BR102018009231A2 - snedds bionanotechnology employed in the transmission of polar extracts of tephrosia toxicaria pers. pesticide / nematicide - Google Patents

Abstract

Biodegradable snedds (autonanoemulsion) products prepared from extracts (aqueous and hydroalcoholic) of the vegetable tephrosia toxicaria (stem, leaves and roots) have been obtained with preferential pesticide / nematicide application. snedds-nano-tt systems (bionano-structured snedds based on t. toxicaria) containing low concentrations polar extracts (0.025% - 0.8%) were evaluated against nematodes meloidogyne enterolobii and meloidogyne javanica. snedds-nano-tt bioproducts are quantitatively effective in preventing egg hatching and juvenile mortality / j2, with optimized bioconcentration around 0.1% (relative to the dilution of snedds system containing 10 mg extract in 1 ml solution). For large-scale production of snedds-nano-tt, the preferred components are: tweens class nonionic surfactant (10% to 16%), low percentage vegetable oil (0.5% to 2.0%) and water bidistilled in high percentages (89.5% to 82.0%); followed by the encapsulation step of t extracts. Toxic. The whole plant has a pesticidal / nematicidal action and can be marketed using snedds biotechnology, with extensive indication for other pesticide activities or pest proliferation control, such as mosquitoes that transmit dengue, zika virus, chikungunya, malaria, filariasis and yellow fever.

Description

(54) Title: SNEDDS BIONANOTECHNOLOGY EMPLOYED IN THE TRANSPORTATION OF POLAR EXTRACTS FROM TEPHROSIA TOXICARIA PERS. WITH PESTICIDE / NEMATICIDE APPLICATION (51) Int. Cl .: A01N 65/20; A01N 25/04; A01N 25/22; A01P 5/00; A01P 17/00; (...)

(52) CPC: A01N 65/20; A01N 25/04; A01N 25/22.

(71) Depositor (s): MARIA APARECIDA MEDEIROS MACIEL.

(72) Inventor (s): FRANCISCO JOSÉ CARVALHO MOREIRA; ADRIANA FERREIRA UCHOA; JOHERBSON DEIVID DOS SANTOS PEREIRA; MARIA APARECIDA MEDEIROS MACIEL.

(57) Abstract: In the present invention, biodegradable products of the SNEDDS type (autonanoemulsion) were obtained, prepared based on extracts (aqueous and hydroalcoholic) of the vegetable Tephrosia toxicaria (stem, leaves and roots), with preferential application pesticide / nematicide. The SNEDDS-Nano-Tt systems (bionano-structured SNEDDS based on T. toxicaria) containing polar extracts carried in low concentrations (0.025% - 0.8%), were evaluated to combat the nematodes Meloidogyne enterolobii and Meloidogyne javanica. SNEDDS-Nano-Tt bioproducts are quantitatively effective in preventing egg hatch and in juvenile mortality / J2, with optimized bioconcentration around 0.1% (relative to the dilution of the SNEDDS system containing 10 mg of extract conveyed in 1 mL of solution). For large-scale production of SNEDDS-Nano-Tt, the preferred components are: non-ionic surfactant of the Tweens class (10% to 16%), reduced percentage vegetable oil (0.5% to 2.0%) and water bidistilled in high percentages (89.5% to 82.0%); followed by the stage of encapsulation of T. toxicaria extracts. The whole vegetable has a pesticidal / nematicidal action and can be marketed using SNEDDS biotechnology, with extensive indication for other pesticide activities or pest proliferation control, such as mosquitoes that transmit dengue, zika virus, chikungunya, malaria, filariasis and yellow fever.

10 20 30 40 50 00? 0 00 90 100

1/18

Invention Patent Specification Report for SNEDDS Bionanotechnology used in the transmission of polar extracts of Tephrosia toxicaria Pers. with pesticide / nematicide application

Field of the Invention and Justification [001] The present invention relates to obtaining bioformulates prepared based on extracts of Tephrosia toxicaria Pers. (Fabaceae). Specifically, polar extracts (aqueous and hydroalcoholic) obtained from the stem, leaves and roots of this plant were used for pesticide / nematicide application. The carrier system of the dry extracts of T toxicaria, corresponds to a nanostructured colloidal system of the SNEDDS type (self-nanoemulsifying-type drug delivery system). The autonanoemulsified carrier (SNEDDS) conveyed the polar extracts of T toxicaria satisfactorily and enabled the bioavailability of this vegetable formulated in a nanotechnological way, as a pesticide / nematicide agent. After incorporating the vegetables, the derivative bioproducts were named SNEDDS-Nano-Tt (bionano-structured SNEDDS type based on Tephrosia toxicaria). Specifically, the action of SNEDDS-Nano-Tt systems to combat nematodes of the species Meloidogyne enterolobii and Meloidogyne javanica was evaluated.

[002] The SNEDDS carrier system selection criterion is based on the following advantages: a) ease of reproduction on a large industrial scale; b) ease of application and stability of bioproducts; c) bioavailability of the active compounds of T toxicaria carried in a nanostructured system resistant to natural actions (rain and sun) for application in different types of soils; d) the SNEDDS carrier system is biodegradable and resists maximum dilutions in aqueous media, and has been shown to be effective in encapsulating T toxicaria extracts ·, e) as it is a carrier system with controlled action, the bioactive compounds of this vegetable act with pesticide / nematicide action over a long period, with controlled release; f) if it comes to be commercialized with this function, the vegetable in its transmitted form (SNEDDS-Nano-Tt) is eco-compatible and acts effectively as a pesticide / nematicide, adding technological value to the agribusiness sector.

Petition 870180037388, of 05/07/2018, p. 8/34

2/18 [003] The present invention, therefore, is associated with the sustainable management of soils infected by nematodes in various crops of agricultural interest exploited economically by man (large crops, vegetables, fibers, floriculture, among other possibilities). In this sense, it is also important to highlight that the SNEDDS-Nano-Tt bioformulates may arouse the interest of companies and institutions linked to organic agriculture. Therefore, it represents a new bio-technological tool for the sustainable management of agricultural pests, as well as for alternative agroecological, organic, biodynamic production, among other possibilities.

Foundations of the invention [004] The genus Tephrosia (Fabaceae) has hundreds of species widely used as natural insecticides, originating mainly from the African continent, and to a lesser extent, occurring in Latin America and Oceania. Phytochemical studies have shown that this genus is rich in rotenoids and flavonoids, citing just one example, the species Tephrosia toxicaria Pers. presents the following flavonoids: obovatin, iso-obovatin, 6a, 12a-dehydro-3toxicarol, 6a, 12a-dehydro-a-toxicarol and α-toxicarol, which confirm the chemotaxonomic profile of the genus Tephrosia [Touqeer, S .; Saeed, Μ. THE.; Ajaib, Μ. A. Review on the phytochemistry and pharmacology of genus Tephrosia, 2013].

[005] Flavonoids and rotenoids are widely present in different types of plant species, including species belonging to the Fabaceae botanical family. In this family, the genera Tephrosia, Derris and Lonchocarpus are the most numerous. The species included in the genus Tephrosia make up a total of 650 plants, in which the plant Tephrosia toxicaria Pers., Popularly known as indigo, indigo bravo, anileira and timbó is found.

[006] Tephrosia toxicaria Pers. it is a small shrub with an erect, semi-woody stem, with leaves composed of 10 to 20 pairs of leaflets, 3 to 4 cm long and 3 to 8 mm wide; they are obtuse and slightly hairy on the inside. The fruit of this plant has a pod shape, 5 to 7 cm long and 1 to 2 cm wide, slightly covered with small rust-colored hairs, containing 10 to 12

Petition 870180037388, of 05/07/2018, p. 9/34

3/18 seeds. It is a plant widely used by indigenous people in fisheries in places with standing water, such as dams and ponds. Milk from the swollen roots (xylopodia) of T toxicaria is used for the purpose of paralysis (ichthyotoxic activity) of fish, which under the action of this vegetable, float and facilitate fishing [Leitão Filho, H. F. Observations on some types of Legumes - Papilionoideae. Campinas: Instituto Agronômico, 2009].

[007] The major compounds (flavonoids and rotenoids) present in extracts of T toxicaria (stems, leaves and roots) have insecticidal, nematicidal, larvicidal, fungicidal and antiprotozoal properties [Touqeer, S .; Saeed, Μ. THE.; Ajaib, Μ. A Review on the phytochemistry and pharmacology of genus Tephrosia, 2013]; [Chen, Y. Yan, T. Gao, C. Cao, W. Huang, R. Natural products from the genus Tephrosia: a review, 2014], [008] The results of tests for larvicidal activity against the Aedes aegypti mosquito, made with extracts and substances isolated from T toxicaria (stems, leaves and roots) reinforce the importance of using this species as an insecticidal agent, and arouses interest for use as an agricultural pesticide. However, botanical pesticides, such as essential oils and extracts, have limitations of formulations and effectiveness of action, including: a) type of formulation and its pesticidal efficacy, evaluated in the soil, depending on the time of action of the phytocomposites; b) effectiveness of action of phytocomposites due to these organic materials degrading in the soil, through percolation, leaching, volatilization, among other factors; c) botanical extracts when applied directly to the soil, without a protective vehicle that acts with release is controlled, lose their nematicidal effects very quickly due to their compounds presenting easy degradation, both due to high temperature, intense sunlight and direct action of the soil microfauna [Vasconcelos, JN Evaluation of the chemical and biological potential of Tephrosia toxicaria Pers. (Fabaceae). Thesis (PhD in Organic Chemistry), Federal University of Ceará, Fortaleza, 2010]; [Touqeer, S .; Saeed, Μ. THE.; Ajaib, Μ. A. Review on the phytochemistry and pharmacology of genus Tephrosia, 2013].

[009] From the biotechnological point of view, the pesticide nanoencapsulation that is being carried out with biodegradable

Petition 870180037388, of 05/07/2018, p. 10/34

4/18 many advantages over conventional formulations, due to the decrease in the amount of product applied while maintaining the pesticidal efficacy; reductions in the number of applications; reduction of percolation / leaching risks; and control of product flow to water sources. In addition, handling the product in low concentrations reduces the exposure time of humans during application [Tsuji, K. Microencapsulation of pesticides and their improved handing safety, 2001].

[010] Among other advantages that are attributed to pesticide nanoencapsulations, the protection of the active principle against its environmental degradation stands out; the reduction of phytotoxicity of seeds and plantations; the high selectivity of non-target organisms; more effective application of the product (reduction of pesticide losses due to degradation, evaporation or dissolution, by temperature and light) [Goertz, Η. M. Controlled release technology, agricultural. In: KirkOthmer (Ed.). Encyclopedia of Chemical Technology, ^4th Edition, John Wiley & Sons, Inc, 2000]; [Tsuji, K. Microencapsulation of pesticides and their improved handing safety, 2001].

[011] Bionanotechnology is a technological area that acts in an inter and multidisciplinary way and is directly related to the creation, use and improvement of nanostructures that are applied in several types of biotechnological processes. Comprising a range of research and applications that deal with the special properties of matter encapsulated in nanometric scales (in the range 1 nm - 100 nm). Among the various fields of research in this area are: synthetic processes; characterizations and applications of nanoparticles with different sizes, shapes and chemical compositions, with wide application in the pharmaceutical, food and agrochemical areas [Suave, J .; Dall’agnol, E. C .; Pezzin, A. P. T .; Silva, D. A. K .; Meier, Μ. M .; Soldi, V. Microencapsulation: innovation in different areas, 2006]; [Lima Brasileiro, J. S. Microencapsulation of bioactive compounds: innovation in different areas. (Integrated Master's Dissertation in Pharmaceutical Sciences). Fernando Pessoa University. 2011].

[012] The most severe restriction on the use of pesticides in field crops is precisely the toxicity of synthetic pesticides. In agriculture and livestock, the use

Petition 870180037388, of 05/07/2018, p. 11/34

5/18 extensive and frequent use of large amounts of chemical pesticides, can develop populations of genetically resistant pests [Khaliq, A .; Khan, A. A .; Afzal, M .; Tahir, Η. M .; Raza, A. M .; Khan, A. M. Field evaluation of selected botanicals and commercial synthetic insecticides against Thrips tabaci Lindeman (Thysanoptera: Thripidae) populations and predators in onion field plots, 2014].

[013] In this sense, plants are important sources of bioactive substances with numerous different chemical structures that act against pathogenic organisms to plants. Historically it is known that pesticides of natural origin were widely used to combat agricultural pests, with great emphasis during the Second World War and in the post-war period, starting in the 1940s [Viegas Júnior, C. Terpenos with insecticidal activity: an alternative for chemical insect control, 2003]. Among the most well-known organic pesticides, whose active substances are derived from plants, the following stand out: the alkaloid nicotine, isolated from Nicotiana spp. of the genera Derrís and Lonchocarpus, and the limonoid azadiractin, obtained from Azadirachta indica [Soloway, SB Environ. Health Perspect, 1976].

[014] The patent BR 102013021210-5 A2 (with international deposit code WO 2014113860 A1) reports the use of Azadirachta indica in the form of powder (macroparticles) and biopolymeric nanoparticles containing oil and extract of this plant species, with indication for several applications . In preparing the nanoparticles, an aqueous solution containing a non-ionic surfactant was used, such as sorbitan monostearate (Span 60) and polysorbate (Tween 80). In another patent (BR 102016002125-1 A2) a biodegradable colloidal product of the self-emulsifying type (SMEDDS system) prepared with the oil of Azadirachta indica A. Juss. Seeds, in the presence of Tween 80 in aqueous medium, claimed for this bioproduct, action pesticide and as pest proliferation control.

[015] The patent BR 102016015370-0 A2 refers to aqueous nanoemulsion from the essential oil of Casearina sylvestris S \ N leaves. for viral inhibition of herpes Simplex Type I. Specifically, the invention features an aqueous nanoemulsion prepared based on the essential oil of the leaves of C. sylvestris, with biomass

Petition 870180037388, of 05/07/2018, p. 12/34

6/18 vegetable rich in non-oxygenated sesquiterpenes (70%) that showed potent antiviral activity for herpes simplex type I (HSV-I).

[016] The patent BR 102016012240-6 A2 refers to a microemulsion containing essential oil of Citrus sinensis to control Aedes eagyti. The invention refers to the production of a bioformulate to control A. aegypti larvae, preventing their proliferation through an eco-compatible system. In this way, it favors the control of a public health problem, and does not cause environmental damage, as well as it values the use of regional biodiversity, as its active ingredient is the essential oil extracted from the peels of pear orange (C. sinensis). Serious diseases such as dengue, zika and chikungunya are combated with a strategy to prevent their vector, the A. aegypti mosquito. In this sense, the invention BR 102016012240-6 A2 has the purpose of acting as a larvicide in drinking water tanks that are places where the larvae tend to proliferate as well as other types of water reservoirs. [017] The patent BR 102015005040-2 A2 refers to a microemulsion containing the essential oil of the leaves of Croton argyrophyllus (Kunt.) As an anti-inflammatory agent. The herbal formulation is indicated for topical and oral use with anti-inflammatory action.

[018] Microemulsions are thermodynamically stable and optically isotropic colloidal systems containing water, oil, surfactant and, if necessary, a co-surfactant. These systems act effectively as protectors against enzymatic hydrolysis and also as reservoirs for drugs, guaranteeing a slow and prolonged release. Nanoenulsions are their equivalents with kinetic stability (DIAS et al., 2012; FORMARIZ et al., 2005; ROSSI et al., 2007). During the stages of the preparation process of a given microemulsified system the main objective is to obtain a critical combination between the components, with maximum solubilization of the dispersed phases. According to the different types of phases that form in microemulsions, a classification was postulated that establishes four main types that were called WINSOR (BASHEER et al., 2013; DAMASCENO et al., 2011). The phase diagram describes the exact experimental condition and indicates whether it is possible or not to obtain the WIV region (WINSOR IV) that corresponds to a single-phase system, on a macroscopic scale, characterized by a single microemulsion phase (DAMASCENO et al., 2011 ;

Petition 870180037388, of 05/07/2018, p. 13/34

7/18

DANTAS et al., 2010; MACIEL et al., 2009; SILVA et al., 2009; LEAL et al., 2007; ROSSI et al., 2007; GOMES et al., 2006).

[019] Colloidal systems (emulsions, microemulsions and nanoemulsions) can be prepared in chemical support (A / O system) or polar (O / A) environments. The varieties SMEDDS (self-microemulsifying-type drug delivery system) and SNEDDS (self-nanoemulsifying-type drug delivery system) are resistant to maximum dilutions in water, without losing the effectiveness of encapsulating the bioactive principle. In this context, some of the many applications of colloidal systems (microemulsions, nanoemulsions, SMEDDS and SNEDDS systems) are highlighted: a) Michaelsen, M. H .; Wasan, K. M .; Sivak, O .; Müllertz, A .; Rades, T. The effect of digestion and drug load on halofantrine absorption from self-nanoemulsifying drug delivery system (SNEDDS), 2016]; b) Silveira, B. Z. Self-emulsifiable systems for modified drug release: development, characterization and applications. Thesis (PhD in Chemical Engineering). Universidade Estadual Paulista, CampinasSP, 2015; c) Saini, J. K .; Nautiyal, U .; Singh, D .; Anwar, F. Microemulsions: A potential novel drug delivery system, 2014; d) Sigward, E .; Mignet, N .; Rat, P .; Dutot, M .; Muhamed, S .; Guigner, J. M .; Scherman, D .; Brossard, D .; Crauste-Manciet, S., Formulation and cytotoxicity evaluation of new self-emulsifying multiple W / O / W nanoemulsions, 2013; e) Mandai, S .; Mandai, S. S., Research paper microemulsion drug delivery system: a platform for improving dissolution rate of poorly water soluble drug, 2011; f) Dantas, T. N. C .; Silva, H. S. R. C .; Dantas Neto, A. A .; Marcucci, M. C .; Maciel, Μ. A. M. Development of a new propolis microemulsion system fortopical applications, 2010; g) Smooth, J .; Dall'Agnol, E. C .; Pezzin, A. P. T .; Silva, D. A. K .; Meier, Μ. M .; Soldi, V. Microencapsulation: Innovation in different areas, 2006.

[020] The toxicity of a chemical substance against agricultural pests (insects, bacteria, fungi and nematodes) does not necessarily qualify it as a pesticide. For this specific action, several properties must be associated, among which are: effectiveness of the product handled in low concentrations of the bioactive principle, with no toxicity for mammals and higher animals and biodegradability [Nath, B. S .; Kumar, R. P. S. Ecotoxicol Environ Saf, 1999]. As examples that best match these conditions, there are compounds of plant origin that have nematicidal activity; are profitable and

Petition 870180037388, of 05/07/2018, p. 14/34

8/18 environmentally safe and can be used on a large scale in agriculture; with emphasis on the following publications: a) Jesus, F. N., Damasceno, J. C. A., Barbosa, D. H. S. G., Malheiro, R., Pereira, J. A., Soares, A. C. F. Control of the banana burrowing nematode using sisal extract, 2015; b) Moreira, F. J. C .; Silva, M. C. B .; Rodrigues, A. A .; Tavares, Μ. K. N. Alternative control of root-knot nematodes (Meloidogyne javanica and M. enterolobii) using antagonists, 2015; c) Moreira, F. J.

C., Ferreira, A. C. S. Alternative control of gall nematode (Meloidogyne enterolobii) with marigold (Tagetes patula L.), incorporated into the soil, 2015; d) Wezel, A., Casagrande, M., Celette, F., Vian, J. F., Ferrer, A., Peigné, J. Agroecological practices for sustainable agriculture: a review, 2014; e) Ntalli, N. G., Caboni, P. Botanical Nematicides: A Review, 2012; f) Chitwood, D. J. Phytochemical based strategies for Nematode control, 2002; g) Oka, Y., Nacar, S., Putievsky, E., Ravid, U., Yaniv, Z., Spiegel, Y. Nematicidal activity of essential oils and their components against the root-knot nematode, 2000; h) Akhtar. M, Mahmood I. Potentiality of phytochemicals in nematode control: a review, 1994.

[021] Among the phytonematodes is the genus Meloidogyne Goeldi (1887), which stands out due to its genetic plasticity, versatility and adaptability. This genus has a high number of species (about 100); high range of hosts (there are more than 2,000 species in more than 30 botanical families); wide geographical distribution; it is present on all continents; it has a high reproductive capacity and resistance to bad weather, managing to survive in a state of dormancy for more than 10 years [Ferraz, L. C. C. B .; Brown,

D. J. F. Plant nematology: fundamentals and importance. L. C. C. B. Ferraz and D. J. F. Brown (Orgs.). Manaus: Norma Editora, 2016]. Phytomatomatids belonging to the genus Meloidogyne are popularly known as gall nematodes, because they cause gall formation in the vegetables they attack. Characteristically, they present obligatory endoparasites of plants of economic interest and interact with other pathogenic organisms. For this reason, they are classified as being one of the main pathogens responsible for significant losses (up to 90%) of agricultural productivity, depending on the degree of infestation, in tropical and subtropical ecosystems [Mitkowski, N. A .; Abawi. G. S. Root-knot nematodes. The Plant Health Instructor, 2003].

Petition 870180037388, of 05/07/2018, p. 15/34

9/18 [022] Over many decades, the control of phytonmatoids was based on soil fumigants and synthetic nematicides, due to the efficiency and ease of use and results obtained. However, several problems have been caused by the use of these pesticides, including contamination of soil, plants and groundwater, as well as toxicological risks for farmers, animals and consumers [Agrios, GN Plant pathology. 5th Ed., Academic Press. 2005; Tihohod, D. Applied agricultural nematology. Jaboticabal: FUNEP, 1993].

[023] These issues motivate the search for clean technologies and enable the development of new eco-compatible products. As examples, we highlight some patents related to the use of plant extracts and essential oils with pesticide / nematicide application, as well as in the control of insects / pests and insects / vectors, as well as microorganisms (fungi, bacteria, among other applications), as shown below.

[024] The patent PI 0511986-3 A refers to the use of a formulation based on the PcMR strain of Pochonia chlamydosporía applied against the gall nematode (Meloidogyne spp.), Which has recommended use due to its high virulence on this phytopathogen.

[025] The patent BR 10 2012 005935-5 A2 refers to the use of jasmonates in pure form or by the use of their fractions and extracts, applied alone or by the association of their chemical analogues in liquid, semi-solid or solid commercial formulations, as potent nematicide or nemastostatic. Its commercial composition has recommended use if the active principle jasmonic acid or methyl jasmonate, with individual or associated use is present in the composition of the formulated.

[026] Patent PI 0712789-8 A2 refers to the preparation of compositions containing essential oils obtained from Tagetes erecta, Ocimum basilicum and Cymbopogon martini, used alone or in a mixture of these or certain components thereof, plus an agriculturally acceptable carrier oil and an emulsifying agent, with nematicidal action.

[027] The patent PI 9508533-5 A refers to the use of bacterial strains, Sphingobacterium spiritiovorun (strain C-926) and Corynobacteríum paurometabolum

Petition 870180037388, of 05/07/2018, p. 16/34

10/18 (strain C-924), without characterized formulation, with nematicidal effect on the gall nematode (Meloidogyne incognita).

[028] The patent CN 201310745538 A refers to a botanical pesticide containing extracts of Tephrosia vogelii and Arthrophyllum spp. and a method of preparation and application of the pesticide in the field for the control of lepdopteras (caterpillars and moths).

[029] The patents CN104560735 A and CN104531543 A refer to endophytic fungi of Tephrosia purpurea (Aspergillus oryzae - strain TPL35 and Penicillium griseofulvum - strain TPL25), with the application of a product of its fermentation that has a high inhibitory activity on the growth of hypha in pathogenic plant fungi, such as rape sclerotinia rot and black smoke cane disease. It was found that these strains have excellent development and are extremely promising as microbial pesticides, being used as a preventive or in the treatment of this disease.

[030] Patent BR 102016002125-1 A2 refers to a self-emulsifying colloidal system based on Azadirachta indica A. Juss., For bioavailability of the oil of this vegetable in pest control. Specifically, the objective was to promote a prudent action in the control or proliferation of mosquito vectors of the following diseases: dengue, zika, chikugunya, malaria, filariasis and yellow fever.

[031] Patent BR 102014004882-0 A2 refers to an aqueous nanoemulsion and production process, applied in pest control. Specifically, a nanoemulsion with bioinsecticidal activity that allows the incorporation of insoluble substances in an aqueous medium, through a stable formulation that presents potential insecticidal action against agricultural pests and / or application in foci of Aedes aegypti larvae proliferation, an important vector of dengue.

[032] In a study by Vasconcelos (2010) it was possible to observe the high larvicidal activity of the extracts obtained from the stems, leaves and roots of T. toxicaria, as well as its flavonoid isolates (iso-obovatin, obovatin, 6a, 12a-dehydro-3-toxicarol, 6a, 12a-dehydro-a-toxicarol and α-toxicarol) and rotenoids (eglutin, 12a-hydroxy-a-toxicarol and tephrosine), which were evaluated in trials

Petition 870180037388, of 05/07/2018, p. 17/34

11/18 biological against the Aedes aegypti mosquito. This study reinforces the use of this species as a source of substances with potential insecticide and also justifies its use as an agricultural pesticide.

[033] According to the literary descriptions, briefly presented in the present patent, there is no record of formulations of the type emulsion, microemulsion, nanoemulsion, SNEDDS or SMEDDS systems prepared with polar extracts (aqueous or hydroalcoholic) obtained from the species T toxicaria for application against some type of pest or agricultural disease.

[034] Having proven that there are no publications that anticipate the proposal of the present invention, which consists of the transmission of polar extracts (aqueous and hydroalcoholic) obtained from the stem, leaves and roots of T toxicaria, carried in a SNEDDS carrier system. (self-emulsifying system), for pesticide / nematicide application. In this sense, it was proved that the SNEDDS-Nano-Tt nanoformules (SNEDDS-type nanostructures based on Tephrosia toxicaria) present inventive activity in relation to the pesticide / nematicide action and novelty in the development of SNEDDS encapsulations based on Tephrosia toxicaria. In addition, the standard SNEDDS system used in the delivery of extracts of this vegetable is eco-compatible, since it comprises aqueous medium in percentage above 80%, extracts of the vegetable transmitted in low percentage (10 mg / mL), and a non-ionic surfactant (non toxic).

Detailed description of the invention [035] The plant Tephrosia toxicaria Pers. was collected (stem, leaves and roots) in the district of São José, in Ibiapina-CE. The identification of the botanical material was carried out at the Herbarium of the Federal University of Rio Grande do Norte (HUFRN), with exsiccata under the fall number UFRN022960. After drying the vegetable biomass, grinding was carried out in a Willye knife mill, STAR FT 50® (Fortinox). The ground materials were placed in polyethylene pots and kept in the refrigerator until the beginning of the subsequent experiments, which comprise: obtaining the extracts, their encapsulations and pesticide testing.

Petition 870180037388, of 05/07/2018, p. 18/34

12/18 [036] We weighed corresponding values for each type of material, which were conditioned in Becker with distilled water in the proportion of 20% (v / w) for 24 hours. After this period, the extracts were filtered on Nalgon® filter paper (5 microns), and subsequently, they were applied to the vases, with the transplant plant seedlings. The hydroalcoholic extracts were obtained via maceration, according to a previously reprotected methodology in which extraction was used with the mixture of polar solvents ethaneLgua (8: 2), having been carried out according to a classic phytochemical procedure (MACIEL et al., 2002).

[037] The dry extracts (aqueous and hydroalcoholic) of the vegetable Tephrosia toxicaria Pers. were carried out in SNEDDS system (autonanoemulsion) obtained through a methodology based on the titration of chemical components, which in this case consists of a non-ionic biodegradable surfactant, an oily phase, and a high percentage aqueous phase (above 80%). The maximum solubility point of the active substance is characterized by the change in the physical aspect of the system obtained, according to previously reported methodology (DANTAS et al., 2010; MACIEL et al., 2009; GOMES et al, 2006). Specifically, in the preparation of the SNEDDS carrier system, a surfactant belonging to the Tweens class was used, in a proportion that is in the preferred range 10% to 16%, a food oil in low concentration (in the preferred range 0.5% to 2%) and bidistilled water (in the preferred range (89.5% to 82.0%). The method used in the preparation of the SNEDDS carrier system is in accordance with the following description: I) Weigh the components on a precision digital scale the system (oil, mixture of surfactant and double distilled water); II) Mix the system components (with specific methodological characteristic), under heating and agitation (magnetic stirrer, and temperature in the preferred range 40 ° C - 50 ° C; III) Centrifuge at a constant speed, 500 rpm, for a specific time (in the preferred range 20 - 30 min.); IV) Incorporate the plant extract in the SNEDDS system (via conventional procedures for solubilizing organic compounds).

[038] The study of the construction of the ternary phase diagram (Figure 1) of the SNEDDS system proved that there was formation of polar nanoemulsion (O / A), which was characterized by the following analyzes: a) D.M.A.M. [maximum dilution of aggregates

Petition 870180037388, of 05/07/2018, p. 19/34

13/18 micellar, made possible by the study of critical micellar concentration (CMC)]; b) surface tension; c) viscosity; d) droplet diameter. The rheological behavior of the SNEDDS nanoemulsion was evaluated from the graph generated by the values of shear stress versus shear rate, using a rheometer in the range between 20 ° C to 30 ° C, with variation for the shear rate (1, 0 s ' ¹ to 1000.0 s' ¹ ), resulting in a linear behavior, with R ² = 0.95509 (Figure 2).

[039] Figure 1. Ternary phase diagram of the SNEDDS carrier system.

[040] Figure 2. Study of the viscosity of the SNEDDS carrier system.

[041] In the flow of a Newtonian fluid there is a proportionality parameter between the shear stress and the shear rate. The viscosity of this fluid is only influenced by temperature and pressure variations (MACHADO, 2002). Therefore, according to the generated graph (Figure 2), the fluid under analysis was classified as Newtonian and the viscosity was determined in the preferred range 2.0 mPa.s to 6.0 mPa.s (equivalent to 2.0 cP at 6 , 0 cP or, 2 x 10 ' ³ Ns / m ² - 4 x 10' ³ Ns / m ² ).

[042] The analysis of the maximum dilution of the nanoemulsified formulation was evaluated by the experimental data of the surface tension. The tests were performed using the Tensiometer, the method description consists in the measurement of the maximum bubble pressure using two capillaries with different diameter orifices, where an inert gas (N2) is pumped at a certain constant pressure (200 kPa). The study of maximum dilutions showed an experimental value in the preferred range 6.0 x 10 ' ³ g / ml - 9.0 x 10' ³ g / ml_). For the initialization of the experimental process, 20 ml_ of the nanoemulsified system was necessary. For each measurement performed, the samples were diluted in distilled water, with variations in the concentrations until reaching surface tension values close to the water (YH2O = 72.1 mN / m), at temperatures ranging from 20 ° C to 30 ° C. The larger capillary has the purpose of measuring the effect of the immersion depth and the smaller capillary measures the surface tension. After successive dilutions in water, the nanoemulsion system was characterized as SNEDDS (self-nanoemulsion drug delivery system).

[043] According to this methodology, from the SNEDDS concentration values versus surface tension it was possible to obtain a graph in which

Petition 870180037388, of 05/07/2018, p. 20/34

14/18 observes the point of intersection of the two lines, which corresponds to the maximum dilution that the surfactant can be subjected to in a given colloidal system, without the loss in its physical-chemical properties (Figure 3). For values below this point, dispersed monomers are found.

[044] Figure 3. Maximum dilution of the micellar aggregates of the SNEDDS system.

[045] To obtain the retraction index, a Brix analog refractometer was used, at a temperature in the range between 20 ° C and 30 ° C.

[046] To determine the diameter of the droplets, measurements were made in triplicates and with a retraction index of 1.4715 with a beam of light of 659 nm, with an incidence angle of 90 °, with an experimental value observed in the range preferred 5 nm -18 nm.

Transmission of polar extracts (aqueous and hydroalcoholic) from Tephrosia toxicaria Pers. in the SNEDDS system [047] The procedure for quantifying solids in the aqueous extracts obtained from the stem, leaves and roots of the vegetable Tephrosia toxicaria, consisted of weighing the tubes (50 ml_) type Falcon® before and after adding 30 ml_ of each type of extract. Then, the tubes were placed in a freezer for 12 h and then, they were placed in a Liotop® Lyophilizer Model L202 lyophilizer (Liobras, São Carlos ,. SP, Brazil), until the materials were completely dehydrated. After this period, the tubes were weighed again, the difference between the first and second weighing, resulting in the dry weight, expressed in mg / ml_.

[048] For hydroalcoholic extracts obtained from the stem, leaves and roots of the vegetable Tephrosia toxicaria, steam-route was used to remove the alcohol, followed by a lyophilization process for total water removal. The solubilization efficacy of dry extracts (aqueous and hydroalcoholic) of Tephrosia toxicaria in the nanoemulsified system (SNEDDS) was evaluated by the method of minimum and maximum solubilities. Each extract was effectively solubilized in a standard concentration of 10 mg / mL [sample mass (mg) per ml of the formulated SNEDDS].

Petition 870180037388, of 05/07/2018, p. 21/34

15/18

Obtaining and multiplying the inoculations of the nematodes Meloidogyne enterolobii and Meloidogyne javanica [049] For the bioassays, eggs and juveniles of second stage (J2) of M. enterolobii and M. javanica were used, extracted from roots of infested and maintained 'Santa Clara' tomatoes in a greenhouse, from IFCE - Campus Sobral using the technique of Hussey & Barker (1973). An aqueous suspension plus kaolin, containing the eggs and juveniles / J2 of nematodes M. enterolobii and M. javanica was subjected to the flotation and centrifugation method proposed by Jenkins (1964), aiming at improving the visualization of this material and counting the number of eggs and juveniles / J2 (Coolen; D'Herde, 1972). Counting was performed with the aid of a Peters Chamber, under a stereoscopic microscope [Jenkins, W. R. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter, Maryland - USA, 1964]; [Coolen, W. A .; D’Herde, C. J. A method for the quantitative extraction of nematodes from plant tissue. State Agricultural Research Center. Ghent, 1972]; [Hussey, R. S .; Barker, K. R. A comparison of methods of collecting inocula of Meloidogyne spp., Including a new tecnique. Plant Disease Reporter, Minnesota - USA 1973]; [Tihohod, D. Applied agricultural nematology. Jaboticabal: FUNEP, 1993].

[050] The bioassays were conducted in different concentrations (0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8%) of each SNEDDS-Nano-Tt system, as follows: I ) SNEDDS-Nano-Tt-Stem system (nanostructured-SNEDDS based on aqueous and hydroalcolic extracts from the stem of T. toxicaria) carried in different concentrations (0.025% - 0.8%) and negative control (distilled water) with six replicates 60 EGGS / J2; II) SNEDDS-Nano-Tt-Leaves system (nanostructured SNEDDS based on aqueous and hydroalcolic extracts of T. toxicaria leaves) carried in different concentrations (0.025% - 0.8%) and 0 negative control (distilled water), with six repetitions of 60 EGGS / J2; III) SNEDDS-Nano-Tt-Roots system (nanostructured-SNEDDS based on aqueous and hydroalcolic extracts from roots of T. toxicaria) carried in different concentrations (0.025% - 0.8%) and the negative control (distilled water) with six repetitions of 60 eggs / J2.

[051] The SNEDDS-Nano-Tt systems (SNEDDS-Nano-Tt-Stem; SNEDDS-Nano-TtFlesh and SNEDDS-Nano-Tt-Roots) evaluated in different concentrations (0.025;

Petition 870180037388, of 05/07/2018, p. 22/34

16/18

0.050; 0.1; 0.2; 0.4; 0.6; 0.8%), received eggs and juveniles / J2 obtained from nematodes M. enterolobii and M. javanica, packed in 48 well acrylic plates, followed by incubation in BOD at 25 ^S C, during the testing period (12 days), according to previously established methodology [Salgado, SML; Campos, VP Natural extracts on the pathogenicity and reproduction of Meloidogyne exigua in coffee and Meloidogyne incognita race 3 in beans, 2003]; [Moreira, FJC; Santos, CDG; Innecco, R. Hatching and mortality of J2 juveniles of Meloidogyne incognito race 2 in essential oils, 2009].

[052] The analysis of hatching and mortality of juveniles / J2, started 48 h after the assembly of the trial until the maximum time of the experiment (12 days). In the evaluations, the number of hatched juveniles / J2 (egg hatched in each 48 h period) and quantification (count) of all juveniles / immobile J2 (mortality in each 48 h period) were counted. To confirm the occurrence of mortality, the specimens were transferred to water, followed by examination on slides, with the aid of an optical microscope with a 40x magnification, to verify the minimum possibility of specimen activity. Juveniles / J2 with straight or slightly twisted appearance were considered inactive (dead). The final number of juveniles / J2 killed in the trial was obtained by adding the six counts.

[053] Figure 4 shows the results obtained with the SNEDDS bioformulate carrying the aqueous extract (EA) obtained from the stem of T. toxicaria SNEDDS-Nano-TtCaule-EA), evaluated at concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8% (corresponding to the dilutions in water of the standard SNEDDS-Nano-Tt-Stem-EA containing 10 mg / mL of aqueous extract), in namaticidal activity via egg hatch (A) and juvenile mortality / J2 ( B) Meloidogyne enterolobii and M. javanica.

[054] Figure 4. Hatching of eggs (A) and mortality of juveniles / J2 (B) of the nematodes M. enterolobii and M. javanica in SNEDDS medium based on the aqueous extract of the stem of T. toxicaria evaluated in different concentrations (0.025 % - 0.8%).

[055] Figure 5 shows the results obtained with the SNEDDS bioformulate carrier of the aqueous extract (EA) obtained from T. toxicaria SNEDDS-Nano-TtFolhas -EA leaves, evaluated in concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8%

Petition 870180037388, of 05/07/2018, p. 23/34

17/18 (corresponding to dilutions in water of the standard SNEDDS-Nano-Tt-Stem-EA containing 10 mg / mL of aqueous extract), in namaticidal activity via study of egg hatch (A) and juvenile mortality / J2 (B ) of Meloidogyne enterolobii and M. javanica.

[056] Figure 5. Hatching of eggs (A) and mortality of juveniles / J2 (B) of nematodes M. enterolobii and M. javanica in SNEDDS medium based on the aqueous extract of leaves of T. toxicaria evaluated in different concentrations (0.025 % - 0.8%).

[057] Figure 6 shows the results obtained with the bioformulate SNEDDS carrier of the aqueous extract (EA) obtained from the roots of T. toxicaria SNEDDS-NanoTt-Root-EA), evaluated at concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8% (corresponding to the dilutions in water of the standard SNEDDS-Nano-Tt-Stem-EA containing 10 mg / mL of aqueous extract), in namaticidal activity via egg hatch (A) and juvenile mortality / J2 ( B) Meloidogyne enterolobii and M. javanica.

[058] Figure 6. Hatching of eggs (A) and mortality of juveniles / J2 (B) of the nematodes M. enterolobii and M. javanica in SNEDDS medium based on the aqueous extract of the roots of T. toxicaria evaluated in different concentrations (0.025 % - 0.8%).

[059] Figure 7 shows the results obtained with the SNEDDS bioformulate carrying the hydroalcoholic extract (EHA) obtained from the stem of T. toxicaria SNEDDSNano-Tt-Stem-EHA), evaluated at concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8% (corresponding to dilutions in water of the standard SNEDDS-Nano-Tt-Stem-EHA containing 10 mg / mL of aqueous extract), in namaticidal activity via egg hatch (A) and juvenile mortality / J2 ( B) Meloidogyne enterolobii and M. javanica.

[060] Figure 7. Hatching of eggs (A) and juvenile mortality / J2 (B) of the nematodes M. enterolobii and M. javanica in SNEDDS medium based on the hydroalcoholic extract obtained from the stem of T. toxicaria evaluated in different concentrations ( 0.025% 0.8%).

[061] Figure 8 shows the results obtained with the SNEDDS bioformulate carrying the hydroalcoholic extract (EHA) obtained from T. toxicaria SNEDDS leaves

Petition 870180037388, of 05/07/2018, p. 24/34

18/18

Nano-Tt-Leaves-EHA), evaluated at concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8% (corresponding to the dilutions in water of the standard SNEDDS-Nano-Tt-Leaves-EHA containing 10 mg / mL of the aqueous extract), in the namaticide activity via study of hatching of eggs (A) and mortality of juveniles / J2 ( B) Meloidogyne enterolobii and M. javanica.

[062] Figure 8. Hatching of eggs (A) and mortality of juveniles / J2 (B) of the nematodes M. enterolobii and M. javanica in SNEDDS medium based on the hydroalcoholic extract obtained from T. toxicaria leaves evaluated in different concentrations ( 0.025% 0.8%).

[063] Figure 9 shows the results obtained with the SNEDDS bioformulated carrier of hydroalcoholic extract (EHA) obtained from the roots of T. toxicaria SNEDDSNano-Tt-Raiz-EHA), evaluated at concentrations 0.025; 0.050; 0.1; 0.2; 0.4; 0.6; 0.8% (corresponding to dilutions in water of the standard SNEDDS-Nano-Tt-Root-EHA containing 10 mg / mL of aqueous extract), in namaticidal activity via egg hatch (A) and juvenile mortality / J2 ( B) Meloidogyne enterolobii and M. javanica.

[064] Figure 9. Hatching of eggs (A) and juvenile mortality / J2 (B) of the nematodes M. enterolobii and M. javanica in SNEDDS medium based on the hydroalcoholic extract obtained from the roots of T. toxicaria evaluated in different concentrations ( 0.025% 0.8%).

[065] According to the results of the evaluations, all bioformulates of T. toxicaria (SNEDDS-Nano-Tt), containing polar extracts obtained from roots, stem and leaves, carried in low concentrations (0.025% - 0.8%), they are effective in preventing the hatching of eggs and in the mortality of juveniles / J2, with optimized bioactive concentration around 0.1% (relative to the dilution of the SNEDDS standard solution containing 10 mg of extract conveyed in 1 mL of solution). Therefore, the entire bush can be commercialized using SNEDDS biotechnology, via the transmission of aqueous or hydroalcoholic extracts, aiming at pesticide / nematicide application. In an expanded form, SNEDDS-Nano-Tt bio-products may be applied to control different types of agricultural pests.

Petition 870180037388, of 05/07/2018, p. 25/34

1/2

Claims (10)

Claims 1. Bioformulations prepared based on Tephrosia toxicaria Pers. purchased commercially or via authorized collection (taking into account the legal provisions of Law n ^s 13,123, of May 20, 2015, and Decree n ^s 8,772, of May 11, 2016) characterized by being a polar colloidal system (The / A) SNEDDS (autonanoemulsion) or SMEDDS (auto-emulsion) type prepared with non-ionic surfactant of the Tweens class (20, 40 or 80, in the preferred range 10% - 16%) containing a reduced percentage vegetable oil (0.5 % - 2.0%) and bidistilled water in the preferred range 89.5% - 82.0%, used as a carrier of polar extracts (aqueous and hydroalcoholic) obtained from the stem, leaves and roots of T toxicaria, for pesticide application / nematicide; 2. Bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with preferential claim 1 that undergoes changes in the percentage scales of its components, as well as by the addition of stabilizers, thickening or gelling agents; 3. Bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with preferential claims 1 and 2, which undergo changes in the chemical classification of the surfactant (which may be ionic or non-ionic) generating colloidal O / A, A / O, A / O / A or O / A / O systems nanostructured or microstructured; 4. Bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with the preferential claims 1 to 3, in the presence of mixtures of surfactants, with or without addition of cotensoative (s); 5. Method of obtaining bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with claims 1 to 4, which undergo changes in the methodology for preparing the standard formulated SNEDDS with variation in temperature, agitation and acquisition time; 6. Agro-industrial use of bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with claims 1 to 5, with encapsulation (in different concentrations) of polar extracts (aqueous and hydroalcoholic) obtained from the stem, leaves and roots of this plant, which have been carried in nanoemulsed polar or support systems Petition 870180037388, of 05/07/2018, p. 26/34 2/2 or microemulsified, as well as in SNEDDS or SMEDDS self-emulsifying polar systems; 7. Agro-industrial use of bioformulations based on Tephrosia toxicaria Pers. characterized by being correlated with claims 1 to 5, depending on the delivery of any type of extract (support or polar, prepared with hexane, chloroform, ethyl acetate, acetone, methanol, ethanol, among other organic solvents, alone or in mixtures ), which was obtained from the stem, leaves and roots of T. toxicaria, and was carried with encapsulation variation, comprising supportive or polar colloidal systems (emulsion, nanoemulsion or microemulsion), as well as in selfemulsifying polar systems (SNEDDS or SMEDDS); 8. Biodevelopment of colloidal formulated carriers of Tephrosia toxicaria extracts Pers. characterized by being correlated with claims 1 to 7, comprising application in the agro-industrial area against gall nematodes (Meloidogyne enterolobii and Meloidogyne javanica), and as a pesticide against any type of nematicidal species; 9. Biodevelopment of colloidal formulated carriers of Tephrosia toxicaria extracts Pers. characterized by being correlated with claims 1 to 7, used as tools for the management of agricultural pests, as alternative agroecological, organic, biodynamic production, among other possibilities that include antimicrobial, insecticide and larvicide activities; 10. Biodevelopment of colloidal formulated carriers of Tephrosia toxicaria extracts Pers. characterized by being correlated with claims 1 to 7, comprising pesticide application or pest proliferation control, such as mosquitoes that transmit the following diseases: dengue, zika virus, chikungunya, malaria, filariasis and yellow fever. Petition 870180037388, of 05/07/2018, p. 27/34 1/5 DRAWINGS