• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Pharmaceutical composition comprising 5-dodecanolide, its preparation and its use. The invention relates to a pharmaceutical composition comprising 5-dodecanolide as an active principle. This active ingredient has been isolated from pork lard through a specific treatment. Thus, the present invention also relates to a method for obtaining the composition of the invention as well as to the use thereof for therapeutic purposes and more particularly for the purpose of treating inflammatory processes. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2623761A1
    申请号:ES201630015
    申请日:2016-01-12
    公开日:2017-07-12
    发明作者:Antoni Pons Biescas；Antoni SUREDA GOMILA；Josep Antoni TUR MARÍ；Miquel MARTORELL PONS；Xavier CAPÓ FIOL
    申请人:Universitat de les Illes Balears；Centro de Investigacion Biomedica en Red CIBER；
    IPC主号:
    专利说明:

    DESCRIPTION
    Pharmaceutical composition comprising 5-dodecanolide, its preparation and its use.
    FIELD OF THE INVENTION
    The present invention belongs to the field of pharmaceutical compositions with anti-inflammatory activity. More particularly it refers to a pharmaceutical composition comprising as active ingredient 5-dodecanolide. This active substance has been isolated from lard through a specific treatment. Thus, the present invention also relates to a process for obtaining the composition of the invention as well as the use thereof for therapeutic purposes and more particularly for the purpose of treating inflammatory processes.
    BACKGROUND OF THE INVENTION
    Inflammation is a tissue process consisting of a series of molecular, cellular and vascular phenomena of defensive purpose against physical, chemical or biological aggressions. The basic aspects that stand out in the inflammatory process are in the first place, the focus of the response, which tends to circumscribe the area of combat against the aggressor. Second, the inflammatory response is immediate and preponderantly nonspecific, although it may favor the subsequent development of a specific response. Third, the inflammatory focus attracts immune cells from nearby tissues. The inflammation causes a large dilation of the blood vessels along with an opening of their pores, increasing their permeability and allowing the passage of fluid, substances and cells from the blood to the tissues, so they increase in volume and temperature. The termination of inflammation is not a passive process, but may be mediated by the synthesis of various molecules derived from the oxidation of polyunsaturated fatty acids such as eicosapentaenoic and docosahexaenoic. Among these molecules are resolvins and hydroxy-derivatives of the oxidation of long-chain omega-3 fatty acids (18, 20, 22 carbons) (US Patent 2009/0180961 A1), or synthetic derivatives thereof (MX Patent 2011010827) . Its composition allows them to be administered in the form of particles and nanoparticles to end inflammation (W02012135032 (A2)).
    Inflammation is already described in Egyptian papyri 3000 years BC. Celsus (Roman writer), during the first century AD, exposes his cardinal signs: Blush, Tumor, Heat and Pain. Next, Virchow presented the fifth sign: functional limitation. In 1793, the Scottish surgeon John Hunter stated that "it is not a disease but a non-specific response that has a healthy effect on the host." In 1839-1884, Julius Cohnheim described the first microscopic findings of this defense response: increased vascular permeability and leukocyte migration. Subsequently, Elie Metchnikoff concluded that the inflammation process generates leukocytes and antibodies for the defense against microorganisms, establishing since then the INFLAMMATION + INFECTION relationship; that is, when bacteria enter the body they produce an infection and the body in its defense generates an inflammation. Moreover, Sir Thomas Lewis, who on the basis of simple experiments of the inflammatory response performed on skin, established that "chemical substances, such as histamine, locally induced by damage, mediate vascular changes in inflammation", this concept it bases the important discovery of chemical mediators of inflammation and the potential use of anti-inflammatory agents.
    In general, we can divide the inflammation into five stages:
    1-Release of mediators. They are molecules, most of them, of elementary structure that are released or synthesized under the action of certain stimuli. Tissues when injured will release mediators of inflammation.
    Mediators include amines such as histamine or serotonin, proteolytic enzymes, nitric oxide, proinflammatory cytokines (such as the chemotactic factor of eosinophil and chemotactic factor of neutrophil) and heparin.
    Other substances of a lipidic nature constitute a second important group of mediators of inflammation. These are de novo synthesized substances derived from arachidonic acid through two metabolic pathways, that of the enzyme cyclooxygenase that determines the production of prostaglandins (PG) and thromboxanes and that of lipoxygenase that leads to the formation of leukotrienes (l T) .
    2-Effect of mediators. Once released, these molecules produce vascular alterations and chemotactic effects that favor the arrival of molecules and immune cells to the inflammatory focus. There is an increase in vascular permeability favoring the arrival to the affected area, from the blood, of molecules and cells of the immune system and the nerve endings of pain are stimulated.
    3-Arrival of molecules and immune cells to the inflammatory focus. From the chronological point of view, the mediators of inflammation will basically produce two effects. In the first initial phase, vascular alterations that facilitate the transfer of molecules from the blood to the inflammatory focus, as well as the production of edema. In a second, later phase, the vascular alterations themselves, as well as the release in the focus of chemotactic factors, determine the arrival of immune cells (basophils, neutrophils, macrophages, lymphocytes and eosinophils) from the blood and surrounding tissues .
    4-Regulation of the inflammatory process. Like most of the immune responses, the inflammatory phenomenon also integrates a series of inhibitory mechanisms aimed at ending or balancing the process. Some of the mediators that produce activation, by varying their concentration or acting on different receptors, will produce inhibition, thereby achieving a balance or modulation of the inflammatory response. The synthesis of molecules, such as resolvins from omega 3 polyunsaturated fatty acids of membrane phospholipids, enters this process of ending inflammation.
    5-Repair. Phase constituted by phenomena that will determine the total or partial repair of the tissues damaged by the aggressor or by the inflammatory response itself. These processes integrate the arrival in the area of fibroblasts that will proliferate and synthesize collagen, proliferation of epithelial cells and proliferation of vessels within the wound.
    To reduce the inflammatory process, non-steroidal anti-inflammatory products (NSAIDs) are basically used. Its operation is based on the inhibition of the enzyme cyclooxygenase reducing the synthesis of prostaglandins. NSAIDs constitute the basic pillar of the treatment of chronic inflammatory diseases, such as the widespread osteoarthritis and arthritis, which, having no cure, require the use of drugs for the treatment of symptoms (symptomatic treatment). This means that stiffness and pain decrease due to its anti-inflammatory and analgesic effect, but they do not cure or modify the disease. Other molecules have been identified, lipid mediators that can contribute to the completion of topical application forms are semi-solid and deformable preparations to spread on the skin or mucous membranes. They are formed by a base or vehicle and the active substance or medication. The bases or vehicles contain the active substance and give the consistency of the presentation, its appropriate choice depends on the type of skin where it is going to be applied and the physical-chemical properties of the medicine to facilitate its release. The purpose of these preparations is the penetration of the active substances through the stratum corneum of the skin with insulating function. Penetration consists of two phases, absorption, which is the passage of the drug through the skin until it reaches the level of the cells. The second phase is diffusion, that is, propagation through tissues.
    the inflammationYwhosefunction,utilization,synthesisYshapefromapplicationhavebeen
    previously patented(PatentUS2009/0180961A1;PatentMX2011010827;
    W020121 35032 (A2) 2012-10-04).
    4
    In the topical treatment the following requirements must be met: intact skin, inflammation of superficial and focal location, and in addition, the application must be continuous. Its use is indicated in trauma, cornering, bruising, tendonitis, etc.
    Within the traditional culture, pork tallow has been used for the treatment of blows and acute inflammatory processes but without knowing the components that may be responsible for these anti-inflammatory effects.
    On the other hand there are examples described in the literature where pork fat has been used in the elaboration of compositions with anti-inflammatory activity.
    For example, document W02009077635 refers to a composition based on olive oil, lard and honey in 1: 1: 1 ratios and its use as an anti-inflammatory, anti-edematous, anti-erythematous and tissue regenerator.
    JP2009149599 also describes a composition of a medicinal oil based on fats and black pig oils fed a diet supplemented with Qtocopherol useful in the cosmetic and nutritional treatment of atopic dermatitis, inflammation, hair loss and eczema.
    For its part, the document ES2087036 aims at a quick-acting ointment against varicose veins, which is based on a mixture of camphor, lard and baking soda.
    US2014377370A1 aims at a homeopathic composition comprising an animal fat, including lard, onion and rosemary for the treatment of
    joints with stiffness, pain, inflammation, cramps, injuries, gout or limited range of motion.
    However, none of the documents described above refers to by which components pork fat exerts an anti-inflammatory effect.
    The authors of the present invention have been able to identify a series of compounds with anti-inflammatory activity from a certain treatment of pig fat. But more surprisingly, they have been able to identify that by means of such treatment a compound appears a compound that is not found in untreated pork fat and that shows a very potent anti-inflammatory effect. This compound identified by the inventors is 5-dodecanolide which is the basis of the compositions of the present invention.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1: Effects of ointment on inflammation induced with zymosan on the diameter of rat legs
    Figure 2: Effects of ointment on inflammation induced with zymosan at 4 hours on the weight of the leg.
    Detailed description of the invention
    A first object of the invention is a pharmaceutical composition comprising 5dodecanolide as an active ingredient and one or more pharmaceutically acceptable excipients or vehicles.
    The composition of the invention can be prepared from the compound obtained commercially or by synthesis or from a process of treatment and purification of lard that is from where the inventors have managed to isolate it and identify its potent anti-inflammatory activity.
    When 5-dodecanolide is obtained from the treatment of lard that will be described later, it is observed that in addition to 5-dodecanolide, the treated shortening has another series of compounds that serve to enhance the anti-inflammatory activity.
    Thus, in a particular embodiment the composition of the invention further comprises 5-dodecanolide, one or more of the compounds selected from octadecenamide, resolvin D1, hexadecanoic acid, 9-octadecenoic acid, 6-octadecenoic acid, 9,12- octadecadienoic acid, octadecanoic acid, 5,8,11, 14-eicosatetraenoic acid, 7hexadecenoic acid, 11,14-octadecadienoic acid, hexadecanamide.
    5-Dodecanolide may be present in the composition of the invention in a range of between 2-6% of the total weight of the composition, preferably between 3-5%.
    In a particular embodiment the composition of the invention comprises ocladecenamide in a percentage of 2-8% of the total weight of the composition.
    In another particular embodiment the composition of the invention comprises resolvin 01 in a percentage of 1-4% of the total weight of the composition.
    Another particular embodiment the composition of the invention comprises hexadecanoic acid in a percentage of 0.01-0.7% of the total weight of the composition.
    Another particular embodiment the composition of the invention comprises 9-octadecenoic acid, 6-octadecenoic acid or both in a percentage of 56-65% of the total weight of the composition.
    Another particular embodiment of the composition of the invention comprises 9,12 octadecadienoic acid in a percentage of 12-15% of the total weight of the composition.
    Another particular embodiment the composition of the invention comprises octadecanoic acid in a percentage of 0.1-0.5% of the total weight of the composition.
    Another particular embodiment the composition of the invention comprises 5,8,11,14eicosatetraenoic acid in a percentage of 5.1-6.3% of the total weight of the composition.
    Another particular embodiment of the composition of the invention comprises 7-hexadecenoic acid in a percentage of 1.2-3% of the total weight of the composition.
    Another particular embodiment of the composition of the invention comprises 11, 14 octadecadienoic acid in a percentage of 5.5-6.1% of the total weight of the composition.
    Another particular embodiment the composition of the invention comprises hexadecanamide in a percentage of 0.7-1.4% of the total weight of the composition.
    A particular embodiment is represented by a composition comprising in percentage by weight with respect to the total composition:
    a) 4.34-4.63% of 5-dodecanolide b) 0.05-0.7% of hexadecanoic acid e) 59.4-63.7% of 9-octadecenoic acid or 6-octadecenoic acid d) 12 , 5-14% of 9,12-octadecadienoic acid e) 0.13-0.47% of octadecanoic acid f) 5.32-6.21% of 5,8,11, 14-eicosatetraenoic acid g) 1, 33-2.83% of 7-hexadecenoic acid h) 5.67-6.00% of 11, 14-octadecadienoic acid i) 0.89-1, 28% of hexadecanamide j) 3.19-6.84% of octadecenamide k) 1.93-3.02% resolvin 01.
    The composition of the invention further comprises one or more pharmaceutically acceptable excipients or vehicles. Depending on the route of administration, one or the other pharmaceutical excipients or vehicles that are widely known to a person skilled in the art can be used such as, for example, support materials, lubricants, fillers, solvents, diluents, colorants, flavor and flavor conditioners such as sugars, antioxidants, waxes or esters of fatty acids, preservatives, emulsifiers and / or binders. The selection of these auxiliary additives or vehicles and the amounts to be used will depend on the form of administration of the pharmaceutical composition.
    The preferred form of administration topically and in this sense the preferred form of the composition of the invention includes ointments, ointments, gels, creams, lotions, suspensions or emulsions. The preferred embodiment of the invention involves the ointment-shaped composition.
    A second object of the invention is a process for obtaining a composition according to the invention comprising:
    a) Extraction of lard b) Crushing of lard e) Cooking of crushed lard d) Separation by sieving of liquid lard from chicharrones e) Optionally, hydroalcoholic extraction of liquid lard
    Before carrying out the first stage of the procedure, the cutting of the pig is carried out.
    The first stage involves the extraction of lard from pork. Pork lard is obtained from bacon and adipose tissue from the region of the omentum. The lean part of the white bacon is separated and the fat attached to the omentum. The feeding of the pig is important in determining the amount and composition of fat accumulated by the animal.
    In a second step, the lard formed by the white bacon and the fat of the omentum are crushed by mechanical means such as, for example, in a meat grinder with a large-hole chopped disc (diameter of about 2 cm). Once crushed, the bacon and the fat of the omentum are mixed until a homogeneous mass is obtained, although not too crushed. Approximately 20 kg of mass per animal are obtained.
    Then the crushed butter is cooked. For this, the butter is preferably introduced into a stainless steel boiler for cooking over high heat. The grease is constantly removed throughout the cooking process with a wooden spoon, to prevent the fat from sticking to the bottom of the boiler and burning. During the cooking process the fat is liquefied and subsequently reaches the boil (50-BODe). The average cooking time for about 20 kg of fat is about 50 minutes over high heat, until the pork rinds are cooked and acquire an intense golden color, which can happen after 90 minutes of cooking. During this cooking the anti-inflammatory principles are produced and released. The production of anti-inflammatory components can be increased, increasing the proportion of protein and using fat from pigs with a diet supplemented with omega 3 polyunsaturated fatty acids.
    Once the butter is cooked, it is removed from the heat and allowed to stand in the same casserole. When it has cooled sufficiently, it goes through a sieve in order to separate the pork rinds from the liquid butter. Subsequently, while the butter is still liquid, it can either be used in the next step of the procedure or stored in glass containers where it finishes cooling and solidifies for optional use in the hydroalcoholic extraction stage.
    Hydroalcoholic extraction and purification of the active ingredients is optional but recommended as it allows a higher concentration of compounds with anti-inflammatory activity. To obtain the hydroalcoholic extract of pork tallow, melt and add a mixture of alcohol: water (65:35, v: v), preferably methanol: water (65:35, v: v) and a concentrated acid preferably Hel 1 M. Stir vigorously with mechanical help and the phases are separated by centrifugation. The anti-inflammatory principles are then purified by a solid phase extraction in which the anti-inflammatory active ingredients are adsorbed to a hydrophobic material that is washed with water, hexane and finally desorbed and released with methylformate.
    The hydroalcoholic extract obtained by this procedure also has 5dodecanolide the following compounds: octadecenamide, resolvin D1, hexadecanoic acid, 9-octadecenoic acid, 6-octadecenoic acid, 9,12octadecadienoic acid, octadecanoic acid, 5,8,11, 14- eicosatetraenoic acid, 7hexadecenoic acid, 11, 14-octadecadienoic acid, hexadecanamide.
    The extract obtained can be used directly as an ointment for topical application or can be processed for formulation as a pharmaceutical composition adapted to any route of administration as explained above.
    On the other hand, it should be noted that both the treated pork tallow and the hydroalcoholic extract contain anti-inflammatory principles that can be used directly or introduced into various application vehicles on inflamed areas. These application vehicles can be ointments, ointments, solutions, sprays, etc.
    Alternatively, the composition of the invention can be prepared using 5dodecanolide synthesized or purified from sources other than pork tallow or a mixture thereof with one or more of the following compounds: octadecenamide, resolvin D1, hexadecanoic acid, 9-octadecenoic acid, acid 6-octadecenoic acid, 9,12octadecadienoic acid, octadecanoic acid, 5,8,11, 14-eicosatetraenoic acid, 7hexadecenoic acid, 11,14-octadecadienoic acid, hexadecanamide.
    A final object of the present invention is represented by the use of the composition of the invention.
    In general, the composition of the invention has its application in the treatment of inflammation.
    More specifically, the composition of the invention can be used for the treatment of inflammation associated with joint diseases such as arthritis or osteoarthritis. So also and, since it has been proven effective in reducing the inflammatory process initiated by fragments of the cell walls of microorganisms such as e. Eoli can also be used for the treatment of inflammation associated with infectious diseases such as that which occurs in mastitis that obstructs a galactophore duct caused by bacterial infections with the ability to make biofilms during breastfeeding. It can also reduce the inflammation associated with acne that is also a consequence of bacterial activity.
    In any case, the composition of the invention is preferably used for the treatment of inflammation topically, either in the form of an ointment, ointment, gel, cream, lotion, suspension or emulsion, and therefore, the composition of the invention. It becomes especially useful in the treatment of superficial level inflammations such as trauma, sprains, bruises or tendinitis.
    EXAMPLES
    The present invention is best illustrated by the following examples, which are not intended to limit its scope. Thus, for example, the specific concentrations and the nature of the ingredients and additives described in the examples can be extended to others and to other concentrations. The breed of pig used to obtain the product as well as its fattening process may vary and are not limiting of the invention. The machinery and instruments used in the examples may vary and are not limiting of the invention.
    EXAMPLE 1
    In this example, an embodiment of how both the treated pork tallow and the hydroalcoholic extract are obtained from this extract is illustrated.
    First, the cutting of the pig was carried out.
    Next, the butter of the bacon and adipose tissue of the region of the omentum was obtained. The lean part of the white bacon is separated and the fat attached to the omentum.
    The lard formed by the white bacon and the fat of the omentum were crushed in a meat crusher with a large hole chopped disc (diameter of about 2 cm).
    Once crushed, the bacon and the fat of the omentum were mixed until obtaining a homogeneous mass, although not too crushed. Approximately 20 kg of mass per animal were obtained.
    The crushed butter was then cooked in a stainless steel boiler for cooking over high heat. The grease was constantly removed throughout the cooking process with a wooden spoon, to prevent the fat from sticking to the bottom of the boiler and burning. During the cooking process, the fat was liquefied and subsequently boiled (60-800 C). The butter was cooked for 90 minutes at which time the pork rinds were cooked and acquired an intense golden color.
    Once the butter was cooked, it was removed from the heat and allowed to stand in the same casserole. When it cooled sufficiently, it was passed through a sieve in order to separate the pork rinds from the liquid butter. While the liquid butter was still stored in glass containers where it finished cooling and solidified. Part of this butter was then used in the rest of the examples and another part was subjected to hydroalcoholic extraction.
    To perform this extraction, we proceeded as described below. 1 g of pork tallow was melted at 50-60 ° C and subsequently 5 mL of methanol: water (65:35, v: v) and 50 ml of 1 M HCI were added. Stir vigorously with the aid of a vortex for 1 minute and then the phases were separated by centrifugation at 3,000 rpm for 5 min. The aqueous phase was passed through a C-18 column (Sep-Pak® Vac 12cc (2g '), and the column was cleaned with 10 mL of water, 6 mL of hexane and finally eluted with 8 mL of methyl formate.The eluate was dried under a stream of nitrogen at 55 ° C and a hydroalcoholic extract of the pork tallow was obtained in solid form.
    EXAMPLE 2
    In this example an experiment was developed that demonstrates the anti-inflammatory properties of ointment (pork tallow). For this, an animal model was used in which acute inflammation was induced, and after which the anti-inflammatory product was tested. Experimental procedure To carry out the experiment, they used Sprague rats weighing 350-400 grams. The rats were divided into two groups (n = 6): a first control group that underwent inflammation, and a second group in which after inflammation it was treated with anti-inflammatory ointment. The animals were anesthetized with sodium pentobarbital. The inflammation was induced by a subplantar injection of one of the hind legs with zymosan (0.1 ml in saline serum of a concentration of 10 mg / ml). Zymosan is formed by yeast cell wall fragments (Saccharomices cerevisiae) and its application generates acute inflammation, generating a model for the study of anti-inflammatory substances. The other leg acted as a negative control and 0.1 ml of saline was injected into it. Through this treatment, an apparent inflammation is obtained in the plantar region with a maximum of edema at the time of injection.
    One hour after the injection, the animals in the first group are given a 0.5 g layer of petroleum jelly that acts as a control, while the second group is given a 0.5 g layer of the ointment. Both products are applied uniformly so that a layer is left covering the entire inflamed area.
    The diameter of the legs is measured at times O (before inducing inflammation), time 1 hour (before putting the ointment or petroleum jelly), and time 2, 3 and 4 hours (see figure 1).
    The diameter of the legs is influenced by the treatment to which it is subjected. The diameter in time O is similar in all animals. One hour after the injection, a differential response is observed between the saline groups (the diameter is virtually unchanged) and the zymosan groups (where the increase in diameter due to inflammation is evident). At times 3 and 4 hours it is observed how the group treated with the ointment recovers from inflammation significantly faster than the group treated only with petroleum jelly. The ointment acts effectively reducing the external symptoms of inflammation.
    EXAMPLE 3
    This example illustrates how the application of ointment in the inflamed area reduces some of the biochemical mediators involved in the inflammatory process.
    The experimental procedure is the same as the one carried out in experiment 2. At 4 hours after generating the inflammation the animals are sacrificed by decapitation, removing the sub-plantar tissue that was weighed (see figure 2) and then processed and collected. blood samples.
    The tissue was homogenized in 50 mM phosphine buffer, pH 7.0 containing 0.5% bromide hexadecyltrimethylammonium. The homogenates were centrifuged at 8,000 9 at 4 ° C and the supernatant was collected. In the supernatant the enzymatic activities of antioxidant enzymes (catalase, and glutathione peroxidase) and myeloperoxidase activity were determined
    (enzyme that is expressed exclusively in neutrophils and is indicative of inflammation). Nitrite levels (indirect indicator of nitric oxide production) and levels of malondialdehyde (lipid damage marker) and carbonyl groups in proteins (protein damage marker) were also determined.
    The neutrophils were purified from the blood. 5 ml of blood were mixed with 5 ml of PBS pH 7.4 and introduced over 4 ml of Ficoll. Samples were centrifuged at 900 9 for 30 minutes at 18 ° C. The supernatant was removed and 10 ml of 5% dextran was added to the precipitates. The samples were allowed to stand 30 minutes at room temperature and finally centrifuged at 750 g, 10 minutes at 4 ° C to obtain a precipitate with the neutrophils. In the neutrophils the activities of antioxidant enzymes and myeloperoxidase were determined.
    Enzymatic determinations The determinations were performed on a Shimadzu UV-2100 spectrophotometer at 37'C.
    Catalase activity was determined by a method based on the decomposition of H20 2. This decomposition is monitored spectrophotometrically at 240 nm. The final concentration of H20 2 was 10 mM. The appropriate sample volume was introduced, and adjusted with 2 ml phosphate buffer. The reaction was started by adding 1 ml of H20 2. The final assay volume was 3 ml.
    The glutathione peroxidase (GPx) activity requires H20 2 as a substrate and the reaction continued to couple the enzymatic reaction catalyzed by glutathione reductase. The final concentrations of glutathione reductase, NAOPH and H20 2 were respectively 0.24 U / mi, 200¡LM and 265¡LL. The total reaction volume was adjusted to 1 ml with 200 mM phosphate buffer with 1 mM EOTA and pH 7. The absorbance was followed at 339 nm, for 3 minutes.
    Myeloperoxidase activity (MPO) monitors the oxidation of guaiac. The reaction mixture contained phosphate buffer pH 7 and 13.5 mM of guaiacol. The reaction began with the addition of H20 2 300 ... ML, monitoring changes at 470 nm.
    Determination of nitrite levels. Nitrite levels were determined as an indirect marker of nitric oxide, using a 96-well microplate calorimetric method.
    The determinations were made in erythrocytes, lymphocytes, neutrophils and in plasma. Samples were deproteinized by adding 1.5 volumes of acetone, and left overnight at -20 ° C. They were centrifuged for 10 minutes at 15,000 9 and 4 ° C, and the supernatants were collected. For the test, 100 JlI of sample was introduced into each well.
    Subsequently, 50 ~ d of a 2% weight / volume sulfanilamide solution in 5% Hel was added and then 50 JlI of a 0.1% weight / volume solution of N- (1-Naphlhyl) ethylenediamine was added in Water. The plates were shaken and read at 540 nm, after 30 minutes of incubation. The determinations were made in duplicate. For the calculations a straight pattern with sodium nitrite was made.
    Determination of MOA levels. The MOA concentration was determined by using a calorimetric kit (Calbiochem) following the manufacturer's instructions.
    Determination of the carbonyl groups. To make the determinations, the samples were precipitated with 30% trichloroacetic acid (TCA). The precipitates were resuspended with 500 µl of a 10 mM solution of 2,4-dinitrophenylhydrazine (ONPH), for 60 minutes at 37 ° C. The samples were then precipitated with 500 JlI of 20% TCA, centrifuged for 10 minutes at 1,000 g at 4 ° C, and the supernatant was discarded. The precipitate obtained was washed twice with 1 ml of ethanol-ethyl acetate (1: 1; v / v) to remove the ONPH that remained free, and centrifuged 10 minutes at 1,000 g at 4 ° C. The final precipitate was resuspended in 1 ml of 6 M guanidine in 2 mM phosphate buffer and pH 2.3. The samples were incubated for 40 minutes at 37 ° C. Finally, the samples were centrifuged for 5 minutes at 3,000 g at 4 ° C to clarify the supernatant. The concentration of carbonyl groups was determined at 360 nm, where ONPH has the maximum absorption.
    All the results obtained in the plantar tissue were corrected by the protein levels determined using a commercial kit (Biorad), while those obtained in neutrophils are expressed corrected by the ONA levels determined fluorometrically.
    The results related to the effect of ointment on zimosan-induced inflammation on the weight of the leg are shown in Figure 2.
    After 4 hours after inducing inflammation, the weight of the leg shows similar 5 values between the two groups of saline. Treatment with the ointment significantly reduces inflammation as evidenced by lower leg weight.
    Table 1 shows the results related to enzymatic activities, oxidative damage markers and nitrite levels in plantar tissue.
    10 Table 1. Enzymatic activities, markers of oxidative damage and nitrite levels in plantar tissue
    Saline Zymosan Salino Zymosan
    ointment ointment
    Enzymatic activities
    MPO nKatlmg prot. 2.79 ± 0.30 3.02 ± 0.28 21, 8 ± 2.4a 17.8 ± 3, Oa
    GPx nKatlmg prot. 0.29 ± 0.02 0.32 ± 0.04 0.38 ± 0.02 0.36 ± 0.06
    Catalase mKJmg prot. 11.3 ± 2.8 12.9 ± 1.2 9.17 ± 1.0 10.7 ± 2.2
    Oxidative damage
    MDA nmol / g prot 523 ± 79 542 ± 86 648 ± 48 591 ± 79
    Carbonyl groups umol / mg prot 15.0 ± 1.1 16.7 ± 1.7 23.9 ± 0.9a 24.2 ± 1.1a
    Nitrite umol / mg prot 5.81 ± 1.06 6.04 ± 0.54 14.1 ± 1.0 '11.6 ± 0.7b
    15 different letters indicate significant differences between treatments (p <0.05, one-way ANOVA)
    16
    The activity of the MPO is used as an indicator of inflammation to be indicative of the infiltration of neulophiles in the studied tissue, being an enzyme that is expressed exclusively in this cell type. Treatment with zymosan produces a marked increase in MPO activity. Ointment does not reduce MPO activity with respect to the group
    5 with petrolatum, possibly because chemotactic signals for neutrophils would be released at the beginning of the inflammation process, before adding the ointment.
    There are no significant differences between the different treatments in the activities of antioxidant enzymes.
    10 The existence of oxidative damage induced by zymosan is only evident in the case of carbonyl groups, without differences between the group treated with the ointment and the group treated with petroleum jelly.
    15 Nitrite is used as a marker for nitric oxide production. Nitric oxide is an important mediator in the inflammatory process. Treatment with zymosan significantly increases nitrite levels, which are reduced by the action of ointment. This reduction would participate in a faster recovery of the inflammatory process.
    twenty
    Table 2 shows the results of enzymatic activity in circulating neutrophils.
    Table 2. Enzymatic activities in circulating neutrophils
    Saline Zyrnosan
    Saline ointmentZymosanointment
    MPO
    nKatlug DNA 4.47 ± 0.823;4.74 ± O, 44a7.73 ± 1.03b4.44 ± 0.6r
    GPx
    nKatlug DNA 0.80 ± 0.060.79 ± 0.060.87 ± 0.00.64 ± 0.04
    Catalase
    Klrng DNA 0.15 ± 0.02 '0.14 ± 0.01 '0.27 ± 0.06b0.16 ± 0.03 '
    25 different letters indicate significant differences between treatments (p <O, 05, ANOVA of a factor)
    17
    The group treated with zymosan and petrolatum has significantly higher MPO and catalase activities than the group treated with zymosan and ointment. In fact, the group treated with ointment has similar activities to the two groups treated with saline. The application of ointment reduces the degree of activation of circulating neutrophils and, therefore, would reduce the risk of oxidative damage induced by the action of neutrophils themselves.
    EXAMPLE 4
    In this example an experiment is developed that demonstrates the anti-inflammatory properties of the hydroalcoholic extract of pork tallow. For this, a cellular model (PBMCs, human peripheral blood mononuclear cells and human neutrophils) was used in which an acute inflammation process was simulated by contacting the cells with a component of the bacterial walls of a lipopolysaccharide nature (LPS) as that of Escherichia coli. The establishment of the inflammatory process was tested by quantifying the production of inflammatory cytokines such as TNF-a, and after which the effect of the hydroalcoholic extract as an anti-inflammatory product was tested.
    Cells were isolated from antecubital vein blood of 8 individuals by puncturing with vacutainers with EDTA as anticoagulant. The PBMCs and neutrophil fractions were purified using a previously described procedure (Boyum 1964; Sureda et al. 2004a). Briefly, blood is introduced into Ficoll in a 1.5: 1 ratio and centrifuged at 900 x g, at 4 ° C for 30 minutes. The fraction of PBMCs is carefully extracted, the sediment, which contains neutrophils and erythrocytes, and the plasma phase are conserved, discarding the intermediate phase of Ficoll. The PBMCs fraction is washed twice with phosphate buffer (PBS), pH 7.4, and centrifuged at 1,000 g, at 4 ° C for 10 minutes, finally obtaining the sediment of PBMCs.
    The sediment containing erythrocytes and neutrophils is incubated at 4 ° C with 0.15 M ammonium chloride to hemolyze the erythrocytes. The suspension is centrifuged at 750 g, at 4 ° C for 15 minutes and the supernatant is discarded. The neutrophil pellet is washed first with 0.15 M ammonium chloride and then with PBS. The last wash with PBS, both of the PBMCS and neutrophil sediment, is performed with a volume of 10 ml. This volume is distributed equally in 5 2 mL aliquots in five tubes so that the same number of cells is in each tube. The tubes with the 2mL aliquots are centrifuged at 1,000 g at 4 ° C for 10 minutes, the supernatant is discarded and a
    precipitate of cells that are used to demonstrate the inflammatory effects of LPS and the anti-inflammatory effects of hidoralcoholic extract of pork tallow.
    PBMCs and neutrophils are resuspended in 2 mL of RPMI-1640 culture medium (Sigma-Aldrich, Spain) alone (control) or with RPMI-1640 culture medium containing the different additives for experimentation. The different cell cultures are incubated at 37 ° C for 2 hours. At the end of the incubation they are centrifuged at 1,000 g, at 4 ° C for 10 minutes, the supernatants are collected and stored at -80 ° C for the subsequent analysis of inflammation markers such as TNF- (l.
    In this experiment the following media / additives were tested 8 times with
    cells from 8 donors in a final volume of 2 mL:
    Control Culture: contains the culture medium and the cells.
    LPS Group: contains the culture medium with 1 ~ g / mL LPS as an additive and the cells
    (Iipopolysaccharides from Escherichia coli 0127: B8; Sigma-Aldrich, Spain). Extract Group: contains the culture medium with a hydroalcoholic extract of pork tallow at a concentration of 1 mg / mL and the cells.
    Extract Group + LPS: contains the culture medium with a hydroalcoholic extract of pork tallow at a concentration of 1 mg / mL and the cells to which LPS (1 g / mL) is added after 30 minutes of incubation.
    LPS + Group Extract: contains the culture medium with LPS 1 IJg / mL and the cells to which a hydroalcoholic extract of pork tallow is added at a concentration of 1 mg / mL after 30 minutes of incubation. The following compounds are determined in the supernatants of the incubation media: TNF-et., IL-6, IL-8.
    The analyzes that were performed using the following ELlSA kits: TNF-a (Diaclone, France, intra-test variation coefficient 3.3%, inter-test 9.0%) IL-6 (Diaclone, France, intra variation coefficient -assay 4.4%, inter-test 9.1%) IL-8 (RayBio, coefficient of variation intra-test 10%, inter-test 12%)
    The differences between the results were analyzed statistically by analysis of the variance of a factor and a post hoc DMS, p <0.05, using the SPSS statistical program, version 21.0.
    The results (see table 3) are expressed as the production rate of the different parameters referred to the cells that produce them (quantity / 103 cel x mUh).
    Table 3. Production rate
    PBMCs
    LPS + Control LPS Extract Extract + LPS Extract
    TNF-a
    (pg / l0 'celx mLlh) 148 ±lla390±32b74.5±26.7c106 ±8a, c133±18a, c
    IL-6
    (pg / l O 'cel x mLlh) 28.1 ±3,2a39.2±5.4b2.71±1, 19c21, 1 ±5.9a2.93±0.57c
    IL-8
    (pg / l O 'cel x mLlh) 857 ±82a1296±162b243±16c433±66c186±71c
    PGE-l
    (pg / l03 cel x mLlh) 62.6±20.5a41, 6±6, to1646±307b2007±385b2005±394b Neutrophils
    IL-6
    (10 'cel x mLlh) 5.70 ± 0.69a 9.66 ± 2.45b 4.24 ± 0.56a 3.89 ± 0.49a 4.91 ± 0.78a Different letters indicate significant differences between groups, p <O, 05.
    5
    The presence of LPS increases the production rate of TNF-a, IL-6, IL-8 by PBMCs, and the production rate of IL-6 in the case of neutrophils. It is revealed that LPS produces an inflammatory effect on PBMCs and neutrophils, probably through activation and translocation to the nucleus of NFK8 that allows the expression of inflammatory genes such as TNF-a, IL-6 and IL-8 . The addition of the pork tallow hydroalcoholic extract already reduces the production rate of TNF-a, IL-6 and IL-8 of the control PBMCs culture and greatly reduces the production rate of these inflammatory factors by activated PBMCs with LPS, showing a potent anti-inflammatory effect of the hydroalcoholic extract of pork tallow. The order in which the extract is entered
    15 hydroalcoholic pork, before or after LPS does not seem to influence the anti-inflammatory response of the hydroalcoholic extract. These results differ in this way from the anti-inflammatory mechanism that some phospholipid oxidation products have.
    natural, which only act if prior to the addition of the inflammatory stimulus they are already present in the environment.
    Likewise, the hydroalcoholic extract of pork tallow significantly increases the production of PGE-1 by PBMCs without the presence of LPS modifying the production rate. It has been indicated that PGE-1 has anti-inflammatory effects. The results obtained show that either the hydroalcoholic extract has high levels of PGE1 or a precursor of its synthesis such as the 9,12octadecadienoic fatty acid.
    EXAMPLE 5
    This example illustrates the different composition of the pork tallow and the hydroalcoholic extract of the pig bait with respect to the composition of the starting material for the preparation of the pork tallow following the procedure described in the present invention. The components with anti-inflammatory activity are present in the already treated pork tallow and in the hydroalcoholic extract, but they are not present in the starting material of the tallow or are in lower concentrations, although some precursor components of anti-inflammatory compounds are present in lower concentration in the starting material.
    To determine the composition of the pork tallow from the starting bacon, an extraction of the total lipid content was carried out with organic solvents, they were derivatized by trans-esterification / esterification to the fatty acid methyl esters, these methyl esters were separated and quantified and other products not methylated by gas chromatography. Likewise, the content of resolvin in the dried organic extract of tallow and bacon was quantified by immunological procedures. The components of the hydroalcoholic extract of pork tallow and bacon were also identified and quantified. The components of the hydroalcoholic extract were trans-esterified / esterified and separated by gas chromatography coupled to a mass spectrophotometer. The identification of the different chromatographic peaks is done by comparison with the mass spectra of pure substances and by the retention time of the chromatographic peaks. In some cases, pure product patterns were used to confirm their identification.
    The procedures followed are summarized below. The lipid content of tallow and previously crushed bacon (5 g) is extracted following a modification of the Folch method (Folch et al., 1957) by chloroform / methanol (2: 1, v: v) with 0.01% hydroxybutylanisol (BHA) as an antioxidant and 2 I-IL of n-heptadecanoic acid (15 mM) as internal standard. The resulting organic phase is evaporated under a stream of nitrogen at 55 ° C. The dried residue is dissolved in 100 IJL of hexane and, subsequently, 25 IJL of derivatization reagent (Meth-PrepTM 11) is added, allowing to react for 30 minutes at room temperature. An aliquot of 1 J..IL is injected into the gas chromatograph using helium as a mobile phase with a flow of 2.17 mUmin, measured at 150 ° C in the column head. The gas chromatograph is an Agilent model 5890 (Agilent Technologies, Santa Clara, CA, USA) with a flame ionization detector (FIO) and a SupelcowaX® 10 Capillary GC column, 30 mx 0.53 mm, df 0, 50! -1m (Supelco, Bellefonte, PA, USA). The temperature ramp starts at 150 ° C with a temperature gradient of 4 ° C / min up to 260 ° C and then an isothermal temperature maintained for 15 minutes. The injector is at 280 ° C and the FID at 300 'C.
    The dry residue of the hidoralcoholic extracts of pork tallow or pork belly obtained according to the described procedure from 10g is dissolved in 100 ~ L of derivatization reagent (Meth-Prep ™ 11) allowing to react for 30 minutes at room temperature . An aliquot of 5 IJL is injected into the gas chromatograph with helium as a mobile phase with a flow of 0.5 mUmin, measured at 150 ° C in the column head. The Agilent model 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) is coupled to an Agilent model 5975 electronic impact mass detector (Agilent Technologies, Santa Clara, CA, USA). The chromatographic column is a Supelcowax® 10 Capillary GC column, 30 m x 0.53 mm, df 0.50 ~ m (Supelco, Bellefonte, PA, USA). The temperature ramp starts at 150 ° C with a temperature gradient of 4 ° C / min up to 260 ° C and then an isothermal temperature maintained for 15 minutes. The injector is at 280 ° C.
    The determination of resolvin 01 is made of the hydroalcoholic extract of 1 g of pork or bacon tallow, by the procedure described above, using an ELlSA kit for the determination of resolvin 01 (Cayman, USA, coefficient of variation intraassay 11, 4 %), following the instructions and with the interferences described there.
    The results referring to the composition of pork tallow and pork belly, taking into account both the components determined in the organic extract and those determined in the hydroalcoholic extract and those determined with the immunological analysis are detailed below:
    Table 4. Composition of treated tallow and starting bacon
    Molecule (TR, min) C12: 0 (6,213) C14: 0 (9,826) C16: 0 (13,785) C16: 1 (14,390) C18: 0 (18,048) C18: 1n9 (18,540) C18: 2n6 (19,577) C18: 3n6 (20,254) C18: 3n3 (20,982) C20: 0 (22,306) C20: 1n9 (22,789) C20: 2 (23,826) C20: 3 (24,409) C20: 4n6 (24,942) C20: 5n3 (25,212) C22: 0 ( 26,398) C22: 1 n9 (26,897) C22: 2 (27,918) C24: 0 (30,603) C24: 1n9 (31, 244) C22: 6n3 (31,437) SFA MUFA PUFA 5-Dodecanolide Unidentified hexadecanamide Sebum ('lo) 0.937 ± 0.064 0.380 ± 0.065 10.2 ± 1.27 3.18 ± 0.09 13.9 ± 1.1 15.6 ± 2.5 3.99 ± 0.429 0.784 ± 0.158 1.67 ± 0.34 0.372 ± 0.044 2.81 ± 0.24 0.784 ± 0.185 0.155 ± 0.033 4.92 ± 2.26 0.414 ± 0.128 2.78 ± 0.79 0.634 ± 0.145 1.97 ± 0.27 4.75 ± 1.00 2.45 ± 0.46 6.80 ± 1.63 Bacon {%) 0.674 ± 0.119 0.465 ± 0.210 4.30 ± 0.09 2.70 ± 0.94 5.43 ± 1.19 8 , 84 ± 0.51 4.09 ± 0.47 0.283 ± 0.070 1.61 ± 0.08 0.204 ± 0.057 10.4 ± 3.1 0.920 ± 0.153 0.385 ± 0.071 2.18 ± 0.63 0.211 ± 0.017 6 , 38 ± 0.31 1.39 ± 0.09 0.565 ± 0.028 6.24 ± 0.31 4.40 ± 0.57 11.4 ± 0.2 33.3 ± 0.8 24.6 ± 2, 5 21, 5 ± 3.1 23.7 ± 1.3 27.7 ± 1.5 21, 7 ± 1.5 0.00543 ± 0.00018 0.00129 ± 0.00025 79.4 ± 2.5 20.6 ± 2.1 NO NO 73.04 ± 1.5 26.96 ± 1.05
    NO not detected. C20: 3 -C20: 3n6 + C20: 3n3. SFA fatty acids
    saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids
    (polyunsaturated fatty acids).
    The fatty acid profile of pork tallow and pork belly is different, implying the effects of tallow production on the fatty acid composition. It is noteworthy that the sebum production process produces new components such as 5dodecanolide, hexadecanamide and octadecenamide, as well as the release of fatty acids
    5 such as hexadecanoic acid, 9-octadecenoic acid or 6-0ctadecenoic acid, 9,12-octadecadienoic acid, octadecanoic acid, 5,8,11, 14-eicosatetraenoic acid, 7-hexadecenoic acid, 11 , 14-octadecadienoic that appear in the composition of the hydroalcoholic extract and that in the composition of tallow and pork belly are included in the respective global values of each of these fatty acids.
    10
    The composition of the hydroalcoholic extract of pork tallow is indicated in the following table
    5. In the hydroalcoholic extract of the bacon these components were not detected except for resolvin D1, which was detected in both the hydroalcoholic extract of tallow (2360 ± 234 pg / g tallow) and that of the bacon (509 ± 20 pg / g bacon).
    fifteen
    Table 5: CG-MASS HYDROALCOHOLIC EXTRACT COMPOSITION OF THE PIG SEED Formula TR Percentage Min-Max Molecule (Certainty,%) molecular (min) (%) (%)
    Hexadecanoic, methyl ester (93%)
    9-0ctadecenoic, methyl ester (99%) 6-0ctadecenoic, methyl ester (99%) 9.12-0ctadecadienoic, methyl ester
    (99%)
    Octadecanoic, methyl ester (85%) 5,8,11, 14-eicosatetraenoic, ethyl ester
    (90%)
    5-Dodecanolide (81%)
    7-hexadecenoic, methyl ester (89%) 11, 14-octadecadienoic, methyl ester
    (93%)
    C17H3402 C1gH360 2 C1gH3402 C1gH380 2 C22H3602 C12H2202 C17H3202 C1gH3402
    4,788 5,632 6,367 7,107 10,929 12,935 13,176 16,956
    0.603 ± 0.096 61.6 ± 2.2 13.3 ± 0.8 0.307 ± 0.167 5.77 ± 0.42 4.49 ± 0.15 2.08 ± 0.72 5.84 ± 0.15
    0.505 0.701 59.463.7 12.5 14.0 0.136 0.478 5.326.21 4.344.63 1.33 2.83 5.67 6.00
    Hexadecanamide (92%) C16HJJNO18,5131.07±0.210.890 1.28
    Octadecenamide (82%) C1sH35NO22,2615.02±0.503.19 6.84
    Resolvin D1 (1-19 / 9 extract) C22H320S 2.86 ± 0.28 1.93-3.02
    The components of the hydroalcoholic extract were identified by mass spectrometry, taking into account the retention time of the compound. The table shows the methylated derivatives of the hydroalcoholic extract compounds, which are those that are separated by gas chromatography. Likewise, there are compounds present that have not been methylated and that are separated in their native form from the hydroalcoholic extract. In brackets, the percentage of similarity of the mass spectrum of the chromatographic peak corresponding to each retention time with that of the pure reference compound is indicated. In the case of 5-dodecanolide and oleamide, standard compounds were used to check their elution at the same retention time and their mass spectrum. The results of the mass spectra are shown below: the chromatographic peaks at the retention times of 12,935 minutes and 22,261 minutes separated from the hydroalcoholic extract have a mass spectrum that includes the mass spectra corresponding to 5-dodecanolide and oleamide ; in turn, these products
    15 cigars have the same retention times.
    Some of these components of pork tallow hydroalcoholic extracts have anti-inflammatory activity.
    20 EXAMPLE 6
    In this example an experiment is developed that demonstrates the anti-inflammatory properties of some of the components of the hydroalcoholic extract of pork tallow. Pure components of the hydroalcoholic extract of pork tallow 25 obtained from commercial chemical houses were used. A cellular model (human neutrophils) was used in which an acute inflammation process was simulated by contacting the cells with a component of the bacterial walls of a lipopolysaccharide nature (LPS) such as Eseherichia eoli. The establishment of the inflammatory process was tested by quantifying the release of catalase and myeloperoxidase into the culture medium as indicators of the degranulation of neutrophils activated by LPS. The effect of 5-dodecanolide, resolvin D1, octadecenamide and the hydroalcoholic extract of pork tallow was tested. The pure products come from resolvin D1, oleamide and 5-dodecanolide come from Quimigen S.L., Spain. Neulophiles were obtained from venous blood from six donors, following the protocol described in Example 4. The hydroalcoholic extract of pork tallow was obtained following the procedure described in this patent. The enzymatic activities of cata lasa and myeloperoxidase were determined following the procedures described in example 2 in the centrifugal supernatants of the neutrophil cultures incubated according to the conditions described in example 3. The concentrations of the different components of the hydroalcoholic extract tested correspond to those provided by the hydroalcoholic extract in the middle of
    10 crop
    Each participant's neutrophils were distributed among the following types of crops: Control Group: RPMI-1640 culture medium (Sigma-Aldrich, Spain). LPS Group: culture medium with Escherichia coli LPS 0127: 88 (Sigma-Aldrich,
    15 Spain) at a final concentration of 1 IJg / mL. LPS group with resolvin: culture medium with LPS (1 IJg / mL) and Resolvine D1 (1 ng / mL). LPS group with oleamide: culture medium with LPS (1 IJg / mL) and Olea mida (0.06
    mg / mL)
    20 LPS Group with 5-dodecanolide: culture medium with LPS (1 IJg / mL) and 5dodecanolide (0.04 mg / mL). LPS Group with Hydroalcoholic Extract: culture medium with LPS 1 IJg / mL and hydroalcoholic extract (1 mg / mL).
    5
    10
    fifteen
    twenty
    ES 2 623761 Al
    Table 6: Cata lasa activity (CAT) and myeloperoxidase (MPO) in the supernatants of the
    incubations of neutrophils in the presence of various components of the pork tallow hydroalcoholic extract
    CATCAT MPOMPO
    (K / 10 ' (%)(nKat / l0 '(%)
    neutrophils)neutrophils)
    Control 53.7 ± 7.2a, e100 ± 13.3 a, e69.1 ± 39.8 a100 ± 57.6a
    LPS 116 ± 19 b215 ± 35.7b177 ± 62.0 b197 ± 86.3b
    LPS + Resolvina 75.2 ± 2.2 b, e139 ± 4.04 b, e7.38 ± 2.03 e10.7 ± 2.93e
    LPS + Olea measure 34.3 ± 13.8 a, e63.7 ± 25.7a, c10.9 ± 3.01 e15.8 ± 4.35c
    LPS + 5-Dodecanolide 51.3 ± 14.2 a, e95.5 ± 26.511.7 ± 1.95 e16.9 ± 2.81c
    a, e
    LPS + tallow extract 20.8 ± 7.4 a38.7 ± 13.8a8.67 ± 1.92 e12.5 ± 2.77c
    Statistical analysis: ANOVA of a factor, Qosthoc DMS, p <O, OS
    The presence of LPS induces an inflammatory response in neutrophils, leading to degranulation and a significant increase in catalase and myeloperoxidase activity in the extracellular environment. Hydroalcoholic extract of pork tallow significantly reduces the inflammatory process induced by LPS, to values even lower than the control itself without activating with LPS. The addition of resolvine D1, 5-dodecanolide and oleamide reduces the extracellular catalase activity and extracellular myeloperoxidase activity at the control level of the sebum hydroalcoholic extract of LPS-activated neutrophil cultures.
    EXAMPLE 7
    In this example an experiment is developed that demonstrates the anti-inflammatory properties of some of the components of the hydroalcoholic extract of pork tallow. One of the components of the hydroalcoholic extract of pork tallow, 5-dodecanolide from Quimigen S. L., Spain, is used. A cellular model (human neutrophils) was used in which an acute inflammation process was simulated by contacting the cells with a component of the bacterial walls of a lipopolysaccharide nature (LPS) such as Eseheriehia eoli. The establishment of the inflammatory process was tested by quantifying the production of the pro-inflammatory cytokine, the tumor necrotic factor alpha (TNF-a, and after which the effect of 5-dodecanolide, one of the components of the hydroalcoholic extract, was tested of pork tallow, at different concentrations.
    27 Neutrophils were obtained from venous blood from nine donors, following the
    protocol described in example 4. TNF-a levels in supernatants are determined
    of centrifugation of neutrophil cultures incubated according to the procedures and
    conditions described in example 3.
    Table 7: TNF-a levels in supernatants of neutrophil incubations in the presence of LPS and different concentrations of 5-dodecanolide Control
    LPS
    LPS + 5-dodecanolide (0.1 mg / mL) LPS + 5-dodecanolide (0.06 mg / mL) LPS + 5-dodecanolide (0.01 mg / mL)
    5-dodecanolide (0.1 mg / mL) 5-dodecanolide (0.06 mg / m L) 5-dodecanolide (0.01 mg / m L)
    TNF-a
    (pg / 10 'neutrophils'mL / h) 15.4 ± 3.05 a, e
    79.1 ± 11.0 b
    9.15 ± 1.53 a 13.5 ± 2.15 a, e
    25.9 ± 3.78 and 7.08 ± 0.73 to 6.87 ± 0.75 to 8.2 ± 1.60 a Statistical analysis: ANOVA 1 factor, gosthoc DMS, p <O, OS LPS triggers an inflammatory response by significantly activating the synthesis of
    TNF-Q that increases about five times. 5-Dodecanolide does not alter TNF production
    to control; however, it totally eliminates the pro-inflammatory effect of LPS in a manner dependent on the concentration of 5-Dodecanolide. The anti-Hamatory effect of 5 dodecanolide, measured as the lowest capacity of TNF-a production by
    LPS activated neutrophils, is about 3 times higher if the concentration of 5-dodecanolide
    is O, 1mg / mL than if it is O, 01mg / mL, although all the concentrations tested
    they reduce at a control level the rate of TNF-a production by the neutrophils activated with LPS.
    权利要求:
    Claims (15)
    [1]
    one. Pharmaceutical composition comprising 5-dodecanolide as active ingredient and one or more pharmaceutically acceptable excipients or vehicles.
    [2]
    2. Composition according to claim 1 which additionally comprises one
    or more of the compounds selected from octadecenamide, resolvin D1, hexadecanoic acid, 9-octadecenoic acid, 6-octadecenoic acid, 9,12octadecadienoic acid, octadecanoic acid, 5,8,11, 14-eicosatetraenoic acid, 7hexadecenoic acid, 11, 14-octadecadienoic, hexadecanamide.
    [3]
    3. Composition according to any of the preceding claims wherein 5-dodecanolide is in a percentage of between 2-6% of the total weight of the composition.
    [4]
    Four. Composition according to any of the preceding claims comprising in percentage by weight with respect to the total composition:
    a) 4.34-4.63% of 5-dodecanolide b) 0.05-0.7% of hexadecanoic acid c) 59.4-63.7% of 9-octadecenoic acid or 6-octadecenoic acid d) 12 , 5-14% of 9,12-octadecadienoic acid e) 0.13-0.47% of octadecanoic acid f) 5.32-6.21% of 5,8,11,14-eicosatetraenoic acid g) 1, 33-2.83% of 7-hexadecenoic acid h) 5.67-6.00% of 11, 14-octadecadienoic acid i) 0.89-1, 28% of hexadecanamide j) 3.19-6.84% of octadecenamide k) 1.93-3.02% resolvin D1.
    [5]
    5. Composition according to any of the preceding claims as an ointment for topical application.
    [6]
    6. Process for obtaining a composition according to any of the preceding claims comprising:
    a) Extraction of lard 29
    ES 2 623761 Al
    b) Crushing of the butter e) Cooking of the crushed butter d) Separation by sieving of the liquid lard from the chicharrones e) Optionally, hydroalcoholic extraction of the liquid butter
    [7]
    7. Method according to claim 6 wherein the butter is extracted from the bacon and the adipose tissue of the omentum.
    [8]
    8. Process according to any of claims 6 or 7 wherein the grinding is done by mechanical means until a homogeneous mass is reached.
    [9]
    9. Method according to any one of claims 6 to 8 wherein cooking involves boiling the fat in the butter for a time of between 50 to 90 minutes.
    [10]
    10. Process according to any of claims 6 to 9 wherein the hydroalcoholic extraction is carried out in an alcohol: water 65:35 (v: v) solution, preferably methanol: water and optionally in the presence of an acid, preferably HCI
    [11 ]
    eleven . Composition according to any one of claims 1 to 5 for use in the treatment of inflammation.
    [12]
    12. Composition for use according to claim 11 wherein the inflammation treated is inflammation associated with joint diseases such as arthritis or osteoarthritis.
    [13]
    13. Composition for use according to claim 11 wherein the inflammation treated is inflammation associated with infectious diseases such as mastitis or acne.
    [14]
    14. Composition for use according to claim 11 wherein the treatment of inflammation is done topically.
    [15]
    fifteen. Composition for use according to claim 13 wherein the topical treatment allows to treat superficial level inflammations such as trauma, sprains, bruises, tendonitis, mastitis or acne.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP3172177B2|2001-06-04|Anti-flame mixtures derived from emu oil
    EP0304603B1|1992-10-21|Polyunsaturated acids having vasokinetic action and pharmaceutical and cosmetic formulations containing them
    KR970010542B1|1997-06-28|Fatty acid composition
    US5767095A|1998-06-16|Anti-inflammatory compositions containing monogalactosyl dieicosapentaenoyl glycerol and methods relating thereto
    Okoli et al.2004|Mechanisms of the anti-inflammatory activity of the leaf extracts of Culcasia scandens P. Beauv |
    JP2009108104A|2009-05-21|Test method of activity of substance which may be effective in inhibiting enzyme activity of phospholipase a2
    Yeşilada et al.1997|Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1α, interleukin-1β and tumor necrosis factor α
    EP1758603A1|2007-03-07|Treatment for asthma and arthritis and other inflammatory diseases
    RO117590B1|2002-05-30|Composition for treating a degeneration, a collagenic or inflammatory disease
    US20100178329A1|2010-07-15|Liposome composition
    ES2309021T3|2008-12-16|FOSFATIDIL-L-SERINA AND PHOSFATIDIC ACID TO TREAT DEPRESSION AND RELIEF STRESS.
    Veerappan et al.2019|Chrysin pretreatment improves angiotensin system, cGMP concentration in L-NAME induced hypertensive rats
    KR20020089275A|2002-11-29|Phamaceutical use for treating allergic rhinitis and atopic dermatitis comprising as main ingredients COCICIS SEMEN, MORI COTEX RADICIS, HOUTTUYNIA CORDATA, PLATYCODI RADIX, REHMANNIAE RADIX, LONICERAE FLOS pharmaceutical preparations containing them
    ES2623761B1|2018-01-19|Pharmaceutical composition comprising 5-dodecanolide, its preparation and use
    Perez Gonzalez et al.2013|Anti-inflammatory activity and composition of Senecio salignus Kunth
    JP2002335996A|2002-11-26|Testing method for substance having latent activity in lipolysis field and its application mainly to cosmetic usage
    US5260067A|1993-11-09|Cytotropic heterogeneous molecular lipids | and process for preparing the same
    US20050037028A1|2005-02-17|Skin perparation for external use containing purpuricenus temminckii frass as the active ingredient
    KR20020070062A|2002-09-05|Agastache rugosa Extract for anti-inflammatory activity and anti-atherogenic activity
    Botha et al.2005|Effect of Boophone disticha on human neutrophils
    Hu et al.2020|A peptide inhibitor of macrophage migration in atherosclerosis purified from the leech Whitmania pigra
    Xin et al.2015|Antipyretic and anti-inflammatory activities of Thais luteostoma extracts and underlying mechanisms
    Lima et al.2002|Walker 256 tumour growth causes marked changes of glutamine metabolism in rat small intestine
    JP2019508485A|2019-03-28|Novel molecules having anticancer activity
    Ramya et al.2017|Effect of anti-inflammatory activity of Hellenia speciose | and Costus pictus |
    同族专利:
    公开号 | 公开日
    WO2017121916A1|2017-07-20|
    ES2623761B1|2018-01-19|
    EP3404054A1|2018-11-21|
    EP3404054A4|2019-11-13|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5128261A|1989-09-28|1992-07-07|Pfw B.V.|Natural delta-lactones and process of the production thereof|
    US5527693A|1992-06-22|1996-06-18|Pernod Ricard|Process for the preparation of saturated delta-decanolide or delta-dodecanolide by biohydrogenation|
    WO1994009646A1|1992-10-30|1994-05-11|Unilever N.V.|Flavoured low fat food products|
    ES2087036A1|1994-10-27|1996-07-01|Aguerri Ignacio Caballero|Fast-acting ointment against varices.|
    WO2009077635A2|2007-12-19|2009-06-25|Menargues Cerezo, Marcelino|Anti-inflammatory, anti-edematous and anti-erythematous composition, method for obtaining same and use thereof|
    US20140377370A1|2013-06-25|2014-12-25|Alan Zurak|Joint pain treatment composition and method of use|
    DE19826499A1|1998-06-13|1999-12-16|Beiersdorf Ag|Use of lactone, thiolactone and/or dithiolactone derivatives|
    EP2008691A1|2007-06-29|2008-12-31|Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.|Vaginal odorants|
    KR101222105B1|2010-05-12|2013-01-15|조선대학교산학협력단|Antifungal compound and use thereof|WO2016205193A1|2015-06-15|2016-12-22|Rush Unversity Medical Center|BRAIN DERIVED PPARα LIGANDS|
    法律状态:
    2018-01-19| FG2A| Definitive protection|Ref document number: 2623761 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180119 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201630015A|ES2623761B1|2016-01-12|2016-01-12|Pharmaceutical composition comprising 5-dodecanolide, its preparation and use|ES201630015A| ES2623761B1|2016-01-12|2016-01-12|Pharmaceutical composition comprising 5-dodecanolide, its preparation and use|
    EP17738236.3A| EP3404054A4|2016-01-12|2017-01-12|Pharmaceutical composition comprising 5-dodecanolide, preparation thereof and use of same|
    PCT/ES2017/070017| WO2017121916A1|2016-01-12|2017-01-12|Pharmaceutical composition comprising 5-dodecanolide, preparation thereof and use of same|
    [返回顶部]