Patent Publication Number: US-2022225619-A1

Title: Method and procedure for obtaining natural pyrethrin microbiocapsules with high stability

Description:
DESCRIPTION 
     TECHNICAL SECTOR 
     The invention belongs to the field of application of the chemical industry sector dedicated to the production of nanobiopesticides, and specifically to methods for the production of pyrethrin nanoemulsions. 
     BACKGROUND OF THE INVENTION 
     With the development of society and the expansion of agricultural areas, there has been a demand for new, more efficient and safe pesticides. Some highly efficient chemical formulations have emerged rapidly, but a high degree of residues appear on analyzing crops. However, with the development of environmental awareness and certification along with advances in biotechnology in recent years, pesticide manufacturers have begun to adhere to safety, efficiency, circular economy and user-friendly guidelines, which have resulted in non-toxic, ecological and zero residue products. Biopesticides have become the priority line of the agrochemical industry. 
     The use of natural pyrethrins as a pesticide has a long history and can be used to control pests that affect both humans and crops. Natural pyrethrin is an insecticide extracted from the pyretheum flower (Chrysanthemum cinerariaefolium) and is easily degradable by light, air, water and microorganisms. Pyrethrin is highly toxic to crop-damaging insects, but is safe for warm blooded animals, is non-polluting, and leaves no residue. 
     For the prevention and control of pests, pyrethrins are usually in the form of solids, oils, wettable powders and emulsifiable concentrations. In the case of pyretheum oil and concentrated emulsions, it is usually mixed with substances and plant derivatives, which are pollutants for water and the environment. The encapsulation of pyrethrins not only allows the need for lower doses of pyrethrin but also protects the environment, reduces production costs, increases the effectiveness of the insecticide in contact with other supplies or other pesticides and blocks the unpleasant odor of the active substances. Pesticide compositions containing a pyrethroid, a vegetable oil, a solvent and an emulsifying surfactant system are known (EP 567,368). Likewise, emulsifiable concentrates containing a pyrethroid, an excipient that can contain vegetable oils, a surfactant system and optionally solvents and stabilizing agents are known (GB 2,058,569). Compositions containing a pyrethroid, a vegetable oil and a surfactant system consisting, for example, of a mixture of non-ionic and anionic surfactants (U.S. 4,617,3318) are also known, such as compositions containing pyrethroids, vegetable oils or other esters such as an organic solvent (ES 2,144,618). However, none of these documents claims, mentions or can be subtracted from the description, the pyrethroid m icroencapsulation. 
     With regard to the procedure for obtaining microcapsules, microencapsulation technologies have been applied to pesticide formulation processes from the early 1970s to the present day. However, the number of products with microencapsulated active substances is quite limited. In 1976 in the United States, Pennwalt launched the first microencapsulated pesticide: Methyl parathion, which significantly reduced the toxicity of the product and prolonged its effects. Pennwalt also encapsulated Pyrethrin ES 8506420 from Pennwalt Corporation by a complex and expensive procedure. Since then, different investigations have been carried out in the field of microcapsule development, and in particular in the field of agriculture, reaching today where there are over 200 agrochemical companies that have research projects in the development and application of microcapsules. Therefore, there is more than 40 years of history in the development of pesticide microcapsules, but compared to developments in the pharmaceutical industry this has been quite slow. As a result, there is little variety of products on the market based on pesticide microcapsules. 
     Current pesticide microencapsulation methods mainly include: polymerization, cohesive separation phases and solvent evaporation. These methods are not only complicated but also expensive. Organic solvents are also used in large amounts which are polluting. 
     Despite all these existing alternatives, the need remains to provide a process for obtaining pyrethrin microbiocapsules that does not affect the insecticidal capacity in such a way that they can be used for pest control. 
     Current pesticide microencapsulation methods mainly include: polymerization, cohesive separation phases and solvent evaporation. These methods are not only complicated but also expensive. Organic solvents are also used in large amounts which are polluting. Despite all these existing alternatives, the need remains to provide a procedure for obtaining pyrethrin microbiocapsules that does not affect the insecticidal capacity in such a way that they can be used for pest control. 
     EXPLANATION OF THE INVENTION 
     The present invention has been designed in such a way as to provide, according to a first aspect thereof, a procedure for obtaining microbiocapsules of one or more pyrethrins as active substance, the process of which comprises three steps: 
     1. Preparation of oil phase, which consists of a dissolution of one or more pyrethrin(s) as active substance with a concentration between 48% -80%, in one or more vegetable oil(s) in a ratio of 1: 20, optionally stirring is carried out, optionally a solvent agent can be added that can be fatty acid(s), alcohol(s), essential oil(s) or a mixture thereof, then stirring is applied to the mixture. 
     2. Emulsifying phase in which an emulsifier and optionally a stabilizing agent and/or a preservative are added, while stirring. Additionally, a co-formulating agent(s) with antioxidant property(ies), preservative(s) and/or anti-foaming agent(s) can be added. 
     3. Phase of obtaining microbiocapsules, wherein by means of a mixing process, preferably using a mixer, such as a mixer with a paddle stirrer, the result is the obtaining of pyrethrin microbiocapsules at 4-25% w/v, preferably between 4-8% w/v with a size between 0.5 to 5 microns, with greater stability. 
     In the present invention, pyrethrin microbiocapsule refers to; micrometric particles with an outer membrane inside which it contains pyrethrin as an active substance. 
     30 
     In phase (1), one or more vegetable oil(s) are added that are selected from the group consisting of sunflower, corn, soybean, avocado, jojoba, pumpkin, grape seed, sesame, hazelnut oils, glycerol tricaprocaprylate, or the vegetable oils of the formula R9 COOR10 in which R9 represents the moiety of a higher fatty acid comprising 7 to 29 carbon atoms and R10 represents a straight or branched hydrocarbon chain containing 3 to 30 carbon atoms in particular alkyl or alkenyl, undecylenic, myristoleic, palmitoleic, oleic, linoleic, linolenic, ricinoleic, eicosenoic or docosenoic acids (found in pine, corn, sunflower, soy, raisin seeds, flax or jojoba). As preferred examples of fatty acids, mention may be made of fatty acids comprising unsaturated molecules, for example of the oleic type, which is oligomerized by a double bond condensation reaction. This reaction results in mixtures that essentially comprise dimers and trimers. By dimers and trimers of fatty acids we mean the oligomers of 2 or 3 identical or different monomers. Advantageously, these fatty acids, saturated or unsaturated, comprise 12 to 100 carbon atoms, and more advantageously 24 to 90. 
     In phase (1) stirring is between 60-150RPM 
     In phase (1) the temperature is 20 to 40 ° C., preferably between 25-35 ° C. 
     In phase (1), a solvent agent selected from the unsaturated fatty acids and their derivatives can be added: linoleic acid, linolic acid, oleic acid, folic acid and their derivatives, ubiquinone and ubiquinol and their derivatives, tocopherols and derivatives. 
     In phase (1) a solvent agent can be added which is selected from lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerol, propylene glycol, dipropylene glycol, and sorbitol; 
     In phase (1) a dissolving agent can be added which is selected from natural or synthetic essential oils such as, for example, the oils of eucalyptus, hybrid of lavender, lavender, vetiver, litsea cubeba, lemon, sandalwood, rosemary, chamomile, savory, nutmeg, cinnamon, hyssop, caraway, orange, geranium, juniper and bergamot. 
     In phase (2) a nonionic emulsifying agent is added which appears in a proportion of 10 to 50% of the composition by weight, and preferably, between 15 to 30% by weight with respect to the total weight of the composition. 
     Nonionic emulsifying agent is understood to mean amphoteric, anionic, preferably cationic. Amphoteric or ampholytic emulsifying agents are understood to be those surfactant compounds that contain, in addition to an alkyl or acyl group with 8 to 18 carbon atoms, at least one free amino group, and at least one -COOH or -SO3H group in the molecule, and are suitable for the formation of internal salts. Examples of suitable ampholytic surfactants are N-alkylglycines, N-alkylpropionic acids, N-alkylaminobutyric acids, N-alkyliminodi-propionic acids, N-hydroxyethyl-N-alkylamidopropylglycines, N- alkyltaurines, N-alkylsarcosines, 2-alkylaminopropionic acids and alkylaminoacetic acids, in each case with about 8 to 18 carbon atoms in the alkyl group. Anionic emulsifying agent is understood to be those having a negative charge, examples of anionic surfactants are, such as alkyl sulfates with a chain length of 8 to 18 C atoms, alkyl or alkylaryl ether sulfates with 8 to 18 C atoms in the hydrophobic moiety and up to 40 ethylene oxide or propylene oxide units, alkyl or alkylaryl sulfonates with 8 to 18 carbon atoms, esters and half esters of sulfosuccinic acid with monovalent alcohols or alkylphenols, alkyl or alkylaryl sulfonates, hydroxylalkanol sulfates, fatty acids of saturated and unsaturated chain of 8 to 24 carbon atoms and their oxy-alkylenated esters and sulfates and phosphates of polyethoxylated alkanols. Cationic emulsifying agents are understood to be those that contain a positive charge in aqueous solution, amines and also quaternary compounds. Examples of cationic emulsifying agents are alkylamines with 7 to 25 carbon atoms, N, N-dimethyl-N- (hydroxyalkyl) ammonium salts with 7 to 25 carbon atoms, mono- and di-(with 7 to 25 carbon atoms)-alkyldimethylammonium quaternized with alkylating agents, the ester quats such as the esterified, quaternary mono-, di- or trialkanolamines, which are esterified with carboxylic acids with 8 to 22 carbon atoms, the imidazolin quats such as the 1-alkylimidazolinium salts of general formulas I or II. 
     In phase (2) the cationic emulsifying agent used is a quaternary compound, it is observed that it increases the insecticidal capacity of the product resulting from the described procedure. 
     In phase (2), optionally, a second emulsifying agent can be added, when using combinations of emulsifying agents, it is advantageous to use a relatively hydrophobic emulsifying agent in combination with a relatively hydrophilic agent, or to use an anionic emulsifying agent in combination with a non-ionic agent. 
     In phase (2), a second emulsifying agent can be added that is present in the composition, generally, in a proportion that can range from 10 to 50% by weight and preferably 15 to 30% by weight with respect to the total weight of the composition. 
     In phase (2), additionally, co-formulating agent(s) can be added that can be selected from antioxidants such as tocopherol and propyl gallate, nitroxides, BHT-butylhydroxytoluene, BHA-butylhydroxyanisole, and/or acidulants such as; citric acid, lactic acid and malic acid, hydrochloric acid, tartaric acid, phosphoric acid, lactic acid, sulfonic acid, and/or anti-foaming agents; at least one silicone-based or fluorine-based fatty acid containing 14-24 carbon atoms. 
     In phase (2) the temperature is between 10-45 ° C., preferably between 25-35 ° C. 
     In phase (3) a stirring of 60-300 RPM is carried out between 1 to 36 hours, preferably between 1 to 4 hours. It has been worked under controlled conditions of density between 0.910-0.980 g/cm 3  and pH between 5-10, preferably between 6.5-8.5. The final result of the described procedure is the obtaining of pyrethrin microbiocapsules of a size between 0.5-5 μm with a regular morphology and a smooth surface. 
     In another aspect, the present invention relates to the use of the microbiocapsules of the present invention as an insecticide. More particularly, the present invention relates to the use of the microcapsules of the present invention for the control of arthropod pests such as insects, preferably aphids and ants. The effectiveness of the insecticidal product obtained by this procedure lasts up to 2 years. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       To complement the description that is being made and in order to help a better understanding of the features of the invention, a set of drawings is attached as an integral part of said description, wherein with an illustrative and non-limiting nature, the following has been represented: 
         FIG. 1 —Shows a chromatographic profile of the resulting composition under normal conditions and 14 days later in the same compound applying the MT 46.3 method. 
         FIG. 2 —Shows a photograph of pyrethrin microbiocapsules wherein an epifluorescence microscope (Axioplan, ZEISS) is observed. A sample drop is placed on a slide (20x40x0.25mm) with a 20x objective. 
         FIG. 3 —Shows a photograph of pyrethrin microbiocapsules wherein their size and monodispersity are indicated: 
       1. Length 5.14 μm 
       2. Length 4.42 μm 
       3. Length 5.48 μm 
       4. Length 5.14 μm 
     
    
    
     PREFERRED EMBODIMENT OF THE INVENTION 
     The invention will be illustrated below by means of tests carried out by the inventors, which show the effectiveness of the procedure of the invention: 
     Example 1 73g of a refined extract of chrysanthemum EDEOIL P60® were mixed as a source of pyrethrins 60% (w/w); Pyrethrin I, Cinerin I, Jasmoline I, Pyrethrin II, Cinerin II and Jasmoline II, the ratio between Pyrethrin I and Pyrethrin II was 1.85, with 557g of soybean oil with 50% linoleic acid and 25% in oleic acid, also containing lauric, myristic, palmitic, stearic and linolenic acid, stirring was carried out at 60rpm in HI 180x-2® at a temperature of 25° C. and 1g of extract rich in tocopherol E306 was added, stirring was continued for 20 minutes, then without stopping stirring, 158g of 99.5% propylene glycol and 185g of 97% polysorbate 80 Sorbilene® were added. Then 15g of D-limonene of 94% concentration (bitter orange) and 8g of vegetable glycerin E-422 were added, acting as a co-formulating agent, improving the tense activity of the formulation. It was stirred at 120 rpm for about 2 hours and the appearance of the microbiocapsules was observed ( FIG. 2 ), then the microcapsules and their shapes were measured according to ( FIG. 3 ). 
     Example 2 
     To determine the stability of the product, obtained by the process of the present invention as described in example 1, the method MT 46.3 (J. Zhang et al. J. of Chromatography A. 1218 (2011) 6621-6629 Dean was used Ban et al. Afr. J. of Biotechnology Vol. 9 (18), pp. 2702-2708, May 3, 2010) by means of heat stimulation to age the formulations and simulate the packaged and stored product ( FIG. 1 ). Formulations that passed the test were considered viable for at least 2 years. 
     
       
         
           
               
             
               
                 TABLE I 
               
             
            
               
                   
               
               
                 Storage stability at 54° C. 
               
            
           
           
               
               
               
               
            
               
                 Assay 
                 0 day 
                 7 days 
                 14 days 
               
               
                   
               
               
                 Physical status 
                 Homogeneous 
                 Homogeneous 
                 Homogeneous 
               
               
                 (Method EPA OPPTS 
                 liquid 
                 liquid 
                 liquid 
               
               
                 830.6303) 
               
               
                 Color 
                 Transparent 
                 Transparent 
                 Transparent 
               
               
                 (Method EPA OPPTS 
                 yellow 
                 yellow 
                 yellow 
               
               
                 830.6302) 
               
               
                 Odor 
                 Typical 
                 Typical 
                 Typical 
               
               
                 (Method EPA OPPTS 
               
               
                 830.6304) 
               
               
                 pH 1/100 (20° C.) 
                 4.66 
                 4.61 
                 4.57 
               
               
                 (Method CIPAC MT 
               
               
                 75.3) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Density (25° C.) 
                 1.0390 
                 g/ml 
                 1.0392 
                 g/ml 
                 1.0386 
                 g/ml 
               
               
                 (Method EPA OPPTS 
               
               
                 830.7300) 
               
            
           
           
               
               
               
               
            
               
                 Acidity (% H2SO4) 
                  0.063 
                  0.065 
                  0.068 
               
               
                 (Method CIPAC MT 
               
               
                 191) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Wettability 
                 &lt;10 
                 sec. 
                 &lt;10 
                 sec. 
                 &lt;10 
                 sec. 
               
               
                 (Method CIPAC MT 
               
               
                 53.3) 
               
            
           
           
               
               
               
               
            
               
                 Suspensibility 
                 96.8  
                 96.2  
                 95.8  
               
               
                 (Method CIPAC MT 
               
               
                 184) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Foam persistence 
                 &lt;2 
                 ml 
                 &lt;2 
                 ml 
                 &lt;2 
                 ml 
               
               
                 (ml in 1 minute) 
               
               
                 (Method CIPAC MT 
               
               
                 47) 
               
               
                 Chemical composition: 
                 4.48% 
                 w/v 
                 4.48% 
                 w/v 
                 4.48% 
                 w/v 
               
               
                 pyrethrins 
               
               
                 (Method HPLC-DAD)