101] Flavanols: which have a double bond between carbons 2 and 3, with a hydroxyl group at C3.
[0102] They are the most ubiquitous flavonoids in food, and quercetin is the most representative.
[0104] Flavones: They have a double bond between carbons 2 and 3, and are the least common flavonoids. They are found in parsley and celery.
[0106] Flavanones: They are characterized by having a saturated chain of 3 carbon atoms and an oxygen atom at carbon 4. They are found only in high concentrations in citrus fruits, although they are also present in tomatoes and in some aromatic plants such as mint .
[0108] Isoflavones: which are structurally similar to estrogens, and in the feet can bind to their receptors, which is why they are also known as phytoestrogens. Soy and its derivatives are the main sources of isoflavones.
[0110] Anthocyanins: which are the pigments responsible for the red, blue and purple coloration of fruits, flowers and other tissues and plant products
[0112] Flavanols: which have a saturated chain of 3 carbons, with a hydroxyl group at C3. They exist as both monomers and polymers, known as catechins and proanthocyanidins, respectively.
[0114] The main flavonoids in the fruit are catechin and epicatechin, while gallocatechin, epi-gallocatechin, and epigallocatechin gallate are essentially found in tea. Catechins are found in fruits like apricots and cherries, and in other products like tea, chocolate, and white wine.
[0115] Phenolic acids: which are divided into two groups, those derived from benzoic acid and those derived from cinnamic acid.
[0117] Hydrobenzoic acids: found in very few plants consumed by humans, and therefore not considered of particular interest for nutrition. Among them, gallic acid or egalic acid stand out
[0119] Hydroxycinnamic acids: which are represented mainly by coumaric acid, caffeic acid and ferulic acid.
[0120] Phenolic Alcohols: Tyrosol and Hydroxytyrosol are the main types. They are found mainly in olive oil. Tyrosol is also found in wine, both white and red, and in beer; hydroxytyrosol, on the other hand, is found in red wine.
[0122] Stilbenes: the main stilbene present in the human diet is resveratrol, although the amount of these compounds ingested in the diet is small. Stilbenes are produced by plants in response to pathogens or certain stress conditions. They have been detected in more than 70 species of plants, such as grapes, berries and peanuts.
[0124] Lignans - Lignans are produced by oxidative dimerization of two phenylpropane units. The main source of these compounds is flax seed.
[0126] Preferably, the polyphenol compound or compounds comprise ellagic acid, flavanols or flavanols or any combination thereof, preferably ellagic acid, flavanols and flavanols. Preferably, the polyphenols comprise catechin, epicatechin or proanthocyanidin or any combination thereof as flavanols, preferably catechin, epicatechin and proanthocyanidin. Preferably the composition comprises as flavanols, myricetin, quercetin or any combination thereof, preferably mycetrin, quercetin and kaempferol.
[0128] One or more sugars or derivatives thereof are preferably the sugars measured by hydrolysis of the alcohol insoluble residue (RIA) of the composition.
[0130] Preferably the sugars comprise neutral sugars and uronic acids. Preferably the neutral sugars selected from the group comprising cellulose, glucose, xylose, galactose, arabinose and mannose and any combination thereof. Preferably, the neutral sugars are selected from the group consisting of cellulose, glucose, xylose, galactose, arabinose, and mannose, and any combination thereof.
[0132] Preferably, the total concentration of dietary fibers in the composition is 50 to 850 mg / g, more preferably 300 mg / g 850 mg / g, even more preferably 550 mg / g 750 mg / g.
[0133] Preferably, the total concentration of one or more polyphenols is between 10 and 100 mg / g, more preferably between 20 and 50 mg / g. The total concentration is measured in mg gallic acid / g.
[0135] Preferably, the total concentration of dietary fibers is at least 3 times higher than the concentration of polyphenols, more preferably between 5 and 30 times higher.
[0137] Preferably, the concentration of neutral sugars is 5 to 20 times higher than the concentration of uronic acids.
[0139] In a preferred embodiment of the invention, the composition comprises the following concentration of polyphenols by weight:
[0140] • ellagic acid: between 0.5 and 10 mg / g of the composition
[0141] • flavanols: between 0.2 and 20 mg / g of the composition
[0142] • flavanols: between 0.11 and 12 mg / g of the composition.
[0144] Preferably, the flavanols comprised by the composition are as follows:
[0145] • catechin: between 0.05 and 5 mg / g of the composition
[0146] • epicatechin: between 0.05 and 5 mg / g of the composition
[0147] • proanthocyanidin: between 0.1 and 10 mg / g of the composition.
[0149] Preferably, the flavanols comprised by the composition are the following:
[0150] • myricetin: between 0.05 and 5 mg / g of the composition
[0151] • quercetin: between 0.05 and 5 mg / g of the composition
[0152] • kaempferol: between 0.01 and 2 mg / g of the composition.
[0154] Preferably, the sugars comprised by dietary fibers are as follows:
[0155] • neutral sugars: between 150 and 550 mg / g of the composition
[0156] • Uronic acids: between 1 and 100 mg / g of the composition
[0157] • Lignin Klason: between 1 and 200 mg / g of the composition.
[0159] Preferably, the neutral sugars comprised by the composition are the following:
[0160] • cellulose: between 0.5 and 30 mg / g of the composition
[0161] • glucose: between 150 and 400 mg / g of the composition
[0162] • xylose: between 0.1 and 5 mg / g of the composition
[0163] • galactose: between 0.5 and 30 mg / g of the composition
[0164] • arabinose: between 0.1 and 10 mg / g of the composition
[0165] • mannose: between 0.5 and 30 mg / g of the composition
[0167] In a preferred embodiment of the invention, the composition comprises the following concentration of polyphenols by weight:
[0168] • ellagic acid: between 0.5 and 10 mg / g of the composition
[0169] • flavanols: between 0.2 and 20 mg / g of the composition
[0170] • flavanols: between 0.11 and 12 mg / g of the composition,
[0171] and where, the flavanols comprised by the composition are the following:
[0172] • catechin: between 0.05 and 5 mg / g of the composition
[0173] • epicatechin: between 0.05 and 5 mg / g of the composition
[0174] • proanthocyanidin: between 0.1 and 10 mg / g of the composition,
[0175] and where, the flavanols comprised by the composition are the following:
[0176] • myricetin: between 0.05 and 5 mg / g of the composition
[0177] • quercetin: between 0.05 and 5 mg / g of the composition
[0178] • kaempferol: between 0.01 and 2 mg / g of the composition.
[0180] In a preferred embodiment of the invention, the composition of the sugars comprised by dietary fibers is as follows:
[0181] • neutral sugars: between 151.7 and 505 mg / g of the composition
[0182] • Uronic acids: between 1 and 100 mg / g of the composition
[0183] • Lignin Klason: between 1 and 200 mg / g of the composition;
[0184] in which the neutral sugars comprised by the composition are the following:
[0185] • cellulose: between 0.5 and 30 mg / g of the composition
[0186] • glucose: between 150 and 400 mg / g of the composition
[0187] • xylose: between 0.1 and 5 mg / g of the composition
[0188] • galactose: between 0.5 and 30 mg / g of the composition
[0189] • arabinose: between 0.1 and 10 mg / g of the composition
[0190] • mannose: between 0.5 and 30 mg / g of the composition
[0192] All of the sugars mentioned are preferably the natural forms of the sugars, eg. RE- Glucose.
[0193] The composition can be part of a functional dietary supplement (nutraceutical).
[0195] The composition of the present invention comprising the grape extract can be administered in any desired and effective way: as pharmaceutical compositions for oral intake or as an ointment or drops for local administration to the eyes (instillation), or for administration parental or other administration in any appropriate manner, such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Furthermore, a compound of the present invention can be administered in conjunction with other treatments. If desired, the grape extract of the present invention can be encapsulated or otherwise protected against gastric or other secretions.
[0197] Although it is possible for the grape extract of the invention to be administered alone, it is preferable to administer the compound in the form of a pharmaceutical formulation (composition) or a nutraceutical. The pharmaceutically acceptable compositions of the invention comprise one or more compounds as an active ingredient in admixture with one or more pharmaceutically acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and / or materials.
[0198] Regardless of the route of administration selected, the grape extract of the present invention is formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
[0200] Pharmaceutically acceptable carriers are well known in the art and include sugars (eg, lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (eg, dicalcium phosphate, tricalcium phosphate, and hydrogen). calcium phosphate), sodium citrate, water, aqueous solutions (eg saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (p eg ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (eg, glycerol, propylene glycol, and polyethylene glycol), organic esters (eg, ethyl oleate and triglycerides), biodegradable polymers (eg, polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed and peanut), cocoa butter, waxes (e.g. suppository waxes), paraffins, silicones, talc, silicon, etc. Each of the pharmaceutically acceptable carriers used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Carriers suitable for a selected dosage form and a Intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using one of ordinary skill in the art.
[0202] The pharmaceutical or nutraceutical compositions of the invention may optionally contain additional ingredients and / or materials commonly used in pharmaceutical or nutraceutical compositions of this type. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose, and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surfactants, (16) dispersing agents; (17) release control agents or absorption retardants, such as hydroxypropyl methylcellulose, other polymeric matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) agents moisturizers; (21) emulsifying and suspending agents; (22) Solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed oils, peanut, corn, germ, olive, castor and sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters; (23) propellants, such as chlorofluoro hydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents that make the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (29) sweetening, flavoring, coloring, perfuming, and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not detrimental to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known. in the art, and ingredients and materials acceptable for a chosen dosage form and method of administration can be determined using one of ordinary skill in the art.
[0204] Pharmaceutical or nutraceutical compositions suitable for oral administration may be in the form of capsules, sachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or nonaqueous liquid, a liquid emulsion of oil in water or of water in oil, an elixir or syrup, a pill, a bolus, an electuary or a paste. These formulations can be prepared by methods known in the art, e.g. eg, by conventional tray coating, mixing, granulating, or lyophilization processes.
[0206] Solid dosage forms for oral administration (capsules, tablets, pills, lozenges, powders, granules and the like) can be prepared by mixing the active ingredient (s) with one or more pharmaceutically acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants and / or coloring agents. Solid compositions of a similar type can be used as fillers in soft gelatin capsules and hard fillers using a suitable excipient. A tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepare using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surfactant or dispersing agent. Molded tablets can be prepared by molding on a suitable machine. Tablets and other solid dosage forms, such as lozenges, capsules, pills, and granules, may optionally be provided with an indentation or prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They can also be formulated so as to provide a slow or controlled release of the active ingredient contained therein. They can be sterilized, for example, by filtration through a filter that retains bacteria. These compositions may also optionally contain opacifying agents and may be of such a composition that they release the active ingredient only or, preferably, in a certain part of the gastrointestinal tract, optionally in a delayed manner. The active ingredient can also be in a microencapsulated form.
[0208] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
[0210] Liquid dosage forms may contain suitable inert diluents, commonly used in the art. In addition to inert diluents, oral compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents. Suspensions can contain suspending agents.
[0212] Pharmaceutical compositions for rectal or vaginal administration can be presented in the form of a suppository or ovules, which can be prepared by mixing one or more active ingredients with one or more suitable non-irritating carriers, which are solid at room temperature, but liquid at room temperature. body temperature and thus will melt in the rectum or vaginal cavity and release the active compound. Pharmaceutical compositions that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or aerosol formulations containing such pharmaceutically acceptable carriers, as known in the art as appropriate.
[0213] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, and inhalation agents. The active compound can be mixed under sterile conditions with a suitable pharmaceutically acceptable carrier. Ointments, pastes, creams, and gels may contain excipients. Powders and aerosols can contain excipients and propellants.
[0215] Pharmaceutically acceptable pharmaceutical compositions suitable for parenteral administration comprise one or more compounds in combination with one or more sterile, pharmaceutically acceptable aqueous and non-aqueous isotonic solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to of use, which may contain antioxidants, buffers, solutes that make the formulation isotonic with the blood of the intended recipient, or suitable suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.
[0217] These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption.
[0218] In some cases, in order to prolong the effect of a drug (eg, pharmaceutical or nutraceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This can be achieved through the use of a liquid suspension of amorphous material that has low water solubility.
[0220] Alternatively, delayed absorption of a parenterally administered drug can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be prepared by forming microencapsulation matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to the polymer and the nature of the particular polymer employed, the rate of release of the active ingredient can be controlled. Injectable depot formulations can also be prepared by trapping the drug in liposomes or microemulsions that are compatible with body tissue. Injectable materials can be sterilized, for example, by filtration through a filter that retains bacteria.
[0222] The formulations can be presented in sealed unit dose or multi-dose containers, eg ampoules and vials, and can be stored in a lyophilized state requiring only the addition of the sterile liquid carrier, eg water for injection, immediately prior to use. Extemporaneous injection of solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.
[0224] Experimental data:
[0226] The term "extract" refers to a dry grape extract. The raw material for the extract was the pomace obtained after the rosé or red winemaking process. The red grapes used in the winemaking process were all from the Tempranillo variety, harvested in vineyards located in the Ribera de Duero Denomination of Origin. The fresh pomace was used as raw material and was subjected to a distillation process to remove aromatic compounds and alcohol. The residue was extracted through the method of traditional solid-liquid extraction using a hydroalcoholic solution (water-ethanol) as solvent. The solid-liquid extraction was carried out by means of the diffusion technique. Once the polyphenolic liquid extract was obtained, the resulting product was centrifuged and stabilized to displace possible solid residues The solution rich in polyphenols was concentrated and finally dried by means of a spray drying process (spray ). Maltodextrin and silicon dioxide were used as encapsulating agents in the spray drying process. The moisture in the extract was around 4.6%. The process can also be carried out as described in ES2319032A1.
[0228] The extract contained high amounts of dietary fiber (659.7 mg / g of extract) and a moderate concentration of total polyphenols (37.44 mg of gallic acid / g of extract) (Table 1). Phenolic compounds include different phenolic acids, flavanols, and flavanols (Table 1). Other chemical parameters analyzed in the extract were moisture (4.6%), total protein (8.7%), fat (0.8%) and ash (20.7%). The extract could be obtained from red grapes used in the wine-making process, as described in GilSánchez, I. (2017) to extract L1. The sugar content based on an alcohol insoluble residue of the extract is shown in Table 2.
[0230] Analysis of flavanols, phenolic acids and flavanols
[0232] The extract (50 mg) was extracted with 50% acetonitrile (1 ml) in an ultrasonic bath for 30 minutes and further centrifuged at 6700xg for 5 minutes; the supernatant was collected and the residue was subjected to the same process another three times. The four supernatants were combined and evaporated to dryness under reduced pressure. The residue was taken up in 1 ml of acetonitrile-water (25:75, v / v) and analyzed by high performance liquid chromatography, by serial diode detection, electrospray ionization / HPLC-DAD-ESI / mass spectrometry. MS on a Hewlett-Packard 1100 chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump and a serial diode detector coupled to a data processing HPChemStation (rev.A.05.04). The HPLC system was connected through the cell output of the serial diode detector to an API 3200 Qtrap mass spectrometer (Applied Biosystems, Darmstadt, Germany), consisting of an ESI source and an ion trap mass analyzer triple-quadruple controlled by Analyst 5.1 software.
[0234] The separation was carried out on an Agilent Poroshell 120 EC-C18 column (2.7 µm, 150 mm x 4.6 mm) at 35 ° C, using (A) 0.1% formic acid and (B) acetonitrile as solvents, using different gradients for the analysis of flavanols and phenolic acids (0% B to 8% B in 5 min and from 8 to 10% during 20 min, 10-14.5% B during 20 min, 14.5-60 % B for 10 min) and flavanols (isocratic 15% B for 5 min, 15-20% B for 5 min, 20-35% B for 10 min, 35-50% B for 10 min, 50-60% B for 7 min) , using in both cases a flow rate of 0.5 mLmin-1. On-line double detection was carried out by DAD at 280 and 370 nm as preferred wavelengths. Mass spectra were recorded between m / z 100 and m / z 1400 in the negative ion mode. Zero grade air served as nebulizer gas (40 psi) and turbo gas (400 ° C) for solvent drying (30 psi). Nitrogen served as curtain at (10psi) and collision gas (medium).
[0236] The quadrupoles were adjusted to unit resolution and the mass spectrometer detector was programmed to perform a series of two consecutive analyzes: a high sensitivity full probe (Enhanced MS, EMS) and a product ion analysis. enhanced (EPI) to obtain the parent ion fragmentation pattern. The EMS parameters were: ion spray voltage 4500V, potential gap (DP) -40V, input potential (EP) -7V and collision energy (CE) -20V; while the EPI adjustments were: DP -40V, EP -10V, CE -25V and CES 0V, for flavanols and phenolic acids; and DP -40V, EP -10V, CE -25V and CES 0V, for flavonols.
[0238] Phenolic compounds were characterized according to their absorption, mass spectra, and retention times compared to a data library, as well as authentic standards where available. The compounds were quantified from the areas of their chromatographic peaks recorded at 280 nm (phenolic acids and flavanols) and 370 nm (flavanols). For compound quantification, calibration curves were obtained for catechin, epicatechin, uercetin, gallic acid, and ellagic acid. Flavanols were expressed as quercetin equivalents and procyanidin dimers as epicatechin. All samples were analyzed in triplicate, and the results were expressed in milligrams of phenolic compound per gram of extract (spray dried product).
[0240] Anthocyanin Analysis
[0242] Samples (50 mg) were extracted using 0.1% trifluoroacetic acid (TFA) -acetonitrile (1: 1, v / v) (1 ml) in an ultrasonic bath for 30 minutes and further centrifuged at 6700xg for 5 minutes; the supernatant was collected and the residue was subjected to the same process another three times. All supernatants were combined and evaporated to dryness under reduced pressure. The residue was taken up in 1 ml of 0.1% TFA-acetonitrile (75:25, v / v) and analyzed by HPLC-DAD-ESI / MS. An AQUA (Phenomenex) C18 column (5 µm, 150mm x 4.6mm) was used at 35 ° C. The solvents were (A) 0.1% TFA and (B) acetonitrile, with the following elution gradient established: isocratic 10% B for 5 min, 10-15% B in 15 min, isocratic 15% B for 5 min, 15-18% B for 5 min, 18-35% B for 20 min, 35-60% B for 7 min. The flow index was 0.5 mLmin-1. DAD was carried out at 280 and 520nm. Mass spectra were recorded between m / z 100 and m / z 900 in the positive ion mode. Zero grade air served as nebulizer (50 psi) and turbo gas (600 ° C) for solvent drying (40 psi). Nitrogen was curtain (10 psi) and collision gas (high). EMS and EPI analysis were also carried out. The EMS mode parameters were: ion spray voltage 5000V, DP 55V, EP 4V and CE 10V; The EPI parameters were: DP 5541V, EP 4V and CE 10V and Collision Energy Dispersion (CES) 0V.
[0244] Analysis of cell wall components
[0246] From each extract, alcohol insoluble residues (RIA) were obtained by immersing the samples in ethanol (96%) at 60 ° C and homogenizing with a mixer for 5 minutes. The materials were filtered through a sintered glass filter (No.3, Scientific Furnishingsltd, Chichester, UK) and re-extracted twice for 10 minutes in 96% ethanol at 20 ° C and filtered again.
[0248] The residues were resuspended in absolute alcohol (twice) and acetone (twice), filtering between times, and then air dried, after which they were stored in a sealed container at room temperature. The results were expressed in milligrams of RIA per gram of extract (spray dried product). The amount of RIA corresponds to the amount of dietary fiber in the extract as used in the present specification.
[0250] The polysaccharides present in the RIAs were extracted and the chemical composition of each fraction was determined. The RIAs were subjected to a treatment of 12molL-1 H2S04 at room temperature for 3 h, followed by dilution to 0.6 molL-1 H2S04 hydrolysis at 100 ° C for 3 h (HF column in table 2), and also to 0.6 molL-1 H2SO4 hydrolysis at 100 ° C for 3 h (HS column in table 2). Both hydrolyzed acids released different components of dietary fiber. The insoluble residues obtained after carrying out acid hydrolysis, using 12 molL-1 H2S04 followed by 0.6 molL-1 H2S04, were gravimetrically quantified as Klason's lignin. The hydrolysates were passed through an AG4x4 ion exchange resin column (Bio-Rad Laboratories, Richmond, CA, USA) to neutralize them. The neutral sugar composition of the hydrolysates was determined by HPLC using a microguard column (Aminex Carbo-P, Bio-Rad) in series with a carbohydrate analysis column (Aminex HPX-87P heavy metal, 300 mm x 7.8 mm, Bio-Rad) operated with a flow rate of 0.5 mLmin-1 using a refractive index detector. Sugar amounts were calculated using System Gold version 7.0 software after calibration with standard sugars (Sigma, St Louis, MO, USA). Before neutralization, erythritol was added as an internal standard.
[0251] The uronic acid content was determined by the mhydroxydiphenyl colorimetric method using D-galacturonic acid as a standard.
[0253] The term "cell culture" refers to a culture of eukaryotic or prokaryotic cells, preferably eukaryotic, more preferably mammalian (eg human, feline, canid, bovine, rodent or porcine cells), even more preferably human. All three Cell types -those derived from ectoderm, mesoderm and endoderm- can be used in the method of the present invention. Preferred cell lines for cell culture in step (a) are those recommended by ISO standards (ISO 10993-5 : 2009) For example, but not limited to, the cell lines CCL 1 (NCTC clone L929; mouse fibroblasts), CCL 163 (Balb / 3T3 clone A31; mouse fibroblasts), CCL 171 ( MRC-5; human fibroblasts), CCL 75 (WI-38; human fibroblasts), CCL 81 (Vero; non-human primate epithelial cells) or CCL 10 [BHK-21 (C-13); hamster fibroblasts] and V -79 379A (hamster fibroblasts) from the "American Type Culture Collection". However, primary cell cultures obtained directly from living tissues are also recommended to carry out the method of the present invention.
[0255] Preferably the cells used in cell culture are cells that can serve to provide the scientific basis for the characteristics of grape extract, and those that are generally useful for the development of products containing said extract. The evaluation of the types of cells where the beneficial effects of the extract are applied, -in this case cyto-toxicity and the antioxidant and antidegenerative capacities- on a regular basis in clinical practice, provides more guarantees on their bio-compatibility, and therefore , about your safety.
[0257] In a preferred embodiment of the method of the present invention, a cell culture of retinal pigment epithelium (RPE) cells is employed. In a more preferred embodiment, the retinal pigment epithelium cells are of human origin. In an even more preferred embodiment, the RPE cells are cells of the ARPE-19 cell line (ATCC CRL-2302), which is a spontaneously generated cell line from human RPE. These cells form a stable monolayer that shows polarity morphological and functional. ARPE-19 cells express specific RPE markers, such as CRALBP and RPE-65, and exhibit morphological polarization. These cells form tight junctions in monolayers with transepithelial resistance. Likewise, other sources of RPE cells and other cells of the retina in culture can be used to carry out the method of the present invention: ganglionic, Müllerian, or of non-retinal origins such as fibroblasts, among others and not only of modified human origin. , but also fresh cells and from other animals.
[0259] The "retinal pigment epithelium" or "RPE" is the layer of pigmented cells located on the outside of the retina that closely interacts with photoreceptors (rods and cones) in maintaining visual function. It is firmly anchored to the underlying choroid by means of its basement membrane and Bruch's membrane. The retinal pigment epithelium is composed of a layer of hexagonal cells that are densely packed and have pigment granules. Viewed in section, each cell consists of an unpigmented outer portion containing a large, oval nucleus, and a pigmented inner portion extending a series of straight threadlike processes between the rods. It also serves as a transport limiting factor that maintains the environment of the retina, supplying small molecules such as amino acids, ascorbic acid and D-glucose, while representing a narrow barrier for substances transported by the blood of the choroid (they form the fundamental structure of the hematoretinal-external barrier). The retinal pigment epithelium also has many other functions such as phagocytosis of the outer segments of photoreceptor cells, the release of growth factors, or the regeneration of photopigment, among others.
[0261] By "retinal pigment epithelium cells" or "RPE cells" is understood any cell type present in said epithelium, preferably epithelial cells. EPR cells can be cultured in the presence of culture media and conditions known in the technical field for epithelial cell culture. Thus, for example, but not limited to, the culture medium can comprise fetal bovine serum (FBS) or human, antibiotics, antifungals, growth factors, etc. The base medium that can be used in the culture medium could be any of those known in the state of the art for in vitro cell culture , such as, for example, but not limited to, the basal medium "Eagle", CRCM-30, CMRL -1066, "Dulbecco's Modified Eagle's Medium” (DMEM), "Eagle's Minimum Essential Medium” (EMEM), "Fischer's Medium”, "Glasgow Minimum Essential Medium”, Ham's F-10, Ham's F-12 (F12), "High Cell Density Medium", "Iscove's Modified Dulbecco's Medium", Leibovitz's L-15, McCoy's 5A, medium 199, "Minimum Essential Medium Eagle", "Alpha Minimum Essential Medium", CnT20, NCTC 109, NCTC 135, RPMI-1640, "WiNiam's Medium E", Waymouth's MB 75211, Waymouth's MB 70511, "Keratinocyte serum-free medium" (KSFM), or any combination thereof. Preferably, the culture medium comprises a DMEM / F12 base medium, supplemented or not with FBS and in the presence of antibiotics and antifungals. In addition, for example, but without limitation, the culture conditions can be standard culture conditions, that is, in the presence of between 5 and 10% CO2, between 36 and 38 ° C and renewing the medium every 48 to 72 hours. . Preferably, said cultivation is carried out as described below in the examples of the present invention.
[0263] In vitro cell culture in the method of the present invention is carried out in 96-well culture plates.
[0265] For the method of the present invention, different concentrations of said extract will be exposed, preferably in an aqueous medium (in this case in the culture medium), until the end of the exposure time.
[0267] The incubation of the cell culture in the presence of the different concentrations of said extract is carried out during a determined exposure time and preferably under the appropriate conditions to allow cell growth or proliferation. Said culture conditions and time will depend on the cell type chosen for culture. Those skilled in the art will recognize said conditions and the incubation time applicable in each case.
[0269] For such cultures, the control cultures comprise cells of the same cell type and origin as the cells grown in the method of the present invention. Said cells from the control cultures are incubated under the same conditions of time, temperature, pH, light / dark cycles, humidity, CO2 concentration, composition of the culture medium, etc., as the culture under study. Control cultures are also processed in the same way after incubation.
[0270] In a preferred embodiment, control cultures comprise cells not exposed to any compound (blank), cells or tissues exposed to resveratrol (negative control), or cells or tissues exposed to a toxic control compound (to benzalkonium chloride; positive control). . The negative control comprises resveratrol (to be studied to help determine that the effects on cells are due to the compound resveratrol which is known to have antioxidant capacity and for which there are numerous published studies).
[0272] Other more preferred embodiments consist of exposing the cultures to the enzyme glucose oxidase to produce chronic damage to the cultures under the same conditions of time, temperature, pH, light / dark cycles, humidity, CO2 concentration, composition of medium. culture, etc., than the culture under study, and also in exposing the cultures to H2O2 to produce the acute damage. Then, the cultures will be exposed to positive and negative controls and the extract at certain times according to the proposed strategies and the protocols described later as study examples.
[0274] In the present invention, several strategies are developed whose advantages, according to the proposed procedures, are, therefore, the following:
[0276] - They allow to establish the solubility of said extract in different organic or inorganic solvents. The detection of solubility of an extract in different organic or inorganic solvents is one of the first characteristics that directs the lines of research and innovation. Aqueous medium and dimethyl sulfoxide (DMSO) are common solvents that are widely used in product development because there are enough published studies in their favor. However, it is necessary to determine its non-cytotoxic and cyto-toxic concentration before its final use. Therefore, the solubility of said extract in the aqueous medium (in this case the culture medium) and the dimethyl sulfoxide (DMSO) must be determined.
[0278] - They allow to confirm the non-cyto-toxic and cyto-toxic concentrations of said extract prepared in the aqueous medium and the dimethyl sulfoxide (DMSO). The detection of cyto-toxicity is another of the first characteristics that directs the lines of research and innovation. The ISO standard recommends several methods to determine cyto-toxicity, one of them being to detect cyto-toxicity by exposing the extract directly in contact with cells. in this case retinal pigment epithelium (RPE). Direct contact between said extract and the cells and / or tissues implies that there is no interference with any other factor, as is the case in indirect contact assays or in extract dilution methods where other factors are involved, such as carriers of extraction, the agar layer on the cell layer, etc., which could affect cell proliferation or growth of tissue structure in culture and consequently the test results. Therefore, the non-cyto-toxic and cyto-toxic concentrations were determined in the RPE cells of said extract in the aqueous medium (in this case the culture medium) and the dimethyl sulfoxide (DMSO). Two tests were used to measure cyto-toxicity: the Alamar Blue test (alamarBlue®) and the MTT® test.
[0279] - They make it possible to confirm the antioxidant capacity of said extract in cellular models of oxidative damage. The detection of the antioxidant capacity is another of the first characteristics that direct the lines of research and innovation for the development of products that act against ROS produced by imbalance in cellular metabolism. Therefore, two cellular models of oxidative damage were proposed; one consisted of treating cells with the enzyme glucose oxidase (GOx) to produce chronic damage and the other is with H2O2, which produces acute damage, and the determination of intracellular oxidant species by using 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA).
[0280] -Allows to confirm in the cellular models of oxidative damage the antidegenerative capacity of said extract. The detection of the antidegenerative capacity is another of the first characteristics to which the lines of research and innovation are directed for the development of products that act against ROS produced by an imbalance in the cellular metabolism that generates cellular degeneration. Therefore, two cellular models of oxidative and degenerative damage were proposed; one consists of treating the cells with the enzyme glucose oxidase (GOx) to produce chronic damage and in the other they are treated with H ² O ² which produces acute damage, and the determination of the metabolic activity of the cells is carried out by the test called MTT®.
[0281] - They allow to confirm the cyto-toxicities of said extract in the cellular models of oxidative damage and other strategies used described in subsequent paragraphs.
[0282] - They allow to confirm the high level of the antioxidant and antidegenerative capacity of said extract in comparison with the comparative control of resveratrol, which is a molecule widely used in food supplements and with demonstrated antioxidant capacity.
[0283] - They allow to confirm the antioxidant and antidegenerative capacity of said extract in various types of treatment strategies:
[0285] i) Previous treatments (PRE)
[0286] In the PRE treatment group, the cultures are exposed for 1 hour to a concentration of 2 mg / ml of grape extract or 10 pM of resveratrol (negative control) (PRE treatment) and subsequently treated for 30 minutes with 10 pM of H2DCF-DA to measure antioxidant capacity. After treating for 24 hours with 11 mU / ml glucose oxidase, at the end of the experiments they were incubated with the compound MTT for 2 hours to measure the antidegenerative capacity.
[0288] ii) Simultaneous treatments (SIMU)
[0289] In the SIMU treatment group, the cultures are treated for 30 minutes with 10 pM of H2DCF-DA to measure the antioxidant capacity and subsequently for 24 hours with 11 mU / ml of glucose oxidase at a concentration of 2 mg / ml of extract of grape or with 10 pM resveratrol (negative control). At the end of the experiments, they were incubated with the MTT compound for 2 hours to measure the antidegenerative capacity.
[0291] iii) Combined treatments (COMB = PRE + SIMU)
[0292] In the COMB treatment group, the cultures are exposed for 1 hour to a concentration of 2 mg / ml of grape extract or 10 pM of resveratrol (PRE treatment). Subsequently, they were treated with 10 pM of H2DCF-DA for 30 minutes to measure the antioxidant capacity and finally the cultures were exposed for 24 hours to 11 mU / ml of glucose oxidase and a concentration of 2 mg / ml of grape extract or 10 pM Resveratrol (SIMU treatment). At the end of the experiments, they were incubated with the MTT compound for 2 hours to measure the antidegenerative capacity.
[0294] - They allow to confirm the cyto-toxicity and the antioxidant and antidegenerative capacity in other groups of PRE, SIMU and COMB treatments. In this group the cultures were not treated for 24 hours with 11 mU / ml of glucose oxidase to produce cell damage.
[0295] These tests and strategies provide scientific data on the solubility, cytotoxicity and antioxidant and antidegenerative capacities and the efficacy of said extract under different conditions (one of them is its comparison with Resveratrol), which will be very useful in the development of products containing this extract.
[0297] The data of the results are compared among them following the standard UNE-EN ISO 10993-5 "C.2.5 Analysis of the data".
[0299] Even if multiple subordination claims are not written, all reasonable combinations of the features in the claims will be described.
[0301] While the present invention has been described and illustrated in conjunction with a number of specific embodiments, those skilled in the art will appreciate that variations and modifications can be made without departing from the principles of the present invention as illustrated herein. as described and claimed. The present invention can be carried out in other specific ways without departing from its spirit or essential characteristics. The described embodiments are considered in all respects illustrative and not restrictive. The scope of the inventions is therefore indicated by the appended claims rather than by the foregoing description. All changes that fall within the meaning and range of equivalence of the claims are to be encompassed within their scope.
[0303] The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
[0305] EXAMPLES
[0307] The invention is illustrated below by means of a series of tests carried out by the inventors, which show the effectiveness of the method described to evaluate the solubility, cytotoxicity and antioxidant and antidegenerative capacities and their efficacy, of said extract on cell cultures of Human retinal pigment epithelium under different conditions developing comparative studies and establishing scientific bases.
[0309] Cell cultures of Retinal Pigment Epithelium (ARPE-19 cell line)
[0310] Culture of the ARPE-19 cell line
[0311] To achieve the main purpose of this study, several experiments were planned using retinal pigment epithelium cell cultures, specifically from the human cell line ARPE-19, from the company "American Type Culture Collection® (ATCC; Manassas, VA, USA). ”This cell line was chosen because it is considered suitable to simulate the clinical conditions of a possible therapeutic use, since it is cells of the retina and also of human origin.
[0313] After thawing the cells, they were cultured in flasks with a surface area of 25 cm2 for cell culture with the culture medium DMEM / F12 (Dulbecco's Modified Eagle Medium and F12 Nutrient Mixture (Ham); Gibco®, Paisley, United Kingdom), supplemented with 10% fetal bovine serum and 1% antibiotic / antifungal (100 U / ml penicillin, 100 µg / ml streptomycin and 0.25 µg / ml amphotericin B, Gibco®). The cells were cultured under conditions of 37 ° C and 5% CO2. The culture medium was renewed every two days until the cells reached 80-90% confluence. When they reached the desired confluence, they were trypsinized and seeded in 96-well plates, where the experiments were carried out, or were cultured in 75 cm2 flasks, to be used in the repetitions of the experiment.
[0315] Seeding 96-well plates for experiments
[0316] After trypsinizing the cells, a cell count was performed with Trypan Blue Solution 0.4% (Sigma-Aldrich®, St. Luis, MO, USA) and subsequently seeded in a 96-well plate (Costar, Corning Inc. ., Corning, NY, USA) at a density of 1 * 104 cells / well. They were incubated at 37 ° C and 5% CO2 with supplemented DMEM / F12 culture medium, until they reached the pre-confluence state. At that time, they were synchronized to the G0 / G1 phase of the cell cycle by culturing them for 24 hours with the DMEM / F12 culture medium supplemented with 1% antibiotic / antimycotic but without fetal bovine serum (incomplete).
[0318] Treatments:
[0319] After synchronization, the cultures were used in the different experiments to achieve the different objects focused on achieving the main object.
[0320] Each experiment is described in more detail below.
[0322] Example 1. Determination of a non-cytotoxic concentration of grape extract by the AlamarBlue® test
[0324] According to previous studies carried out with this extract in other areas of study, it is a hydrophilic product. Therefore, initially it was decided to use the culture medium to prepare the different concentrations of the extract. Three concentrations (20, 2 and 0.2 mg / ml) were prepared. The possible cytotoxicity of these concentrations of the grape extract was evaluated by incubating it for 24 hours with the ARPE-19 cultures and measuring the cytotoxicity by means of the Alamar Blue test (alamarBlue®).
[0326] In the Azul Alamar (alamarBlue®) assay, the active ingredient in Azul Alamar is resazurin, a non-toxic compound that is permeable to cells and colored blue. Upon entering the cells, resazurin is reduced to resorufin by oxide reductases that are mainly found in the mitochondria of living cells and diffuses into the environment and initially, as it is not concentrated inside the cell, it does not induce death, as it does. in other techniques. Resorufin produces a very bright red or pink fluorescence. Viable cells perform this reaction and thus generate a quantitative measure of viability or cytotoxicity. The study working solution (Alamar blue 10% dissolved in culture medium) was prepared. At the end of the experiment, the culture medium was removed and the cells were washed. Then 200 µl of the working solution was added per well. Subsequently, they were incubated under standard culture conditions (37 ° C and 5% CO2) for 5 hours. After 5 hours the medium was collected from each well and deposited in another 96-well plate. The fluorescence intensity of the medium in each well was read using a plate reader at an excitation intensity of 560 nm and an emission intensity of 590 nm.
[0328] The experimental groups were the following:
[0329] - Culture control group: cells were incubated with the supplemented DMEM / F12 culture medium. Two control groups were arranged: one on the left side (control 1) and the other on the right side (control 2) following the recommendations of the ISO standards.
[0330] - Group of grape extract: the cultures were incubated with different concentrations of grape extract.
[0331] o Subgroup 20 mg / ml grape extract: cultures were incubated with 20 mg / ml of grape extract.
[0332] o Subgroup 2 mg / ml grape extract: cultures were incubated with 2 mg / ml grape extract.
[0333] o Subgroup 0.2 mg / ml grape extract: the cultures were incubated with 0.2 mg / ml of grape extract.
[0335] Results:
[0336] Once the solution of 20 mg / ml of grape extract in culture medium had been prepared, it was filtered using a 0.2 µm filter. From the filtered solution, two additional solutions of 2 mg / ml and 0.2 mg / ml were prepared.
[0337] Observation:
[0338] It was found that, with filtration, much volume of the 20 mg / ml solution is lost. It was observed that the grape extract particles did not fully dissolve in the culture medium and, for this reason, a considerable amount of the prepared solution was lost during filtration. In relation to this result, it may be necessary to use another sterilization method, such as exposure to UV light for a time to be determined.
[0339] Result and discussion:
[0340] The data obtained are quite homogeneous and acceptable to be able to carry out the corresponding statistical analysis. Statistical analysis of the data (figure 1) has shown that:
[0341] - The mean value of viability (%) of cultures of the control groups sown both on the left and right side of the rest of the groups was not more than 15% of the mean value of viability (%) of cultures of all control wells. The assay meets the ISO standard criteria to ensure that the number of cells is consistent in all wells of the cell culture plate. It is appreciated that the cultures maintain the morphology of the ARPE-19 cells. It is appreciated that it is a healthy crop. The data obtained from the control groups were established as 100%.
[0342] - Most of the cells incubated with the grape extract at 20 mg / ml died (3.68% viability), but those incubated with the 2 and 0.2 mg / ml solutions grew well and maintained the same range of values of OD that were obtained in the controls (98.33% and 104.32% viability).
[0343] Conclusion:
[0344] The conclusion of this first study is that the 20 mg / ml concentration of the extract is cytotoxic, while the 2 and 0.2 mg / ml solutions are not, although they do not significantly improve the proliferation capacity of ARPE cells. 19.
[0346] Figure 1 shows the effect of three different concentrations of grape extract on the retinal pigment epithelium cell cultures, specifically cells of an ARPE-19 cell line after a 24-hour exposure and measured by the assay of Alamar Blue. The abscissa axis represents the cell viability in percentage after treatment with the different concentrations of extract, as well as the untreated cells (control). The average of the percentages of viability obtained from the results of the developed experiments is shown. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0348] Example 2. Determination of a non-cytotoxic concentration of grape extract by the MTT® test
[0349] Taking into account the results of the previous study, in this experiment six concentrations of the grape extract (2, 5, 10, 12, 15 and 20 mg / ml) were prepared in a culture medium. Based on the results of the previous study, it was calculated that the IC50 of this product could be 12 mg / ml, and for this reason this dose was included in this experiment. The possible cytotoxicity of these concentrations of the grape extract was evaluated by incubating them for 24 hours with the ARPE-19 cultures and measuring the cytotoxicity by the MTT assay.
[0351] The MTT® assay is a colorimetric technique, which mainly measures the activity of the enzyme succinic dehydrogenase, which is located in cellular mitochondria. But cytosolic reductases or reductases from other subcellular compartments can also intervene. The compound MTT is taken up by cells and reduced by the succinic dehydrogen enzyme to its insoluble form formazan. The product of the reaction, the formazan is retained in the cells and can be released by solubilization in an organic solvent, and its color is measured by means of a plate reader. At the end of the experiment, all treatments are removed from all wells and washed with culture medium without phenol red. 50 uL of the previously prepared 10% MTT solution is added. I know incubate for 2 hours in the incubator under standard culture conditions of 37 ° C and 5% CO2. The crystals formed are resuspended with 100 uL of a 10% solution of HCl in Isopropanol. Pipet into each well to resuspend the crystals. The absorbance is read with parameters of 570 nm and 650 nm.
[0353] The experimental groups were the following:
[0354] - Culture control group: The cells were incubated with the DMEM / F12 culture medium supplemented. Two control groups were placed: one on the left side (control 1) and the other on the right side (control 2) following the recommendations of the ISO standards.
[0355] - Group of grape extract: the cultures were incubated with different concentrations of grape extract.
[0356] o Subgroup 2 mg / ml grape extract: cultures were incubated with 2 mg / ml grape extract.
[0357] o Subgroup 5 mg / ml grape extract: the cultures were incubated with 5 mg / ml of grape extract.
[0358] o Subgroup 10 mg / ml grape extract: the cultures were incubated with 10 mg / ml of grape extract.
[0359] o Subgroup 12 mg / ml grape extract: the cultures were incubated with 12 mg / ml of grape extract.
[0360] o Subgroup 15 mg / ml grape extract: the cultures were incubated with 15 mg / ml of grape extract.
[0361] o Subgroup 20 mg / ml grape extract: cultures were incubated with 20 mg / ml of grape extract.
[0362] o Positive control group: cultures were incubated with a 0.001% benzalkonium chloride solution.
[0363] o Negative control group: cultures were incubated with culture medium with 8.7% PBS. The PBS was used in the preparation applied to prepare the previous solution, and for this reason this control was included, to determine that the possible effect was due to benzalkonium chloride and not to the carrier.
[0364] Results:
[0365] Six concentrations (2, 5, 10, 12, 15 and 20 mg / ml) of the grape extract were evaluated following the same method described in the previous section, except that the MTT test was used to determine the cytotoxicity of said concentrations.
[0366] Observation:
[0367] The observations from the previous experiment are repeated again confirming that the grape extract does not completely dissolve in the culture medium. It is required to use another more appropriate solvent or another sterilization method.
[0368] Result and discussion:
[0369] The statistical analysis of the data obtained (figure 2) has shown that:
[0370] - The mean viability value (%) of cultures of the control groups of the culture medium, sown both on the left and on the right side of the rest of the groups was not more than 15% of the mean viability value (% ) of cultures from all control wells. The assay meets the ISO standard criteria to ensure that the number of cells is consistent in all wells of the cell culture plate. It is appreciated that the cultures maintain the morphology of the ARPE-19 cells. It is appreciated that it is a healthy crop. Control group data were set at 100%.
[0371] - In the positive control group, the percentage of viability was 3%, confirming that the culture responds perfectly to cytotoxicity.
[0372] - In the negative control group, the percentage of viability was 97% confirming that the culture responds perfectly under non-cytotoxic conditions. The amount of PBS (8.7%) added in the culture medium did not produce a significant difference in the viability of the culture, confirming that this amount can be applied in the following studies.
[0373] - In the group with grape extract, the percentages of viability were 100%, 98%, 69%, 54%, 23% and 7% for concentrations of 2, 5, 10, 12, 15 and 20 mg / ml of grape extract.
[0374] Conclusion:
[0375] The conclusion of this second study is that the concentrations of 10, 12, 15 and 20 mg / ml of the grape extract are cytotoxic, and the solutions of 2 and 5 mg / ml were not, but neither did they significantly improve cell proliferation. . The IC50 concentration of the extract is between 12 and 13 mg / ml of extract because the viability of the culture is 54% with the concentration of 12 mg / ml and is reduced to 23% by increasing the concentration of the product to 15 mg / ml.
[0376] Figure 2 shows the effect of different concentrations of grape extract on the retinal pigment epithelium cell cultures, specifically the cells of an ARPE-19 cell line after a 24-hour exposure and measured by the MTT assay. . The abscissa axis represents the cell viability in percentage after treatment with the different concentrations of extract, as well as the untreated cells (Control). The average of the percentages of viability obtained from the results of the developed experiments is shown. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0378] The concentration of 2 mg / ml was chosen for the following experiments because the viability of the culture was 104% in experiment 1 (measured by the Alamar Blue® test) and 100% in experiment 2 (measured by the test of the MTT®).
[0380] Example 3. Determination of the antioxidant and antidegenerative capacities and their efficacy of the grape extract in a model of cellular damage produced after a treatment with glucose oxidase
[0381] Once the non-cytotoxic concentration of the grape extract had been determined, a study was planned in which the crops were exposed to different treatments. The purpose of this study was to evaluate the effect of grape extract on a culture damaged with glucose oxidase (which induces chronic damage to RPE cultures).
[0383] Three types of treatments were compared:
[0385] i) Previous treatments (PRE)
[0386] In the PRE treatment group, the cultures were exposed for 1 hour to a concentration of 2 mg / ml of grape extract or 10 ^ M of resveratrol (negative control) and subsequently treated for 30 minutes with 10 ^ M of H2DCF- GIVES. After treating for 24 hours with 11 mU / ml of glucose oxidase, they were incubated with the MTT compound for 2 hours at the end of the experiment.
[0388] ii) Simultaneous treatments (SIMU)
[0389] In the SIMU treatment group, the cultures were treated for 30 minutes with 10 ^ M H2DCF-DA and subsequently for 24 hours with 11 mU / ml of glucose oxidase at a concentration of 2 mg / ml of grape extract or with 10 ^ M resveratrol (negative control). They were incubated with the MTT compound for 2 hours at the end of the experiment.
[0391] iii) Combined treatments (COMB = PRE + SIMU)
[0392] In the COMB treatment group, the cultures were exposed for 1 hour to a concentration of 2 mg / ml of grape extract or 10 µM of resveratrol (PRE treatment). Subsequently, they were treated with 10 ^ M H2DCF-DA for 30 minutes and finally the cultures were exposed for 24 hours to 11 mU / ml of glucose oxidase and a concentration of 2 mg / ml of grape extract or 10 uM of resveratrol (treatment SIMU). They were incubated with the MTT compound for 2 hours at the end of the experiment.
[0394] In other PRE, SIMU and COMB treatment groups, the cultures were not treated for 24 hours with 11 mU / ml of glucose oxidase to produce cell damage. The duration of 24 hours and the concentration of 11 mU / ml of glucose oxidase were chosen taking into account previous results obtained in the laboratory in the development of other studies.
[0396] The duration of 30 minutes and the concentration of 10 ^ M of H2DCF-DA have been chosen because it is the one used in the experiments that appear in the articles published in this field of study, in addition to being the one used regularly in previous studies obtained in the laboratory in the development of other projects.
[0398] The concentration of grape extract and resveratrol was prepared in the solvent DMSO.
[0399] The experimental groups were the following:
[0400] - Culture control group: cells were incubated with the supplemented DMEM / F2 culture medium. Two control groups were used: one on the left side (control 1) and the other on the right side (control 2) following the recommendations of the ISO standards.
[0401] - Group of grape extract: the cultures of this group were incubated with a concentration of 2 mg / ml of grape extract.
[0402] o PRE grape extract subgroup: the cultures were incubated following the PRE treatment protocol with or without the presence of glucose oxidase.
[0403] o SIMU grape extract subgroup: the cultures were incubated following the SIMU treatment protocol with or without the presence of glucose oxidase. o COMB grape extract subgroup (PRE + SIMU): the cultures were incubated following the COMB treatment protocol with or without the presence of glucose oxidase.
[0404] - Control group: several controls were used consisting of cultures incubated with culture medium, with DMSO and with resveratrol (PRE, SIMU and COMB treatment) with or without the presence of glucose oxidase.
[0405] After incubation, DCF fluorescence and culture viability of each group were measured using the MTT assay.
[0407] Results
[0408] Observation:
[0409] The grape extract and the resveratrol control dissolve completely in DMSO, which facilitated its filtration using a 0.2 µm filter for sterilization. The DCF fluorescence (method of detection of antioxidant capacity) was measured after 1, 3, 5, 8 and 24 hours of duration after treatment, but only the analysis of the data obtained after 24 hours is presented (since it was the group in which the best fluorescence value was obtained, shown in Figure 5, compared with the fluorescence value obtained after 1 hour). In addition, Figures 3 and 4 present the analysis of the data obtained after 24 hours by the MTT test (the antidegenerative capacity).
[0411] Result and discussion:
[0412] The statistical analysis of the data (figures 3 and 4) obtained has shown the relationship between the antioxidant and antidegenerative capacities in each crop group:
[0413] - In the Control group with culture medium, it is appreciated that the cultures maintained the morphology of the ARPE-19 cells. It is appreciated that it is a healthy crop. The data of this group were established as 100% to be able to compare the results obtained for both the antidegenerative capacity (viability) and for the antioxidant capacity. When the control group was treated with glucose oxidase (11 mU / ml), the viability was 92.17% with respect to the control without damage (100%) confirming that the glucose oxidase concentration chosen for this study produces less damage (8%) to culture.
[0414] - In the control group with 0.5% DMSO but not treated with glucose oxidase, the viability was 115.85%, confirming that the concentration of 0.5% DMSO is not cytotoxic for the culture. The DCF fluorescence of the culture (measured with the parameters (nm) of the DCF detection at 24 hours) was 186.81%, confirming that the presence of DMSO increased the in vitro oxidation conditions of the culture cells but did not produce cell degeneration (oxidative condition versus degenerative condition). The control group, when treated with glucose oxidase, presented a viability of 52.69%, confirming that the 0.5% of DMSO and the 11 mU / ml of glucose oxidase together produce a damage close to 50%, which is considered desirable (cell degeneration). It should be noted that the degenerative damage effect was produced at 50% when DMSO and glucose oxidase acted together with the cells. A damage of almost 50% in a crop is recommended for this type of study. It should be noted that DMSO (0.5%) individually did not damage the culture and glucose oxidase (11 mU / ml) produced only 8% damage. Furthermore, the DCF fluorescence has increased (3991.29%), which is 21 times more than the DMSO group without glucose oxidase (186.81%). These findings confirm that the cell degeneration of the culture occurs by oxidation.
[0415] PRE group
[0416] - In the resveratrol PRE group, the viability was 111.81%, confirming that the concentration of 0.8% of DMSO used to dissolve the resveratrol and the concentration of 10 ^ M of resveratrol, after 1 hour of exposure, are not cytotoxic for culture. The DCF fluorescence of the culture was 59.51%. When the culture of this group was treated with glucose oxidase, after one hour of pretreatment with resveratrol, the viability was 115.54%, which confirms that the damage produced by glucose oxidase has been inhibited in this group (116, 54% compared to 92.17% of the glucose oxidase group, and 111.81% of the resveratrol PRE group without glucose oxidase). It should be noted that there is no group with one hour of pretreatment with DMSO and subsequently exposed to glucose oxidase for 24 hours, because the objective of the study was to look for differences between treatments (grape extract or resveratrol) without or with glucose oxidase. The DCF fluorescence was 59.75%, the same as in the pretreated resveratrol group but without glucose oxidase (59.75% compared to 59.51%). The antioxidant effect of resveratrol cannot be confirmed by the absence of a group pretreated for one hour with DMSO and subsequently exposed to glucose oxidase for 24 hours. It can only be confirmed that the one hour pretreatment with resveratrol improves viability compared to the group with glucose oxidase (115.54% compared to 92.17%), although the presence of DMSO for one hour prior to glucose oxidase has increased the oxidation condition in vitro (59.75% compared to 16.65% of the glucose oxidase group).
[0417] - In the PRE grape extract group, viability was 117.14%, confirming that the concentration of 0.8% of the DMSO used to dissolve the grape extract and the concentration of 2 mg / ml of the grape extract, after 1 hour of exposure, they are not cytotoxic for culture. The DCF fluorescence of the culture was 183.72%. When the culture of this group was treated with glucose oxidase after one hour of pretreatment with the grape extract, the viability was 98.78%, which confirms that the damage produced by glucose oxidase has been inhibited in the group (98 , 78% compared to 92.17% of the glucose oxidase group, and 117.14% of the PRE grape extract group without glucose oxidase). It should be noted that there is no group with one hour of pretreatment with DMSO and subsequently exposed to glucose oxidase for 24 hours because the objective of the study was to look for differences between the treatments (grape extract or resveratrol) without or with glucose oxidase. The DCF fluorescence was reduced to 101.35%, compared to 183.72% in the group of grape extract without glucose oxidase. The antioxidant effect of the grape extract cannot be confirmed due to the absence of a group pretreated for one hour with DMSO and subsequently exposed to glucose oxidase for 24 hours. It can only be confirmed that the one hour pretreatment with grape extract improves viability (antidegenerative capacity) compared to the group with glucose oxidase (98.78% compared to 92.17%), although damage due to glucose oxidase is observed. (98.17% of the group with glucose oxidase compared to 117.14% of the group without glucose oxidase) and the presence of DMSO for one hour before the glucose oxidase had increased the oxidation condition in vitro (101.35% compared to 16.65% of the group with glucose oxidase).
[0418] SIMU Group
[0419] - In the resveratrol SIMU group, the viability was 96.02%, which confirms that the concentration of 0.8% of DMSO and 10 ^ M of resveratrol after 24 hours of exposure, are not cytotoxic (96.02% with respect to 100% of the Control Group). The DCF fluorescence measured in this group was 93.40%, which is 50% lower than what was detected in the group with 0.5% DMSO (93.40% compared to 186.81% in the group with DMSO ). The presence of DMSO, both in the control group and in the resveratrol SIMU group, was 24 hours. Resveratrol is confirmed to have inhibited 50% of the in vitro oxidative condition of cultured cells. When the culture of this group was treated with glucose oxidase together with the resveratrol for 24 hours, the viability was 16.65%, which is 3 times lower than the DMSO group with glucose oxidase (16.65% compared to 52.69%) and 6 times lower than the group with glucose oxidase ( 16.65% compared to 92.17%). This result has confirmed that, although the applied resveratrol concentration is not cytotoxic (demonstrated by the group without glucose oxidase), it was not able to recover the damage produced to the culture by DMSO and glucose oxidase. The DCF fluorescence was 3091.21%, which is a reduction of 0.8 times compared to the control (3091.29% compared to 3991.29%), which confirms that resveratrol has produced a partial inhibition in degeneration oxidative of the culture.
[0420] - In the SIMU grape extract group, viability was 104.28%, which confirms that the concentration of 0.8% of DMSO and 2 mg / ml of grape extract after 24 hours of exposure are not cytotoxic (104.28% compared to 100% of the control group). The DCF fluorescence measured in this group was 89.44%, which is 48% less than what was detected in the group with DMSO (89.44% compared to 186.81%). The presence of DMSO, both in the control group and in the SIMU grape extract group, was 24 hours, which confirms that the grape extract inhibited 48% of the in vitro oxidative condition of cultured cells. When the culture of this group was treated for 24 hours with glucose oxidase and grape extract, the viability was 16.28%, which is 3 times less than the DMSO group with glucose oxidase (16.28% with respect to the 52.69%), and 6 times less than the group with glucose oxidase (16.65% compared to 92.17%). These results confirm that, although the grape extract is not cytotoxic (demonstrated by the group without glucose oxidase), it was not able to recover the damage produced by DMSO and glucose oxidase to the culture. The DCF fluorescence was 694.80%, which represents a reduction of 6 times compared to the control (694.80% compared to 3991.29%), and which confirms that the grape extract has produced a partial inhibition in the oxidative degeneration of the culture.
[0421] It should be noted that in both groups, both resveratrol and grape extract, the antioxidant capacity (viability) was the same, but resveratrol reduced DCF fluorescence by 0.8 times and grape extract 6. These data confirm that the antioxidant capacity of grape extract is more potent than that of resveratrol (efficacy).
[0422] COMB Group
[0423] - In the resveratrol COMB group, viability was 89.05%, which confirms again that the concentration of 0.8% of DMSO and 10 ^ M of resveratrol, after 1 hour of pretreatment and 24 hours with glucose oxidase, did not it is cytotoxic (89.09% compared to 100% control). The 6% reduction in viability compared to the resveratrol SIMU group (in the culture without pretreatment with resveratrol, it was 89.05% compared to 96.02%) could be due to the incubation for one more hour with DMSO (resveratrol PRE). After one hour pretreatment with 10 µM resveratrol, the culture was washed, labeled with H2DCF-DA, washed again and finally incubated for 24 hours with 10 µM resveratrol. The DCF fluorescence of this group was 215.04%, which is 1.5 and 3.6 times more than that detected in the control group with DMSO (215.04% compared to 186.81%) and the group of resveratrol SIMU (215.04% compared to 59.51%). This result has confirmed that there is inhibition of the degeneration of the culture by oxidation produced by DMSO (antidegenerative effect). When the culture of this group was treated with glucose oxidase for 24 hours together with resveratrol (1 hour of resveratrol PRE and later 24 hours of resveratrol SIMU), the viability was 24.48%, which is 3.8 and 2 , 1 times less than that detected in the Control Group for glucose oxidase (24.48% compared to 92.17%) and DMSO with glucose oxidase (24.48% compared to 52.69%) respectively , and 1.5 times more than what was detected in the resveratrol SIMU group (24.48% compared to 16.65%). This result confirms that resveratrol is not cytotoxic as demonstrated by the group without glucose oxidase and was not able to recover the damage produced in the culture by DMSO and glucose oxidase. However, the result of the resveratrol COMB group has presented an increase of 1.5 times compared to the resveratrol SIMU group (24.48% compared to 16.65%), which is the opposite compared to the group without glucose oxidase ( where a reduction of 6% was obtained; that is, 89.05% compared to 96.02%). The DCF fluorescence was 3223.23%, which is a reduction of 0.8 times compared to the control (3223.23% compared to 3991.29%), and confirms that resveratrol produced a partial inhibition in oxidative degeneration when culture and improved viability compared to the resveratrol SIMU group without glucose oxidase.
[0424] - In the COMB grape extract group, viability was 95.47%, which confirms that the concentration of 0.8% of DMSO and 2 mg / ml of grape extract, after 1 hour of pre-treatment and 24 hours with glucose oxidase, is not cytotoxic (95.47% compared to the 100% control). The 9% reduction in viability compared to the SIMU grape extract group (without pretreatment with grape extract, 95.47% compared to 104.28%) could be due to having had one more hour of incubation with DMSO (extract group grape PRE). After one hour pretreatment with 2 mg / m of grape extract, the culture was washed, labeled with H2DCF-DA, washed again and finally incubated for 24 hours with the concentration of 2 mg / ml of grape extract . The DCF fluorescence of this group was 90.16%, which is 2.1 times lower than the fluorescence detected in the control group with DMSO (90.16% compared to 186.81%) and similar to that detected in The group of SIMU grape extract (90.16% compared to 89.44%). This result has confirmed that, when a PRE and SIMU treatment is carried out, the grape extract inhibits the oxidation in vitro of the cells of the culture, but an inhibition is not so powerful as to block the degeneration of the culture (a reduction of 9 % regarding the viability of the SIMU grape extract group). When the culture of this group was treated with glucose oxidase for 24 hours together with the grape extract (1 hour of PRE grape extract and later 24 hours of SIMU grape extract), the viability was 30.35%, which It is 3 and 2 times lower than the viability detected in the glucose oxidase control group (30.35% compared to 92.17%) and DMSO with glucose oxidase (30.35% compared to 52.69%) respectively, and 2 times higher than the viability detected in the SIMU grape extract group (30.35% compared to 16.28%). This result has confirmed that the grape extract is not cytotoxic (as demonstrated by the group without glucose oxidase) and was not able to recover the damage produced to the culture by DMSO and glucose oxidase. However, the result of the resveratrol COMB group has presented a 2-fold increase compared to the SIMU grape extract group (30.35% compared to 16.28%), contrary to what was observed in the group without glucose oxidase (a reduction of 9%, that is, 95.47% compared to 104.28%). The DCF fluorescence was 684.94%, which is a reduction of 6 times compared to the control (684.94% compared to 3991.29%), which confirms that the grape extract has produced a partial inhibition in the degeneration oxidative of the culture.
[0425] It should be noted that in both groups, both resveratrol and grape extract, the difference between antidegenerative capacities (viabilities) is only 6% (30.35% of the grape compared to 24.48% of the resveratrol) but resveratrol has reduced DCF fluorescence 0.8 times and grape extract 6. These data confirm that grape extract has a higher antioxidant capacity than resveratrol (efficacy).
[0427] Conclusion:
[0428] The conclusion of the third study is that this grape extract could be used as an antioxidant treatment against the damage caused by glucose oxidase (chronic degenerative damage) in the retinal pigment epithelium cells, since the extract inhibits chronic damage produced in the culture and at the same time the fluorescence is inhibited. The results obtained show that 2 mg / ml grape extract is more effective than resveratrol (10 ^ M).
[0429] Figure 3 shows the evaluation of the cytotoxicity and the antioxidant and antidegenerative capacities of different treatments (PRE treatment, SIMU treatment, COMB treatment (PRE + SIMU) and controls) on the retinal pigment epithelial cell cultures, specifically cells of the ARPE-19 cell line. After 24 hours from the end of the treatment, cytotoxicity and antidegenerative capacity (viability) were measured by the MTT assay and the antioxidant capacity by the assay with the H2DCF-DA marker. The abscissa axis represents the cell viability and the antioxidant capacity detected in percentage after the treatments, including the controls. The average of the percentages of cell viability and the antioxidant capacity obtained from the results of the developed experiments is shown. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0431] Figure 4 shows the evaluation of cytotoxicity and antidegenerative capacity (viability) and antioxidant capacity of different treatments (PRE treatment, SIMU treatment, COMB treatment (PRE + SIMU) and controls) on pigment epithelial cell cultures of the retina, specifically the cells of the ARPE-19 cell line (damaged with glucose oxidase). 24 hours after treatment, cell viability was measured by the MTT assay and antioxidant capacity by the H2DCF-DA marker assay. The abscissa axis represents the cell viability and the antioxidant capacity detected in percentage after the treatments, including the controls. The average of the percentages of cell viability and the antioxidant capacity obtained from the results of the developed experiments is shown. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0433] Figure 5 shows the evaluation of the antioxidant capacity of different treatments (PRE treatment, SIMU treatment, COMB treatment (PRE + SIMU) and controls) on the cultures of the retinal pigment epithelium cells, specifically the cells of the cell line ARPE-19 (undamaged or damaged with glucose oxidase). The antioxidant capacity was measured after 1 and 24 hours post-treatment using the H2DCF-DA marker assay. The abscissa axis represents the antioxidant capacity detected in percentage after the treatments, including the controls. The average of the percentages of antioxidant capacity obtained from the results of the developed experiments. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0435] The enzyme glucose oxidase catalyzes a reaction by which H2O2 is constantly generated from the glucose in the culture medium in the culture cells, causing chronic oxidative damage in the culture. This study with cells with chronic damage has shown that grape extract has a greater antioxidant capacity than resveratrol, but the differences between antidegenerative capacity were not significant. It was then decided to apply another method to verify that the grape extract has a more powerful antioxidant and antidegenerative capacity than resveratrol. H ² O ² was added directly to the culture to generate a direct and acute oxidative condition.
[0437] Example 4. Determination of the antioxidant and antidegenerative capacities of the grape extract in a model of cellular damage produced after a treatment with H2O2
[0438] Once the non-cytotoxic concentration of the grape extract was determined, a study was planned in which the crops were exposed to different treatments. The purpose of this study was to evaluate the effect of grape extract on a culture damaged with H2O2 (which produces acute damage to the RPE culture and therefore cell degeneration).
[0439] To optimize the oxidative damage protocol with H2O2 and its detection, the effect of different combinations of H2O2 (0.125 mM, 0.250 mM, 0.500 mM and 1 mM) and H2DCF-DA (5 and 10 ^ M) was studied. The results have shown that the culture loaded with 10 µM H2DCF-DA (45 minutes of incubation) and subsequently incubated with 0.250 mM of H ² O ² (1 hour of incubation) is suitable for this type of study. These concentrations have been applied in the following studies.
[0441] Three types of treatments were compared:
[0443] - Post treatments (POST)
[0444] They were incubated for one and two hours with H ² O ² , subsequently one hour with the grape extract, resveratrol and controls.
[0445] The experimental groups were:
[0446] - Culture control group: cells were incubated with the supplemented DMEM / F12 culture medium.
[0447] - Group of grape extract: cultures were incubated with a concentration of 2 mg / ml of grape extract after incubation with H2O2 (POST treatment)
[0448] - Group of controls: several controls were used consisting of the cultures incubated with the culture medium; with DMSO and with resveratrol with or without the presence of H ² O ² .
[0449] After incubation, DCF fluorescence and culture viability of each group were measured using the MTT assay.
[0451] Results
[0452] Results and discussion: The statistical analysis of the data (figures 6 and 7) has shown the relationship between cell viability, antioxidant and antidegenerative capacities in each study group as follows:
[0453] 1 hour incubation with H202:
[0454] In the H2O2 group, viability was 90.20%, which confirms that e1H2O2 in one hour of incubation has produced a damage of only 10% to the culture (90.20% compared to 100% of the control). The DCF fluorescence was 87.75% (a difference of 12% from the control). When the culture was incubated for one hour with 0.8% DMSO and H2O2, the cell viability was 60.95% and the DCF fluorescence detected was the same as in the H2O2 group (87.50% compared to 87, 15%), which confirms that this condition is not adequate to produce 50% damage to the crop. These data have confirmed that one hour of incubation with DMSO and H2O2 produces an intense oxidative condition in vitro of the cultured cells (60% of the damage produced). When the culture was incubated for one hour with 10 ^ M resveratrol after one hour of incubation with H2O2, the cell viability detected was 62.75%, which confirms an almost 40% reduction in viability compared to the control ( 62.75% compared to 100% control), 30% compared to the H2O2 group (62.75% compared to 90.20%) and an increase of 2% compared to the H2O2 group with DMSO. It should be noted that resveratrol was dissolved in 0.8% DMSO. The DCF fluorescence was 86.23%, the same as that detected in the H2O2 group (86.23% compared to 87.15%) and the H2O2 group with DMSO (86.23% compared to 87.60%). These data have confirmed that resveratrol has not been able to inhibit the intense oxidative condition of degeneration (60% damage). When the culture was incubated with 2 mg / ml of the grape extract, the detected cell viability was 81.27% and the DCF fluorescence was 72.65%. In the presence of grape extract, cell viability has increased by 20% compared to the DMSO and H2O2 group (81.27% compared to 60.95%), which confirms the antidegenerative capacity of the grape extract and DCF fluorescence was reduced by 14% compared to controls (72.65% compared to 86.23%, 87.50% and 87.15%) which confirms the antioxidant capacity of grape extract. Resveratrol was not able to inhibit, but grape extract was, and CFD fluorescence was reduced and crop viability increased, confirming again that grape extract has a higher antioxidant and antidegenerative capacity than Resveratrol. The conclusion of this study confirms that although in the previous study the antidegenerative capacity did not seem considerable, this study has confirmed the antioxidant and antidegenerative capacities of grape extract.
[0456] 2 hours of incubation with H2O2: -In the H2O2 group, the viability was 82.92%, which confirms that e1 H2O2, after two hours of incubation, has produced 18% damage to the culture (82.92% with respect to 100% of the control) and 8% more than what was detected after one hour of incubation (90.20%). The DCF fluorescence was 93.68% (a difference of 7% compared to the control), which confirms the presence of a lower oxidative condition, which has reduced the viability of the culture. When the culture was incubated for one hour with only 0.8% DMSO, cell viability and DCF fluorescence have been detected 53.03% and 104.58% respectively, which confirms that 0.8% DMSO only generates degeneration. cell in this study. A powerful oxidative effect is seen that has reduced viability by 50% (53.03% compared to 100% control). When the culture was incubated for two hours with H2O2 and subsequently with 0.8% DMSO, the viability and fluorescence detected were 54.33% and 107.63%, the same as that detected in the DMSO group, confirming the presence of a powerful oxidative condition. It should be noted that in the previous experiment, in which the culture was exposed for one hour with H ² O ² and later one hour with DMSO, the oxidative condition was less powerful, and the cell viability was 81.21%. When the culture was incubated for two hours with H ² O ² and later for one hour with resveratrol, the viability and fluorescence detected were 57.58% and 100.39%, which confirms a reduction in the oxidative condition in vitro. of cultured cells, which improves cell viability. When the culture was incubated for two hours with H2O2 and then one hour with grape extract, the viability and fluorescence detected were 74.94% and 79.98%, which confirms a greater reduction in the oxidative condition in vitro. of cultured cells, which improves cell viability. The comparison between the resveratrol group and the grape extract confirms again that the grape extract has a higher antioxidant and antidegenerative capacity than resveratrol.
[0457] Conclusion: The conclusion of the third study is that this grape extract could be applied as an antioxidant and antidegenerative treatment against the damage produced by H2O2 in the cells of the retinal pigment epithelium because the extract inhibits the acute damage produced in the culture and at at the same time, it inhibits DCF fluorescence related to the determination of antioxidant capacity and is also more effective than resveratrol. Therefore, it can be concluded that the results obtained show that 2 mg / ml grape extract is more effective in antioxidant and antidegenerative capacities than resveratrol (10 ^{| j} M).
[0459] Figure 6 shows the evaluation of the antidegenerative (cell viability) and antioxidant capacities of different treatments on the retinal pigment epithelial cell cultures, specifically the cells of the ARPE-19 cell line (damaged with H2O2 for one hour). After 1 hour incubation, cell viability was measured using the MTT assay and DCF fluorescence using the H2DCF-DA marker assay. The abscissa axis represents the antidegenerative capacity (cell viability) and the antioxidant capacity detected in percentage after the treatments, including the controls. The average of the percentages of cell viability and antioxidant capacity obtained from the results of the developed experiments is shown. Because the results are presented as a mean of percentages, the standard deviation values are not shown.
[0461] Figure 7 shows the evaluation of the antidegenerative (cell viability) and antioxidant capacities of different treatments on retinal pigment epithelial cell cultures, specifically the cells of the ARPE-19 cell line (damaged with H2O2 for two hours). After 1 hour of incubation, cell viability were measured using the MTT assay, and DCF fluorescence using the H2DCF-DA marker assay. The abscissa axis represents the anti-degenerative capacity (cell viability) and the antioxidant capacity detected in percentage after the treatments, including the controls. The average of the percentages of cell viability and the antioxidant capacity obtained from the results of the developed experiments is shown. Because the results are shown as a mean of percentages, the standard deviation values are not shown.
[0462] Thus, taking into account the results obtained in the previously exposed examples, it can be concluded that:
[0464] Considering the differences observed in terms of cell viability (antidegenerative capacity) in the cell culture of the retinal pigment epithelium ARPE-19 (cell line) and the fluorescence of DCF (antioxidant capacity) in comparison with the controls used in the experiments, have obtained the following conclusions regarding said grape extract:
[0465] - DMSO is the best solvent.
[0466] - the optimal non-cytotoxic concentration is 2 mg / ml.
[0467] - It has been shown to have antioxidant capacity.
[0468] - it has been shown to have antidegenerative capacity (oxidation does not always produce cell degeneration as has already been demonstrated in this study).
[0469] - It has been shown that it has a higher antioxidant capacity than that of resveratrol.
[0470] - It has been shown to have a higher antidegenerative capacity than that of resveratrol.
[0471] - It has been shown that it can act as an antioxidant and antidegenerative against acute and chronic damage.
[0472] -It has been shown that it can act as an antioxidant and antidegenerative against acute and chronic damage, more efficiently than that of resveratrol.
[0473] Table 1
[0475] Composition PHENOLIC COMPOSITION (mg / g extract) Total Polyphenols (mg gallic acid / g) 37.44 Phenolic Acids
[0476] Ellagic acid 5.64 ± 0.65 Flavanols
[0477] Catechin 0.28 ± 0.07 Epicatechin 1.03 ± 0.25 Proanthocyanidin 4.62 Flavonols
[0478] Myricetin 0.26 ± 0.01 Quercetin 0.34 ± 0.03 Kaempferol 0.16 ± 0.00 RESIDUAL INSOLUBLE ALCOHOL COMPOSITION
[0479] (mg / g RIA)
[0480] RIA Total Yield 660 Neutral Sugars 294.7 Uronic Acids 32.3 ± 1.0 Total Sugars 328.2 Klason Lignin 84.3 ± 1.1 Table 2
[0481]

权利要求:
Claims (9)
[1]
1. Procedure to identify properties of a grape extract, which comprises the following steps:
a) provide a grape extract;
b) determining the solubility of the extract in one or more solvents;
c) determining the non-cytotoxic and cytotoxic concentration of said extract for the cells to be tested;
d) determining the antioxidant and antidegenerative capacity of said extract based on a non-cytotoxic concentration determined in step a) for the cells to be tested.
[2]
2. Method according to claim 1, characterized in that the determination of the antioxidant and antidegenerative capacity of the extract comprises the oxidative treatment of the cells with the enzyme glucose oxidase and / or the treatment of the cells with H2O2.
[3]
3. Method according to any of claims 1 or 2, characterized in that the cells to be tested are retinal pigment epithelium (RPE).
[4]
4. Process according to any of claims 1 to 3, characterized in that the antioxidant and antidegenerative capacity of the extract is measured in at least one of the following treatment strategies:
i) exposure of the cells to the extract and subsequently measurement of the intracellular oxidant species and cell viability with and without oxidative treatment (PRE treatment);
ii) measurement of the intracellular oxidant species and cell viability during the treatment with the extract with and without oxidative treatment (SIMU treatment);
iii) exposure of the cells to the extract and subsequently measurement of the intracellular oxidant species and cell viability with and without oxidative treatment during the additional treatment with the extract (COMB = PRE SIMU treatment).
[5]
Method according to any one of claims 1 to 4, characterized in that the viability of the cells is measured after step d).
[6]
6. Composition comprising the treatment or improvement of an ophthalmological disorder, comprising a grape extract, characterized in that the grape extract shows a better performance than 5 to 20 µM of resveratrol in the process according to one of the following claims 1 to 5.
[7]
Composition according to claim 6, characterized in that the grape extract in a concentration of 1 to 5 mg / l shows a better performance than 10 µM of resveratrol.
[8]
Composition according to claim 7, characterized in that the grape extract in a concentration of 2 mg / l shows a better behavior than 10 µM of resveratrol.
[9]
9. Composition for treating or ameliorating an ophthalmological disorder, wherein the composition comprises as active ingredients a grape extract comprising as active ingredients the following components:
a) one or more polyphenols at a concentration between 0.5 and 100 mg / g of the composition; b) dietary fibers, wherein the dietary fibers comprise one or more sugars or derivatives thereof at a concentration of 50 to 600 mg / g based on composition.

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同族专利:

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公开号 | 公开日

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ES2888499A2|2022-01-04|

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WO2020169870A1|2020-08-27|

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ES2779984B2|2021-10-08|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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ES2319032A1|2007-03-12|2009-05-01|Carlos Moro Gonzalez|Procedure for the extraction of polyphenols from grape orujo from distillation. |

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EP2752195A1|2011-08-10|2014-07-09|Abro Biotec, S.L.|Grape extract, nutritional supplement comprising grape extract, and the use thereof as a functional ingredient|

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ES202190047A| ES2888499A2|2019-02-20|2020-02-20|Method for identifying properties of grape extract|