Formulation comprising extract of Crynodendron patagua and essential oil of Satureja montana for the

专利摘要:
Formulation comprising Crynodendron patagua extract and Satureja montana essential oil for the prevention and treatment of diseases caused by Pisciricketsia salmonis in individuals of the genus Salmo. This invention relates to a formulation comprising a mixture of a plant extract of the plant variety Crynodendron patagua and essential oil of the plant variety Satureja montana and also to its use, either directly or through a composition comprising it., in the prevention and treatment of diseases caused by the bacterium Pisciricketsia salmonis, in individuals of the genus psalm, and more preferably in trout and salmon. (Machine-translation by Google Translate, not legally binding)
公开号:ES2769918A1
申请号:ES201931162
申请日:2019-12-27
公开日:2020-06-29
发明作者:Navarro Gustavo Emilio Zuniga；Soto Ruben Rodrigo Vidal；Coloma Azuzena Gonzalez；Yeves María Fe Andres；Hernandez Carmen Elisa Diaz；Alquezar Jesus Burrillo；Rocha Juliana Navarro
申请人:Universidad de Santiago de Chile；Centro De Investig Y Tecnologia Agroalimentaria De Aragon Cita；Consejo Superior de Investigaciones Cientificas CSIC；
IPC主号:

专利说明:

[0002] Formulation comprising extract of Crynodendron patagua and essential oil of Satureja montana for the prevention and treatment of diseases caused by Pisciricketsia salmonis in individuals of the genus Salmo
[0004] The present invention is included in the aquaculture sector and refers to a formulation comprising a mixture of a plant extract of the plant variety Crynodendron patagua and essential oil of the plant variety Satureja montana, which is used in the prevention and treatment of diseases caused by the bacterium Pisciricketsia salmonis in individuals of the genus Salmo, and more preferably, in trout and salmon.
[0006] BACKGROUND OF THE INVENTION
[0008] Aquaculture remains the fastest growing animal food producing sector, accounting for around 46% of the total fish supply for food to meet the protein needs of the world's growing population. There has been a general increase on all continents in the share of aquaculture in total fish production. China is reported to contribute more than 60% of global aquaculture production and also to administer large amounts of antibiotics to ensure adequate productivity and the management of diseases caused by bacterial microorganisms.
[0010] Antimicrobial regimens are used prophylactically and therapeutically to control these diseases, as well as to enhance growth promotion. In aquaculture, antibiotics are generally administered in surface-coated or pelletized foods or by other routes, such as immersion in water or injection. Global use of antimicrobials is increasing dramatically, estimated at 63,151 tonnes in 2010, and is projected to increase by 67% in 2030. Antibiotics have not always been used responsibly in aquaculture, and control over their use has not has provided an adequate guarantee of risk prevention for humans. Responsible use of antibiotics requires clear instructions from drug manufacturers, proper handling and distribution by distributors, and veterinary supervision of management by aquaculturists, respecting withdrawal periods before slaughter. The unregulated use of antibiotics in the healthcare industry Aquaculture could pose health and food security problems that are largely unaddressed in most developing countries of the world. The presence of antibiotic residues in aquaculture products could result in the development of bacterial resistance and toxicity for consumers that can lead to morbidity and / or death.
[0012] As an example, note that sixteen types of antibiotics are used in Chile (world power in the aquaculture field), compared to three in the United States (USA) and four in Norway. Returning to Chile, antibiotics were mainly used in the cultivation of Atlantic salmon in seawater, which represented 80% of the total use of antibiotics used for 2015, followed by 11% for coho salmon, 9% for bow trout iris and 0% for Chinook salmon. The consumption of antibiotics in the salmon industry in Chile has increased 56% from 2005 to 2015, with an increase in production in those years of 23.48%. The highest consumption was recorded in 2014, with a total use of 563.2 tons of antibiotics with 955,179 tons of salmonids produced. In 2016, there was a 30.66% decrease in the use of antibiotics compared to 2015, using a total of 382.5 tons of antibiotics to produce 727,812 tons of fish. The most widely used antibiotics in the salmon industry in Chile are florfenicol and oxytetracycline. The use of florfenicol has increased steadily since 2013 and represented 87% and 82.50% of the total antibiotics used in 2015 and 2016, respectively.
[0014] In addition to the use of antibiotics for the treatment of the most common pathologies or diseases in aquaculture, there are other commonly used alternatives, such as the application of monovalent, polyvalent and autovaccine vaccines and the application of immunostimulants that are also used as a prophylactic measure seeking to stimulate mechanisms of innate immunity.
[0016] Especially relevant is the pathogenic bacterium Pisciríckettsia salmonis, identified in the late 1980s, and which is the cause of septicemia due to Rickettsial salmonids (SRS), also known as piscirickettsiosis or rickettsial salmonidea septicemia, which is a fatal disease in salmonids. Strategies for the use of antibiotics and vaccines have been mainly tested against this disease, although, however, the intracellular nature of this bacterium, the existence of different strains of P. salmonis and the low immunological memory in salmonids, makes treatments are not effective enough resulting in the deaths of millions of aquaculture salmonids every year. As an example, since the initial isolation, P. salmonis has been the main cause of industry mortality in Chile. During 1995, more than 10 million salmon died during marine farming operations. Most of the losses were attributed to P. salmonis, with an estimated economic impact of $ 49 million.
[0018] Furthermore, the use of natural products with therapeutic properties is as old as human civilization and, for a long time, the main sources of drugs were mineral, vegetable and animal products. About 25% of prescription drugs worldwide come from plants, and 121 of these active compounds are currently used. Of the 252 drugs considered basic and essential by the World Health Organization (WHO), 11% are exclusively of plant origin and a significant number are synthetic drugs obtained from natural precursors.
[0020] In direct relation to the treatment of SRS in salmonids, it is possible to find a patent document that protects the use of active ingredients of plant origin, see for example:
[0022] - WO2011 / 119047A1 which claims the use of a composition comprising a propyl-methyl-phenol compound which is preferably selected from thymol and carvacrol, or a mixture of both, for use in the treatment and / or in the prevention of SRS and diseases caused by P. salmonis, but also for use in the treatment and / or prevention of diseases caused by the salmon anemia virus, including infectious salmon anemia; or
[0024] - CL20180000674 that discloses natural antibacterial extracts from Crynodendron patagua and Anstotelia chilensis plants or mixtures thereof, which are used as a food formulation for fish and used in the treatment or prevention of bacterial infections, especially infection by Piscirickettsia salmonis , responsible for Rickettsial salmon sepsis (SRS).
[0026] Based on the foregoing background, the development of environmentally friendly biotechnological alternatives is necessary to efficiently combat bacterial infections caused by P. salmonis and specifically SRS, and reduce the use of antibiotics.
[0027] DESCRIPTION OF THE INVENTION
[0029] The present invention is based on the observation that a formulation comprising a combination of compounds of plant origin, specifically C. patagua biomass plant extract and S. montana biomass essential oil , hereinafter formulation of the invention, is effective sample in inhibiting the pathogenic bacterium Piscirícketsia salmonis in in vitro tests (see Example 5), and therefore it could be applied to trout and salmon populations affected by SRS, estimating that they could reduce up to 30% mortality.
[0031] Additionally, the intake of the formulation of the invention should not affect feed conversion rates, unlike what happens when using other conventional treatments such as antibiotics.
[0033] In the present invention, "food conversion rate or TCA” is understood to be the relationship between the quantity of food supplied and the growth of the individual, or in other words, the weight produced per kg of food supplied. The supply of antibiotics, in addition to its lethal effect on the pathogenic bacteria affects the subject's intestinal flora, in this case a fish, impacting the general state of health and therefore, reversing lower rates of food conversion.
[0035] Preferably, the formulation of the invention uses plant materials and controlled procedures that allow the production of biomass in a standardized way and that ensures stable levels of phytochemicals in the extracts.
[0037] In a first aspect, the invention relates to a formulation comprising an ethanolic extract of biomass from Crynodendron patagua and essential oil from biomass from Satureja montana .
[0039] In a particular embodiment, the formulation of the invention consists of an ethanolic extract of biomass from Crynodendron patagua and essential biomass oil from Satureja montana.
[0041] In the present invention, "ethanolic extract" is understood to be the product obtained after exposing biomass to an alcohol-based organic solvent, which is preferably ethanol and is characterized by having a significant content in compounds phenolic. The ethanolic extract of the invention can be presented in a liquid state but also in a solid state, for example using lyophilization techniques.
[0043] Other alcoholic-based organic solvents having similar extraction capacity for phenolic compounds could be used within the scope of the invention.
[0045] The ethanolic extract used in the invention uses the biomass variety Crinodendron patagua, or patagua or patahua, which is a perennial tree species of the elaeocarpaceas family of the genus Crinodendron, and which is endemic to Chile.
[0047] Preferably, the C. patagua ethanolic extract is characterized by comprising phenolic compounds that are selected from quercetin, gallic acid and chlorogenic acid.
[0049] By "essential oil" is understood the product obtained after the hydro-distillation of biomass and that has a significant content of terpenic compounds.
[0051] The essential oil used in the invention comes from biomass of the species Satureja montana, which is a phanerogamic perennial botanical species in the Lamiaceae family, native to temperate and warm regions of southern Europe.
[0053] Preferably, the essential oil of S. Montana is characterized by comprising terpenic compounds that are selected from carvacrol, p-cymene, and -terpinene.
[0055] In a particular embodiment, the formulation of the invention is characterized by comprising chlorogenic acid and carvacrol.
[0056] In another particular embodiment, the formulation of the invention comprises quercetin, gallic acid, chlorogenic acid, carvacrol, p-cymene and y-terpinene.
[0058] Preferably, the Crynodendron patagua ethanolic biomass extract and Satureja montana biomass essential oil are present in the formulation of the invention in a 1: 1 ratio (by weight or by volume), although they can also be present in a ratio from 1: 2 to 2: 1 (by weight or by volume) or even in a ratio from 1: 3 to 3: 1 (by weight or by volume).
[0059] As demonstrated in the examples, it is the combination of the extract of C. patagua and the essential oil of S. montana, and therefore of the compounds they comprise, that cause an unexpected synergistic effect that exceeds the mere additive effect of each of the components separately.
[0061] In a second aspect, the invention relates to a process for the production of the formulation of the invention, comprising:
[0062] a) starting from biomass of C. patagua to obtain an ethanolic extract,
[0063] b) starting from biomass of S. montana to obtain an essential oil by hydro-distillation,
[0064] c) combining the ethanolic extract of biomass from C. patagua with the essential oil from biomass S. montana in a ratio ranging from 1: 3 to 3: 1 (by weight or by volume).
[0066] In a particular embodiment, the biomass of C. patagua consists of shoots obtained by in vitro multiplication starting from C. patagua buds .
[0068] In another particular embodiment, the biomass of S. montana comes from the Martin variety, with application number A201804150 before the Community Plant Variety Office (CPVO) Registry of Community Protection of Plant Varieties on December 19, 2018.
[0070] In a particular embodiment, the ethanolic extract and essential oil are combined in a 1: 1 ratio by weight or by volume.
[0072] In a third aspect, the invention relates to a composition comprising the formulation of the invention, hereinafter composition of the invention.
[0074] In a fourth aspect, the invention relates to a veterinary pharmaceutical composition comprising the formulation of the invention.
[0076] The veterinary pharmaceutical composition is any substance or combination of substances that is presented as possessing curative or preventive properties with respect to animal diseases or that can be administered to an individual in order to restore, correct or modify their physiological functions exerting a pharmacological action. , immunological or metabolic, or to establish a veterinary diagnosis. I also know "Veterinary feed premixes" prepared to be incorporated into a feed shall be considered veterinary pharmaceutical compositions.
[0078] In a particular embodiment, the veterinary pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier and / or excipient.
[0080] The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, to allow a better dosage and administration or to give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance that is used in the medicine to dilute any of the components of the veterinary pharmaceutical composition of the present invention to a determined volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and shape to the medicine. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.
[0082] The term "excipient" refers to a substance that aids in the absorption of any of the components of the composition of the present invention, stabilizes said components, or aids in the preparation of the veterinary pharmaceutical composition in the sense of giving it consistency. Thus, the excipients could have the function of keeping the components together such as starches, sugars or cellulose, dye function, protection function of the medicine such as to isolate it from air and / or humidity, filling function of a tablet, capsule, pellet, granule or any other form of presentation such as, for example, dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that matter that, included in the galenical forms, is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physico-chemical properties of the pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, to be compatible with said components.
[0084] Furthermore, as the person skilled in the art understands, the excipient and the vehicle must be pharmacologically acceptable, that is, that the excipient and the vehicle are allowed and evaluated so as not to cause harm to the organisms to which it is administered.
[0085] The veterinary pharmaceutical composition can be presented under any suitable administration form and in a therapeutically effective amount, preferably in a form adapted for oral administration. The veterinary pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, pellet, granule, solution, injection, gel or microsphere. The form adapted for oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, emulsion, extemporaneous suspension, drinkable vial, tablet, capsule, powder, granule, seal, pill, tablet, pill, troche or lyophilisate.
[0087] In the present invention, the term "therapeutically effective amount" refers to that amount of the component of the pharmaceutical veterinary composition that when administered to a fish is sufficient to produce prevention and / or treatment, as defined below, of a disease or pathological condition of interest in fish. The therapeutically effective amount will vary, for example, according to metabolic stability and duration of action of the formulation of the invention; age, body weight, general health, the sex and diet of the fish; the mode and time of administration; the rate of excretion, the drug combination; the severity of the particular disorder or disease condition; and the subject undergoing therapy.
[0089] By "medicated feed" is meant a combination of the formulation of the invention, which may comprise excipients and / or vehicles, and a conventional feed for administration to a fish.
[0091] The "conventional feed for administration to a fish" will generally be present in the form of pellets or granules and may include, by way of example, fish meat, fish oil, vegetable proteins, monosaccharides or polysaccharides, such as mannans, glucans or alginates and other excipients such as pigments, vitamins, minerals, binders and the like.
[0093] In a particular embodiment, the veterinary pharmaceutical composition is presented in a form adapted for oral administration. The form adapted for oral administration refers to a physical state that can allow oral administration. Said form adapted for oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granule, pellet, seal, pill, tablet, pill , troche or lyophilized.
[0094] In another particular embodiment, the veterinary pharmaceutical composition is presented in a form adapted to the application by means of a medicinal bath, in which the fish is placed in a container that comprises it, and maintained for a period of time (from minutes to several hours). ).
[0096] In another particular embodiment, the veterinary pharmaceutical composition is presented in a form adapted for parenteral application, for example by injection. The injectable formulations can also be in the form of a powder, pellet or granules that can be reconstituted in a suitable solvent before use, or also as solutions prepared for use.
[0098] In another particular embodiment, the veterinary pharmaceutical composition is presented in a form adapted to its combination with conventional fish feed.
[0100] In a particular embodiment the medicated feed is specially adapted for oral administration in liquid, granule or pellet form, so that the conventional feed is capable of absorbing or being combined with the formulation of the invention.
[0102] The formulation of the invention can be incorporated into the feed before pelletizing. Alternatively, the pellets or granules can be coated with the formulation of the invention. For example, commercial pellets or granules could be coated with the formulation of the invention and one or more excipients such as starch, fumed silica (Aerosil®), microcrystalline cellulose, lactose or the like.
[0104] However, the formulation of the invention can be encapsulated using for example lyophilization drying methodologies using encapsulating materials.
[0106] In a particular embodiment, the formulation of the invention is encapsulated, using materials that are selected from gum arabic (GA) or maltodextrin (MD) that can later be lyophilized and provided with the most convenient dimensions.
[0108] Additionally, the medicated feed or encapsulated material may include a preserving agent.
[0110] The amount of formulation in the medicated feed, preferably will not exceed 20% by weight of the total weight of the product.
[0111] As an alternative to the veterinary pharmaceutical composition, the composition of the invention can also be a nutritional composition.
[0113] The term "nutritional composition" of the present invention refers to that food that, independently of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so as to provide a better state of health and well-being. As a consequence, said nutritional composition can be used for the prevention and / or treatment of a disease or the causative factor of a disease. Therefore, the term "nutritional composition" of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food.
[0115] In a fifth aspect, the invention relates to a nutritional composition comprising the formulation of the invention.
[0117] In a particular embodiment, the nutritional composition is a food, a supplement, or a nutraceutical.
[0119] The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement"; or "food supplement" is a "food ingredient" intended to supplement food. Some examples of dietary supplements include, but are not limited to, vitamins, minerals, botanicals, amino acids, and food components such as enzymes and glandular extracts. They are not presented as substitutes for a conventional food or as a single component of a meal or diet but rather as a supplement to the diet.
[0121] The term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in dosages that have a beneficial effect on health.
[0123] In a particular embodiment, the feed is "conventional feed for administration to a fish" as defined above.
[0125] In a sixth aspect, the invention relates to the veterinary pharmaceutical composition for use as a veterinary drug.
[0126] In a seventh aspect, the invention relates to the veterinary pharmaceutical composition for use as a veterinary drug in the treatment and / or prevention of salmonid rickettsial sepsis (SRS) in individuals of the psalm genus.
[0128] The use of the veterinary medicine as an adjuvant to enhance the use of another medicine is also included within the scope of this invention.
[0130] In an eighth aspect, the invention relates to the veterinary pharmaceutical composition for use as an adjunct to other drugs in the treatment and / or prevention of salmonid rickettsial septicemia (SRS) in individuals of the genus Salmo.
[0132] In a particular embodiment, the formulation of the invention is used as an adjuvant to an antibiotic that is selected from among florfenicol, oxytectracicline, flumequine, oxolinic acid, erythromycin, amoxicillin.
[0134] In the present invention, the term "treatment" refers to combating the effects caused as a consequence of a disease or pathological condition of interest in a subject, preferably a fish, including:
[0135] (i) inhibit the disease or pathological condition, that is, stop its development;
[0136] (ii) alleviating the disease or the pathological condition, that is, causing the regression of the disease or the pathological condition or its symptoms;
[0137] (iii) stabilize the disease or pathological condition.
[0139] In the present invention, the term "prevention" refers to preventing the occurrence of the disease, that is, preventing the disease or disease condition from occurring in a subject, preferably a fish.
[0141] Within the scope of the present invention, individuals of the genus Salmo are selected from trout and salmon, and preferably from Atlantic salmon, rainbow trout ( Oncorhynchuss mykiss), coho salman salmon ( Oncorhynchus kisutch) and salvelino ( Salvelinus alpinus).
[0143] In the present invention, Piscirickettsia salmonis is understood as the bacterial agent that causes piscirickettsiosis or salmonid rickettsial syndrome or rickettsial salmonid septicemia (SRS) and which is one of the main pathogens, responsible for more than 50% of mortality at sea.
[0144] The formulation of the invention and the composition of the invention can be used effectively in any of the stages of development of individuals of the genus Salmo.
[0146] In a ninth aspect, the invention relates to a disinfecting treatment to eliminate the presence of the Pisciríckettsia salmonis bacteria on surfaces or devices normally used in aquaculture facilities and which comprises applying the formulation of the invention in an effective dose to eliminate the bacteria. .
[0148] BRIEF DESCRIPTION OF THE FIGURES
[0150] Figure 1. Major phenolic compounds present in the C. patagua extract . Figure 2. Major terpenic compounds present in S. montana oil . Figure 3. Microcapsules of the mixture of C. patagua ethanolic extract: S. montana essential oil (1: 1 by weight) in gum arabic.
[0152] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
[0154] EXAMPLES
[0156] Example 1: Obtaining the plant material
[0158] C. patagua (patagua) plants were cultivated in vitro starting from buds of adult plants in a Murashige and Skoog (MS) medium supplemented with the following vitamins and hormones: biotin (1 mg / L), kinetin (0.2 mg / L), thiamine (2 mg / L), naphthalenacetic acid (0.2 mg / L) and benziladenine (0.3 mg / L). They were stored for 30 days at 23 ° C ± 2 ° C, with a photoperiod of 16 light hours and 8 hours dark. The generated shoots were removed from the culture medium to be dried at 37 ° C ± 2 ° C, until constant weight. Subsequently, the bud tissue was ground and kept in a desiccator until use.
[0159] For S. montana , aerial parts of a domesticated variety, specifically the Martin variety, were used. The plant material was dried at 37 ° C ± 2 ° C until constant weight. It is a domesticated variety that was acclimatized in 2 different geographical areas in Spain: in the province of Almería (southeast) and in Ejea de los Caballeros (Zaragoza, northeast) and for which protection has been requested as a plant variety with a number of application A201804150 with the Community Plant Variety Office (CPVO) Registry of Community Protection of Plant Varieties on December 19, 2018.
[0161] Example 2: Obtaining the ethanolic extract of C. patagua and the essential oil of S. montana
[0163] For the preparation of the C. patagua extracts, 1 gram of the dried plant material was mixed as indicated in Example 1, with 100 mL of 85% V / V ethanol. The solution was sonicated at a frequency of 50 Hz, at a temperature of 25 ± 2 ° C for two hours (Contreras, RA, Kohler, H., Pizarro, M. and Zúñiga, GE (2015). In vitro cultivars of Vaccinium corymbosum L. (Ericaceae) are a source of antioxidant phenolics. Antioxidants, 4, 281-292). The solution was sterilized through filtration, with a filter with a pore size of 0.22 pm and the extracts were stored at 4 ° C until use.
[0165] The dried plants of S. montana were extracted by hydro-distillation on a laboratory scale (Clevenger, EO) to obtain essential oils from all the samples according to the method recommended by the European Pharmacopoeia ( 6th edition, 2008), obtaining, by decantation , essential oil or using a large-scale plant (semi-industrial, PEO) steam entrainment in the pilot stainless steel distillation plant equipped with a 100 Kg distillation chamber and a 500 L beaker, using a pressure range between 0.5-1.0 bar, and obtaining, by decanting, the essential oil (PEO).
[0167] Example 3. Analysis of compounds in the ethanolic extract of C. patagua and in the essential oil of S. montana .
[0169] In order to determine the profile of phenolic compounds present in the ethanolic extract, an LC-MS kit (Agilent LC-MS / MS 6410, Agilent Technologies, Santa Clara, CA, USA) was used. The analyzes were performed by injecting a volume of 20 pL of extract, through a Reodyne valve and an elution flow of 0.3 mL / min was used, in a gradient of acetonitrile and 0.1% V / V formic acid, with a pressure 75 bar at the start of the program to room temperature, a Xorbax RP-C18 fast resolution packed column (150x4.6mm, 5 pm, XDB-C18, Agilent Technlogies, Santa Clara, USA) with a 1.8 pm pore and a length of 5 cm. The tricuadrupole mass detector was used under the following conditions: electrospray ionization (ESI) in negative mode at a N ² temperature of 330 ° C, N ² flow of 9 L / min, nebulization pressure of 45 psi , the capillary voltage of 4000 V, in sweep operation mode, negative ion and the mass range between 50 and 1000.
[0171] The major compounds present in the C. patagua extract were; Quercetin (28%), Gallic Acid (8%) and Chlorogenic Acid (5.5%) (Fig. 1).
[0173] For its part, the essential oil of S. montana was analyzed by gas chromatography on an Agilent 7890 A equipment interconnected with an Agilent 5975C mass spectrometer with electronic impact ionization (70 eV). An HP-5MS silica capillary column (30m 250mm id), coated with 5% phenylmethyl silox (phase thickness 0.25mm) was used. The oven temperature was programmed as follows: 60 ° C for 8 min, increasing at 180 ° C at 3C / min, 180-250 ° C at 20C / min, then at 250 ° C for 10 min. Injector and detector temperatures were maintained at 250 ° C and 280 ° C, respectively. The samples were injected in split mode with a split ratio of 1:55. 0.2 ml of each sample was injected. The carrier gas was helium with a flow rate of 1 ml / min and an interface temperature of 250 ° C. The analyzed mass range was between 40-450 m / z. The components of the linear retention indexes of the volatile extracts calculated from their retention times and homologous series of n-alkanes C8-C20 (Fluka Analytical) were used for the identification compared to those of the pure standards. The fragmentation patterns of the mass spectrum components were also compared to those stored in the GC-MS library.
[0175] The main compounds present in the essential oil of S. montana were Carvacrol (40%), p-cymene (16%) and gamma terpinene (15%). (Fig. 2)
[0177] Example 4: Preparation of the formulation of C. patagua ethanolic extract and S. montana essential oil . and the antibiotic:
[0179] Mixtures of the ethanolic extract of C. patagua and the essential oil of S. montana were made in different proportions, from 1: 3 to 3: 1 (by weight). The extract mixture Ethanolic / essential oil was diluted in 85% ethanol to reach concentrations of 10, 8, 6 and 3% of the formulation.
[0181] In parallel, a control of the antibiotic florfenicol was prepared using 95% ethanol as solvent and diluted in sterile distilled water. A stock solution of 5120 pg / ml was used as a starting point, to prepare serial dilutions in base 2 up to the 1/64 dilution.
[0183] Example 5. Effect of the formulation comprising the extract of C. patagua and the essential oil of S. montana in the bacterium Piscirickettsia salmonis
[0185] Preparation of the inoculum of Piscirickettsia salmonis:
[0187] The P. salmonis inoculum used corresponded to a soil isolate obtained from an outbreak in Salmo salar. An inoculum was adjusted to a concentration of approximately 0.5 Macfarlane, to obtain a bacterial concentration close to 1x108 bacteria / ml. The purity of the inoculum was evaluated by Gram staining, IFAT and PCR.
[0189] Culture:
[0191] Both in the maintenance passages and in the antibiograms, P. salmonis was cultivated in a blood agar medium with the following characteristics: heart infusion broth 1.5%, glucose 1%, cysteine 0.1%, agar 1.5% , Hemoglobin 0.2%, Lamb's Blood 5%, SFB 3% and at an incubation temperature of 18 ° C.
[0193] Diffusion test on well agar:
[0195] 0.1 ml of the P. salmonis bacterial suspension was spread evenly on the surface of the blood agar, ensuring that the water was completely absorbed by it. Subsequently, perforations of approximately 5 mm in diameter were made in the agar using a punch, to deposit 120 µl of each extract and its dilutions in each well formed. One of the wells was filled only with solvent, to evaluate the effect of this on the growth of the bacteria. As a positive control, wells were used with concentrated florfenicol and dilutions of florfenicol solubilized in 95% ethanol and diluted in water, as well as essential oil of S. montana at 100% and ethanolic extract of C. patagua at 100%. All plates were subsequently incubated for four days at 20 ° C.
[0196] Finally, the mean diameter of the inhibition halo formed around each well was measured.
[0198] The test results are shown in Table 1.
[0200] Table 1. Antibiotic effect of the formulation comprising C. patagua ethanolic extract and S. montana essential oil against P. salmonis bacteria and other compounds used as controls.
[0204] * DT: standard deviation
[0206] In addition to the evident antibiotic activity of the formulation comprising the combination of the extract of C. patagua and the essential oil of S. montana , it is also seen that the combination of the extract and the essential oil cause a synergistic effect that improves the addition of the effects of each separately. Additionally, it can also be seen how the combination of the formulation with extract and essential oil with a small proportion of antibiotic achieves very similar results to those obtained with the antibiotic.
[0208] Example 6: Microencapsulation of the formulation .
[0210] The encapsulating material, gum arabic (GA) or 20% W / V maltodextrin (MD) was mixed with the formulation comprising the combination of the extract of C. patagua and the essential oil of S. montana in different amounts and homogenized. The samples were frozen at -20 ° C, and then lyophilized (Christ, Alpha 1-2). The lyophilized sample it was ground and sieved (pore size 500 microns). The powder obtained was stored in a desiccator until it was used. Figure 4 shows an example of the obtained microcapsules.

权利要求:
Claims (18)
[1]
1. Formulation comprising a mixture of ethanolic extract of biomass from Crynodendron patagua and essential biomass oil from Satureja montana.
[2]
2. Formulation according to claim 1 consisting of a mixture of C. patagua biomass ethanolic extract and S. montana biomass essential oil .
[3]
3. Formulation according to any of claims 1 to 2, where the C. patagua ethanolic biomass extract and the S. montana biomass essential oil are combined in a ratio from 1: 3 to 3: 1 (by weight or volume ).
[4]
4. Formulation according to claim 3, where the ratio in which the C. Patagua biomass ethanolic extract and the S. montana biomass essential oil are combined is 1: 1 (by weight or volume).
[5]
5. Process for the production of the formulation according to any of claims 1 to 4, comprising the following steps:
a) starting from biomass of C. patagua to obtain an ethanolic extract, b) starting from biomass of S. montana to obtain an essential oil by hydro-distillation,
c) combine the ethanolic extract of biomass from C. patagua with the essential oil from biomass S. montana in a ratio from 1: 3 to 3: 1 (by weight or volume).
[6]
6. Process according to claim 5, wherein the ethanolic extract of C. biomass .
Patagua and S. montana biomass essential oil are combined in a 1: 1 ratio (by weight or volume).
[7]
7. Process according to any of claims 5 to 6, wherein the biomass of C.
Patagua is obtained from shoots obtained by in vitro multiplication starting from C. patagua buds .
[8]
8. Process according to any of claims 5 to 7, wherein the biomass of S.
montana comes from the Martin variety.
[9]
9. Composition comprising the formulation according to any of claims 1 to 4.
[10]
10. Composition according to claim 9 wherein said composition is a nutritional composition.
[11]
11. Composition according to claim 10, wherein the nutritional composition is a food, a supplement or a nutraceutical,
[12]
12. Composition according to claim 9, wherein said composition is a veterinary pharmaceutical composition.
[13]
13. Composition according to claim 12, wherein said composition comprises at least one pharmaceutically acceptable vehicle and / or excipient.
[14]
14. Formulation according to any of claims 1 to 4, or composition according to any of claims 9 to 13, for use as a veterinary drug.
[15]
15. Formulation according to any of claims 1 to 4, or composition according to any of claims 9 to 13 for use in the treatment and / or prevention of rickettsial septicemia in individuals of the genus Salmo or as an adjuvant in treatment and / or prevention of rickettsial sepsis in individuals of the genus Salmo.
[16]
16. Formulation for use according to claim 15, or composition for use according to claim 15, wherein individuals of the genus Salmo are selected from salmon and trout.
[17]
17. Formulation for use according to claims 16, or composition for use according to claim 16, wherein the salmon is selected from Atlantic salmon (Salmo salar), Coho salmon ( Oncorhynchus kisutch) and trout is rainbow trout ( Oncorhynchuss mykiss ).
[18]
18. Use of the formulation according to any of claims 1 to 4, as a disinfectant against P. salmonis bacteria .

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同族专利:

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公开号 | 公开日

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CL2018003878A1|2019-05-17|

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ES2769918B2|2021-10-15|

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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WO2011119047A1|2010-03-26|2011-09-29|Ewos Innovation As|Carvacrol/thymol composition for the treatment of salmonid rickettsial septicemia and infectious salmon anemia|

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WO2013100774A1|2011-12-28|2013-07-04|University Of Belgrade|Pharmaceutical compositions containing herbal based active ingredients for application in human and veterinary medicine|

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US20180289759A1|2015-04-08|2018-10-11|Maqui New Life S.A.|Veterinary composition of marine algae and andrographis sp extracts, which can be used to treat infections in fish|

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CL2018000674A1|2018-03-13|2018-06-08|Univ Santiago Chile|Natural antibacterial extracts of crynodendron patagua, aristotelia chilensis or mixtures thereof, grown in vitro, food additive for fish diet that includes them, food formulation for fish comprising said additive and its use in the treatment or prevention of bacterial infections, in especially, an infection with piscirickettsia salmonis, responsible for rickettsial salmon syndrome .|

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优先权:

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申请号 | 申请日 | 专利标题

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CL2018003878A|CL2018003878A1|2018-12-28|2018-12-28|Formulation comprising extract of crynodendron patagua and essential oil of satureja montana for the prevention and treatment of diseases caused by piscirickettsia salmonis in individuals of the genus Salmo.|

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