WO2019109155A1 - Biodegradable particle and use - Google Patents

Abstract

The present invention describes a biodegradable particle with proven larvicidal properties to combat the Aedes aegypti mosquito and as a result of this fact it has come to the fore as an economically viable and also sustainable alternative for the elimination of this vector. The particle is dried and upon immersion in water it swells and allows the release of the oil, thereby killing all the larvae in a specific concentration.

Description

 BIODEGRADABLE PARTICLE AND USE

 Field of the Invention:

 The present invention is in the field of bioccids containing material obtained from plants and describes a biodegradable particle which comprises the starch encapsulated thyme essential oil (Thymus vulgaris).

 The particle of this invention has demonstrated larvicidal properties against the Aedes aegypti mosquito and, in view of this fact, appears as an economically viable alternative, besides sustainable, for elimination of this vector.

 The particle is dehydrated and when immersed in water, it swells, leaches and allows the release of the oil, causing the death of all the larvae in a certain concentration.

 Fundamentals of. invention:

 Mosquito-borne diseases such as malaria, yellow fever, dengue fever, filariasis and leishmaniasis are public health problems in countries with a tropical climate, such as Brazil. More specifically, dengue, yellow fever, zika and chikungunya are diseases that can be transmitted to humans by the bite of an infected Aedes aegypti mosquito.

 [005] billions of cases of diseases transmitted by the Aedes aegypti mosquito are registered in Brazil and the annual increase in cases of deaths is noted. Only the awareness of the population to avoid the accumulation of still water to avoid mosquito proliferation has not been sufficient.

[006] The consequences of yellow fever and dengue fever are already known, however, the effects of zika virus and chikungunya were recently discovered. Specifically, the relationship between infection caused by zyka virus in pregnant women is the birth of children with microcephaly, which has attracted worldwide attention.

 [007] Although there are already vaccines for some diseases, it does not yet exist for all. In addition, viruses responsible for the transmission of such diseases may mutate and new diseases may occasionally arise.

 [008] Aedes aegypti infections are caused by the fact that this is a home mosquito, which is housed in or near houses, especially in urban and local areas with high population density.

 Contamination outbreaks usually occur in the summer in tropical and subtropical regions due to the combination of high temperature and high intensity of rain showers.

 [010] Aedes aegypti is a mosquito that reproduces in still waters and presents daytime habits. A female mosquito averages 120 eggs at a time and these can retain viability for more than a year if kept in the dry state. When in contact with water, eggs hatch and develop into larvae, becoming adult mosquitoes after a period of 5 to 18 days on average.

 [011] The larval stage is the feeding and growing period. The duration depends on factors such as temperature, availability of nutrients and amount of larvae, and can vary from 5 days to several weeks.

The pupal phase is the last stage of development in an aqueous environment and lasts for about two to three days and corresponds to the metamorphosis for the adult stage. After this period, they become adult mosquitoes (terrestrial environment), living on average 45 days,

 The mosquito develops in two environments: the aquatic (stage larva with stages L1, L2 and L3 and pupal phase) and terrestrial (adult phase). To prevent the mosquito from becoming a vector of diseases, it is necessary to act in the larval phase (in the aqueous environment), because the virus responsible for the diseases will not be able to be transmitted.

 [014] Eliminating the mosquito in its adult phase is much more difficult due to the large area to be applied the insecticide, in addition to several reapplications for the effect to continue being observed. Today, many synthetic insecticides and repellents are marketed. However, repeated use of these causes problems such as resistance, pollution of the environment, destabilization of ecosystems and intoxication of non-target organisms, including us, humans.

 [015] Substances naturally present in plants (extracts or essential oils) have many advantages compared to synthetic insecticides because they are made up of several substances. Depending on the stress conditions, the plant may produce secondary metabolites to attract insect pollinators or repel predatory insects. The identification and quantification of these substances is important as a parameter of the insecticidal efficacy of the palm.

It is pointed out that a single compound is not necessarily responsible for the biological activity of the plant, so that a synergism between the potential substances may occur! effect. [017] Synthetic larvae are generally composed of isolated chemicals. In this way, its cost of production is high, since it is necessary to isolate the active compound more and more frequently, due to the rapid adaptation. Since there is no synergistic effect, the development of resistance is potentiated, necessitating the frequent switching of the synthetic larvicide type to have the desired effect.

 [018] In addition to extracts and oils from renewable sources, the development of mosquito resistance occurs more slowly or almost non-occurring, since the substances are composed of complex associations of active principles (synergistic effect).

 [019] While the most diverse compounds can be identified in plant extracts or essential oils, the principal component generally determines the biological properties.

 The active compound may be isolated, however, it is often observed that an isolated compound is less effective than the mixture of the complex from which it is isolated, down to the synergistic effect.

 [021] Encapsulation emerges as a method of protecting the asset, in addition to improving handling and transport, releasing gradually and at the desired time and reducing the amount of compound needed to achieve the desired activity.

Accordingly, the present invention relates to a biodegradable particle comprising essential oil of thyme encapsulated in corn starch which is capable of killing the larvae of the Aedes aegypti mosquito. concentration. [023] Particle encapsulation was performed by the thermoplastic extrusion process. The same was chosen because it did not use chemical solvents; be continuous, fast and with high yield; have a low residence time; do not generate chemical effluents; be scalable; to present operational versatility and to obtain a product with a low moisture content.

 State of the art:

 [024] Some prior art documents describe the use of vegetable oils as insecticidal agents as well as the encapsulation thereof in starches.

 The document "Encapsulation - a convenient way to extev.d the persistence of the effect of ecofrlendly mosquito larvicides" describes a method of microencapsulation with starch, which consists of the application of supercritical fluid to encapsulate essential oils that can be used as larvicides.

 [026] The use of supercritical fluid requires high pressure production, being a high cost, dangerous and low yielding process. The production of particles by the extrusion technique, as proposed herein, is a rapid, continuous, rel. it does not use chemical solvents, does not generate effluents and operates on an industrial scale.

 [027] Monoterpenes from thyme (Thymus vulgaris) as potential mosquito repellents, are pure compounds (isolated monoterpenes, such as thymol) from thyme oil, which have been added to a base repellent composition.

[028] Unlike the present invention, repellents act on the mosquito in its adult phase. In addition, the compounds present in the thyme essential oil, such as described in this invention, may act synergistically with the larvae to eliminate the mosquito in its initial phase, differently from the isolated compounds, as provided for in the above-mentioned article.

 CN 101420818A discloses the production of a gel for encapsulating essential oils, including thyme oil, for use in controlling / reducing an insect population, such as A. aegypti.

 The encapsulation method consists in the formation of an emulsion, which may not be stable over time, resulting in a moist particle and of questionable stability at room temperature.

 In EP 2004099536, the production of adhesive particles for application was fibers (textile, or paper) to act as insect repellents, using the starch as a reticulant agent to form particles in ge 1.

 The thyme oil particle obtained, as described in the present invention, acts on the. egg and larval phases of the mosquito, eliminating it was its initial phase (aqueous environment) and preventing the vector from proliferating diseases. Repellents, as described in the Japanese application, act on the mosquito in its adult phase and require several applications to continue having the desired effect.

 US 2008/0131538 Al, in turn, describes the production of starch microparticles having an extremely porous structure, which are loaded by adsorption.

[034] In this way, there is no guarantee that the oil will be totally inside the particle (since it is adsorbed), nor is it known how it will be released.

 BRIEF DESCRIPTION OF THE INVENTION:

 [035] This invention describes a biodegradable particle comprising the corn starch encapsulated thyme oil.

 The particle of this invention has demonstrated larvicidal properties against the Aedes aegyptx mosquito and, in view of this fact, appears as an economically viable alternative, besides sustainable, for elimination of this vector.

 [037] The particle is dehydrated and when immersed in water, it swells, bleaches and allows the release of the oil, causing the death of all larvae in a certain concentration.

 Brief description of the figure:

 Figure 1 graphically shows the kinetics of leaching, swelling and oil release of the best particle.

 Figure 2 shows the particles obtained with different fillings (free oil and emulsified oil) and the oil release mechanism on coming into contact with the "

 Detailed description of. Invention:

 [040] This invention describes a biodegradable particle comprising the thyme oil encapsulated in corn starch, yielding particles of 0.8 to 0.32 cm in diameter.

 [041] The particles were produced without filling and filling (liar oil and emulsified oil with modified starch, less than 10% dry basis).

[042] The particle has larvicidal properties against the Aedes aegypti mosquito and, in view of this fact, arises an economically viable and sustainable alternative for the elimination of this vector.

[043] Commercial thyme free oil results in CL ₉₉ (lethal concentration to eliminate 99% of the population) equal to 94 μg / ml in 24 h, indicating to be an efficient compound to the desired end. According to the volatile chemical profile analyzed by gas chromatography, the thymol is the major compound of the thyme oil, corresponding to 53% of its total.

 [044] Corn starch has been chosen as a wall material because it is readily available, abundant, inexpensive, non-toxic and because it swells when it comes in contact with water (thus favoring the release of the active substance).

 [045] Three types of corn starch with different amylose contents ranging from 1.8 to 71% were used. The particles were produced with the respective pure amides and with two intermediate mixtures between them.

 [046] These particles were obtained by thermoplastic extrusion in a single-screw and double-screw extruder, with a temperature lower than that generally used to expand the expanded extrudates (150 ° C), in order to reduce the volatilization and degradation of the active coroldo.

 [047] Other advantages of the process of encapsulation by extrusion of the thyme oil with corn starch are:

- reducing the moisture content of the particle by reducing microbiological deterioration and allowing the particles to be stored at room temperature (increasing stability and shelf life, which facilitates their commercialization); and - gradual swelling and leaching of the particles due to the gradual absorption of water and the consequent release of the active substance.

 [048] Thyme oil still acted as a plasticizing agent, participating in the physico-chemical reactions and as an enhancer during the extrusion process.

 The particle obtained is dehydrated (moisture content between 3 to 11% depending on the formulation) and, when immersed in water, it swells and allows the release of the oil, due to the diffusion of the oil to the aqueous phase through the pores of the wall material, thus causing the death of all larvae in a given concentration.

 Characterization of the particles:

 Encapsulation Efficiency:

 [050] The process of encapsulation via thermoplastic extrusion provided the maintenance of the biological activity of the thyme oil.

 [051] Depending on the amylose content and the type of filling, the encapsulation efficiency of the particles was between 50 and

70%.

 Swelling and leaching:

[052] Swelling is the result of the entry of water into the matrix and indicates that the molecular structure and that the ^: starch chains are being modified. The water absorbed reduces the hydrogen bond between the starch chains and increases the molecular motility thereof. Further water absorption leads to the formation of channels in the hydrated matrix, permitting the diffusion transport of the active compound.

[053] As mentioned above, the release of highly hydrophobic actives such as thymol is also controlled by leaching of the matrix (loss of solids). The degree of leaching is an important parameter correlating with the release of the atrium.

 [054] Figure 1 shows the relationship between swelling kinetics (water absorption), leaching (loss of solids from wall material) and oil release. There was a great correlation between the kinetics of swelling and release. The maximum swelling, leaching and release occurs within the first 24 hours.

 [055] Depending on the type of filling and the amylose content, swelling, leaching and release have been found to vary between the different formulations.

 [056] The degree of leaching will depend on the composition of the starch and its molecular organization. Typically, a short linear amylose chain will leach more easily in solution than highly branched and high molecular weight amylopectin molecules.

 [057] In addition, fully gelatinized amorphous starches are expected to disperse more easily than native, partially crystalline or retrogradated starches.

 Mechanism of release:

 [058] With respect to the wall material, the leaching further weakens the particle structure, favoring the exit of the active compound, whereas swelling determines the formation of the pores within the particle. In relation to the active, the partition coefficient limits the amount that may be available in aqueous medium.

 [059] The particle release behavior is sustained, since thymol is highly hydrophobic.

[060] Its partition coefficient ilog P = 3.17) imposes thermodynamic limitations so that it does not leave the solid phase completely.

 [061] The best formulation resulted in oil release equal to 18%. This means that the oil undergoes small partitioning into the aqueous phase, representing an incremental potential greater than 80% for release into water after further exposure and / or dilution of the system. This result is extremely relevant because it indicates that the roatrix can maintain the oil for a long time and release it was enough to kill the larvae for long periods of time.

 [062] Figure 1 indicates the potential of the encapsulation technique, avoiding the volatilization of the active compounds and allowing a slow and controlled release of the active.

 [063] Figure 2 illustrates the different mechanisms for releasing particulate fill when they come into contact with water.

 Larvícld activity:

 [064] According to the kinetics of swelling, leaching and release, the larvicidal activity of the different particles obtained also varied in a certain concentration.

[065] It has been found that the immersion time and the type of filling influences the desired effect. Particles with free oil as fill showed larval mortality between 23-86% after 24 h depending on the amylose content, while after 48 h, mortality increased to 77 to 100%. On the other hand, the particles with emulsified oil as a filling presented mortality between 12 to 34% in 24 h and. between 47 to 82% in 48 h depending on the amylose content. [066] According to larval mortality and release kinetics, the oil-containing emulsion-containing particle probably presented a protective layer to the oil molecule (as shown in Figure 2) which hindered its release, thereby reducing at its own risk,

 [067] As can be seen in Figure 1, the oil is released due to swelling of the matrix, since the degree of leaching is very low. Thus, the oil released into the water, if in free form, can act immediately against the larvae.

 Ovicidal activity:

 [068] The most efficient particles against mosquito larvae were also tested against Aedes aegypti eggs.

 [069] Egg hatching was around 60% after 48 h in contact with water, however, all larvae (100%) died when in contact with the particles containing the free thyme oil.

 Lethal Contention:

The particle that eliminated 100% of the larvae was 48 h had a lethal concentration equal to 31 μg / mL, that is, three times less than the lethal concentration of the unencapsulated oil (CL ₉₉ equal to 94 pg / mL).

 [071] The particles are ideal for outdoor applications, especially in solid waste containing standing water, such as tires and cups or indoors such as plant pots.

 [072] The concentration of oil released from the particle is much lower than that considered toxic.

[073] Thermoplastic extrusion encapsulation helped to protect the essential oil from thyme, to release it gradually and at the most desired moment, provided the reduction of the oil concentration necessary to eliminate mosquito larvae, as well as facilitating the transportation and commercialization, from a fast, economically feasible, sustainable encapsulation method with biodegradable wall material, cheap e. abundant and with natural assets.

Claims

 A biodegradable particle characterized in that it comprises starch-encapsulated thyme essential oil, which comprises araylose contents ranging from 1.8 to 71%, obtaining particles of 0.18 to 0.32 cm in diameter, ranging from 3 to 11% unit and with less than 10% active on dry basis.  Biodegradable particle according to claim 1, characterized in that it is obtained by thermoplastic extrusion.  3. Use of the biodegradable particle, as defined in claims 1 and 2, characterized in that it is as larvicide against the Aedes aegypti mosquito.