Products derived from Azadirachta indica (A. Juss) (Neem) lead a prominent role in a select group of environmentally correct natural products which are commercially available for controlling insects and pests. Neem is a plant which is tolerant to the most adverse growth conditions, rapidly spreading throughout the globe. Literature reports identify 500 insect species which are sensitive to some kind of Neem extract action.
Currently, biopolymeric nanoparticles make up a more sophisticated agrochemical formulation approach, which renders them suitable for application in the search for novel properties for the correct use of Neem. Among their main advantages in the field, emphasis can be given to their capability of controlling active ingredient rate and release conditions, increasing solubility, reducing contact with active ingredients by farmworkers, and environmental advantages such as reduction of drainage rates.
Such technology allows for managing the external casing properties of a capsule so as to control the moment of release of active substance.
Many papers describe the use of Neem for obtaining metal nanoparticles, and for preparing powder microparticles made with Neem. Studies which use Neem for obtaining metal nanoparticles use only Neem in the process; however, the objective is to provide metal products completely unrelated to the present process.
Agriculture is normally seen as comprising three types of systems: economic, social, and ecologic (or environmental). All the three of them are interconnected, and interactions between agriculture and the environment are complex. Environmental problems such as soil degradation, desertification, destruction of tropical forests, and the resulting decrease in wildlife and pollution of water sources relate to inappropriate agricultural practices or to the intensive use of agricultural inputs.
From Roman times (1st century B.C.) up to the middle of the 20th century, insect control was performed by products derived from plants such as pyrethrin, rotenone, and nicotine (bioinsecticides or natural insecticides). Discovery of DDT (dichlorodiphenyltrichloromethane), initially believed as the solution to insect attacks, was proven a disaster after a long period of use. That gave rise to a search for safe synthetic compounds based on chlorinated hydrocarbons. Such compounds, however, were proven extremely toxic and ecologically disastrous, in addition to rendering insects resistant thereto.
Synthetic pesticides have been used for over 50 years, which makes them the main insecticidal tool. Even though their use has been efficient at controlling some pest species—making it possible to significantly increase food production—, extensive, and sometimes careless, use thereof has triggered many social- and environment-related problems, including contamination of soil, air, water, fish, and man himself; reduction of biodiversity, of the population of natural enemies, of the population and number of bees and other pollinator species, in addition to pest resistance and emergence of secondary pests.
It is estimated that the damage caused by pests in the world production of food, in spite of the efforts made, is still at least one-third of the production.
Another problem to be dealt with is the existent phytosanitary barriers. They are of great importance for the independence of countries regarding crop protection and the population's food safety. Phytosanitary restrictions have imposed food exporting countries the need to master agricultural production technology and to control all steps in the agro-production chain. Brazil, as one of the biggest food producers in the world, has phytosanitary products as one of the essential instruments to the promotion of plant defense.
Nowadays, natural insecticides have become an option and/or a complement to pest control, reducing or eliminating the use of synthetic agrochemicals. Such properties make natural insecticides an important tool for many pest management programs. However, significant production, regulations and application problems must be solved first, in order to enable such products to be reliably purchased in the market.
Among the main limiting problems to the use of natural insecticides, emphasis can be given to the need to identify and study plant species which allow for sustainable exploitation, the influence of seasonal factors and weather conditions, the lack of quality control and reproducibility of the insecticidal action, and the lack of stabilizing mechanisms for the correct use and handling of the active compounds.
Several factors may change the stability of a naturally occurring active product or compound. Each component, either active or inactive, can impact stability depending on the amount.
Other factors, named extrinsic, such as temperature, radiation, light, air (specifically oxygen, carbon dioxide, and water vapors), moisture, harvesting and storage place and time also change stability and content of active compounds.
Furthermore, there are intrinsic factors such as incompatibilities, pH, hydrolysis, racemization e oxidation. For example, rapid degradation of A. indica compounds makes them unsuitable for some cultures such as fruit production and gardening (since residual effect normally lasts only from 4 to 8 days). Studies have shown that azadirachtin activity can be reduced to nearly 60% after 4 hours of sunlight exposure, decaying to nearly 50% after 15 hours. Results obtained in the field indicated that A. indica extracts applied over the cultures may remain active only for about three days.
In the search for natural insecticides, the Meliaceae family has been identified as one of the most promising groups, once most of its species exhibits multiple actions in pest control. Inside the Meliaceae family, one species in particular is worth mentioning: A. Jussieu's Azadirachta indica, popularly known as Nimtree or Neem.
Neem, plant which is original from Myanmar and the arid regions of the Indian subcontinent, present from India to Indonesia, has been introduced throughout the tropical region of the globe. It can currently be found in Asian and African countries, in Australia, tropical North America, Central America, and South America.
Neem is a plant which has been extensively used in several areas, such as medicine, veterinarian medicine, farming, pharmacy, cosmetic and furniture industry, plant reforestation, and the like. It is a tree which is tolerant to the most adverse culture conditions (high temperatures, poor and saline soils, etc.), which is one of the factors that justifies its quick spread all over the world. Among such properties, the greatest current interest involving Neem, mainly in the western world, lies in the insecticidal properties thereof. About 500 insect species have been currently reported as being sensitive to some kind of Neem extract action.
Neem properties are due to a large number of secondary metabolites, mainly triterpenes and limonoids, available in many parts of the plant. Among such compounds, emphasis is given to azadirachtin (FIG. 1), main constituent of the plant and of commercial formulations.
Azadirachtin is a limonoid which is mostly concentrated in the fruits, although it can be found in lesser amounts all over the plant.
Said substance has several biosynthetic oxidations which form many functional groups comprising oxygen, which tends to make this compound more reactive both chemically and biologically.
Previous studies have shown many stability problems and degradation in naturally occurring active compounds. For example, even when stored under optimal conditions, away from humidity, light and temperature, the azadirachtin content in commercial Neem oils decays with time.
FIG. 2 illustrates the decay curve for azadirachtin.
Neem oil samples subjected to accelerated aging process by UV radiation allow for observing the constant degradation of azadirachtin.
In a closed system, performed under ultraviolet radiation, degradation rate in oil/water samples was approximately 108 times higher than that observed for oil samples without water.
Such results show the degradative action of humidity on the natural product in an ultraviolet radiation catalyzed hydrolysis mechanism. The mechanism is illustrated in the following Equation 1.

The presence of functional groups in Neem metabolites such as, for example, epoxide, ether and ester groups, conjugated systems, and the like present in the azadirachtin molecule is liable for its low environmental endurance, the main problem being its sensitivity to photodegradation. Azadirachtin's rapid activity loss limits its use in farming. An insecticide must be persistent enough in order to kill or control insects and pests.
Lack of monitoring methods and quality control has become another limiting aspect to the development and reliable use of natural products. It is impossible to ensure reproducibility of a product's expected action in the absence of production protocols and quality control methods.
Concurrently to the development of (natural or synthetic) biocides, there has been the need for a wide variety of types of formulations, additives and technological processes which enable formulation of active ingredient pesticides with different physical and/or chemical properties.
Formulations aim to promote convenient and safe use of a product which will not deteriorate in a period of time, and which will obtain the maximum activity inherent to an active compound.
More sophisticated formulations based on powerful surfactants and other additives, and better comprehension of the principles of colloid and surface chemistry as to increasing stability and biological activity are in accordance with the operator's needs, environmental safety requirements, and improvement of active compound activity and endurance.
The most common pesticide active ingredient formulations mentioned by GCPF—Global Crop Protection Federation (CropLife International) include granulates (GR), concentrated solutions (SL), emulsible concentrates (EC), wettable powders (WP), concentrated suspensions (SC), oil/water emulsions (EW), microcapsules (CS), etc.
A more sophisticated approach to the formulation of agrochemicals in nanometric scale involves nano- and microencapsulation. Among the main advantages thereof in the field, emphasis can be given to capability to control the conditions in which the active ingredient is released, increased solubility, reduced contact with active ingredients by farmworkers, extended patent validity, and environmental advantages such as drainage rate reduction. Such technology allows for managing the external casing properties of a capsule so as to control the moment of release of active substance.
A large number of different strategies has been proposed in order to modify nano- and microparticle physical-chemical characteristics, and thus their interaction with the biological medium. For example, it is already possible to modify the chemical nature of the particle's polymer matrix by changing some characteristics such as biorecognition, biodistribution, bioadhesion, biocompatibility, mobility, and biodegradation.
Polymeric nanoparticles can be defined as colloidal polymeric particles comprising active compounds. The nanoparticles can be classified in two categories: nanocapsules and nanospheres.
Nanocapsules are carrier compounds formed by an oily core coated by a polymeric wall, the active compound being either in said core or adsorbed in the polymeric wall.
On the other hand, nanospheres consist of a solid polymer matrix which does not have oil in its composition, the compounds being trapped and/or adsorbed.
Both colloids are stabilized by surfactants in the particle/water interface.
Different nano- and microparticle production processes are available and may develop and/or improve physical-chemical characteristics such as size, structure, morphology, surface texture and composition. On that matter, see the publications Soppimath, K. S., et al. Biodegradable polymeric nanoparticles as drug delivery devices. J. Control. Release 70, 1-20, 2001; Couvreur, P. et al., Nanocapsule technology. Crit. Rev. Ther. Drug Carrier Syst. 19, 99-134, 2002; Tice, T. R.; Gilley, R. M. Preparation of injectable controlled-release microcapsules by solvent-evaporation process. J. Control. Release 2, 343-352, 1985; Ibrahim, H.; et al., Aqueous nanodispersions prepared by a salting-out process. Int. J. Pharm. 87:239-246, 1992; Caliceti, P. et al., Effective protein release from PEG/PLA nanoparticles produced by compressed gas anti-solvent precipitation techniques. J. Control Release 94, 195-205, 2004; Galindo-Rodriguez, S. et al., Physicochemical parameters associated with nanoparticle formation in the salting-out, emulsification-diffusion, and nanoprecipitation methods. Pharm Res 21, 1428-1439, 2004.
Colloidal release systems show a great and efficient potential as release systems for one or a mixture of active compounds (plant extracts) at specific action sites (delivery systems), and control over the release rate, thus minimizing undesirable toxic effects and enhancing their physical-chemical stabilities, see Tse, G. et al., Thermodynamic prediction of active ingredient loading in polymeric microparticles. J. Control. Release 60, 77-100, 1999.
Biodegradation may occur in a biological system through polymer chain relaxation, break of the monomer unit located at the end of the chain (erosion), or even through random bond breaking at some position along the polymer chain (degradation).
Studies related to nanotechnology described in literature for the species Azadirachta indica (A. Juss) (Neem) are as diverse as the nanotechnology theme is vast.
Among the studies which are closest to the research which resulted in the present application is the article by Riyajan, As-Ad.; Sakdapipanich, J. T. Encapsulated neem extract containing Azadiractin-A within hydrolyzed poly(vinyl acetate) for controlling its release and photodegradation stability. Chemical Engineering Journal 152, 591-597, 2009. The authors used polyvinyl acetate meshed with 5% (w/v) glutaraldehyde for preparing powder microcapsules containing Neem extracts using the Spray-Drying technique. As a result, powder microparticles were obtained having average diameter greater than 10 μm, about 80% encapsulation efficiency, with enhanced stability (efficiency) against photodegradation.
Another relevant article is the one by Kulkami, A. R. et al., Glutaraldehyde crosslinked sodium alginate beads containing liquid pesticide for soil application. J. Control. Release 63, 97-105, 2000. In this article, the authors describe Neem oil encapsulation in particles formed by sodium alginate polymers meshed with glutaraldehyde. Particle diameter and encapsulation efficiency ranged between 1.01-1.68 and 72-90% respectively.
In the article by Shankar, S. S. et al., Rapid synthesis of Au, Ag, and bimetallic Au core-Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J. Colloid Interface Sci. 275, 496-502, 2004, an environmentally correct extracellular synthesis method is described for the production of metal silver and gold nanoparticles using Azadirachta indica leaves.
Tripathy, A. et al., Process variables in biomimetic synthesis of silver nanoparticles by aqueous extract of Azadirachta indica (Neem) leaves. J. Nanopart. Res. 12, 237-246, 2010 used aqueous A. indica leaf extracts for producing silver crystal nanoparticles in biomimetic processes.
Silver nanoparticles have also been produced by Prathna, T. C. et al., Kinetic evolution studies of silver nanoparticles in a bio-based green synthesis process. Colloids Surf.A:Physicochem.Eng.Aspects, 2011, doi:10.1016/j.colsurfa.2010.12.047 using Neem leaf extracts applied in kinetic studies.
Electroanalytical measurements of adenosine and adenosine-5′-triphosphate have been determined by Goyal, R. N. et al., Simultaneous Determination of Adenosine and Adenosine-5′-triphosphate at Nanogold Modified Indium Tin Oxide Electrode by Osteryoung Square-Wave Voltammetry. Electroanalysis 19, 575-581, 2007 using modified gold nanoparticles in biological systems such as A. indica extracts.
Particles containing powder Neem leaves, calcium alginate, kaolin, and bentonite have been prepared in order to control release kinetics, toxicity, and properties of the fungicide Thiram®. In this study, Singh, B. et al., Controlled release of thiram from neem-alginate-clay based delivery systems to manage environmental and health hazards. Applied Clay Science, 47, 384-391, 2010 demonstrate an application of Neem in preparing controlled release systems with particles of approximately 1 mm and encapsulation efficiency for Thiram® close to 100%.
There are several studies with patent applications for products and processes involving Neem.
Patent applications in progress at the Instituto Nacional da Propriedade Industrial (INPI)—National Institute for Industrial Property—are related to fertilizers, biological activities (germicides, insecticides, bactericides, fungicides, etc.), Neem oil and extract production techniques, granular formulations, repellents, stability studies, deodorization, analytical techniques, etc. However, none of those studies focuses on polymeric nano- and microparticle production techniques applied to increase stability of Neem-derived chemical constituents, or to control the release kinetics thereof in biological medium, which are the objective of the present application.
Patent applications which were filed at INPI involving the species Azadirachta indica include the following Published Brazilian Application Nos.: BR0804594-1 A2, BR0804546-1 A2, BR0702226-3 A2, BR0701347-7 A2, BR0502588-5 A2, BR0508296-0 A2, BR0000809-5 A2, BR9307044-6 A2, BR9302976-4 A2, BR9202628-1 A2, BR0502772-1 A2, BR0109913-2 A2, BR0700034-0 A2, BR9305223-5 A2; Utility Model MU8602632-1 U2, and Brazilian Patents BR9302980-2 B1 e BR9302981-0 B1.
Published Brazilian Application No. BR0702226-3 A2 describes the preparation of a Neem emulsible oil using tall oil extracted from Pinnus as emulsifier.
Published Brazilian Application No. BR0508296-0 A2 describes a granular formulation having systemic action when distributed in the rhizosphere. The formulation contains Neem extracts, hydrocarbon wax, clay, and colorant. The azadirachtin content in said study was monitored and controlled according to its concentration in the extracts. Such formulations produced an stabilizing effect in storage and control over field release of azadirachtin.
Brazilian Patent No. BR9302980-2 B1 proposes a method for preparing Neem extracts containing high levels of azadirachtin, which is more stable in said extract.
Among the several diverse extract preparation techniques, Utility Model No. MU8602632-1 U2 introduces an extraction technique which uses ball mills for micronizing Neem seeds in the presence of organic solvents, affording Neem seed microparticles, in fact, ground and micronized seeds.
Published Brazilian Application No. BR0109913-2 A2 describes a completely water soluble organic solvent-free solid form pesticide with Neem extract release system, formulated with saccharides (methylcellulose, sucrose), affording pellets.
International patent libraries also list a great number of Neem-related studies, such as North-American U.S. Pat. Nos. 4,515,785, 4,537,774, 4,556,562, 4,902,713, 4,943,434, 4,946,681, 5,001,146, 5,001,149, 5,047,242, 5,110,591, 5,124,349, 5,229,007, 5,281,618, 5,298,247, 5,298,251, 5,352,672, 5,352,697, 5,356,628, 5,368,856, 5,370,873, 5,371,254, 5,372,817, 5,391,779, 5,395,951, 5,397,571, 5,405,612, 5,409,708, 5,411,736, 5,420,318, 5,472,700, 5,501,855, 5,503,837, 5,602,261, 5,626,848, 5,635,193, 5,663,374, 5,679,662, 5,695,763, 5,698,423, 5,730,986, 5,736,145, 5,756,773, 5,827,521, 5,856,526, 5,900,493, 6,193,974, 6,294,571, 6,312,738, 6,340,484, 6,545,167, 6,602,823, 6,660,291, 6,703,034, 6,703,347, 6,733,802, 6,734,198, 6,746,988, 6,773,727, 6,811,790, 6,824,787, 6,835,719, 6,849,614, 6,855,351, 6,855,351, 6,875,885, 6,930,076, 6,991,818, 7,083,779, 7,112,553, 7,132,455, 7,182,952, 7,186,891, 7,194,964, 7,204,994, 7,250,175, 7,250,396, 7,320,966, 7,345,009, 7,345,080, 7,351,420, 7,361,366, 7,390,480, 7,476,397, 7,514,464, 7,530,196, 7,531,189, 7,534,447, 7,537,777, 7,618,645, 7,622,641, 7,655,597, 7,655,599, 7,674,807, 7,687,533, 7,696,232, 7,722,695, 7,754,655, 7,803,832, 7,803,992, 7,807,679, 7,823,323, 7,867,507, 7,871,645, 7,872,067, 7,887,827 and H1.541.
In general, a lot of those processes relate to extract preparation, azadirachtin extraction, increase in stability, biological activities, fertilizers, formulation, and structural modifications.
Among application results in searches, no technique, process, or formulation component is similar to those described in the methodology which is the object of the present application.
U.S. Pat. No. 6,340,484 describes saccharide pellets saturated with completely water-soluble organic solvent-free Neem extracts, which affords stability and control over the release kinetics.
U.S. Pat. No. 5,856,526 describes the preparation of powder azadirachtin having a purity content close to 90%, and an emulsible concentrate comprising about 30 wt % azadirachtin. According to this patent document, powder azadirachtin was prepared using classic extraction and fractionation phytochemical techniques involving maceration, concentration and chromatographic separation steps, and the emulsible concentrate prepared by dissolving the azadirachtin rich fraction in organic solvents which comprise emulsifiers and sun blocks or not.
Something similar is described in U.S. Pat. No. 5,736,145 for production and purification processes of powder azadirachtin, and for preparation of a stable aqueous composition for storage. For the emulsion formulation, a mixture of alcohol and water, oleic acid, emulsifier, Neem oil and extract, and p-aminobenzoic acid as sun block is used.
U.S. Pat. No. 6,193,974 describes the preparation of microemulsions using non-ionic surfactants for providing the emulsion also comprising p-aminobenzoic acid as sun block. As a result, a stable aqueous composition is obtained, where, depending on the amount of Neem oil and dilutions, different contents of azadirachtin can be found.
Preparation processes for azadirachtin rich extracts and fractions, and stable emulsions for storage are also described in U.S. Pat. Nos. 6,811,790, 5,827,521, 5,695,763, H1541, 5,420,318, 5,411,736, and others.
Despite the great number of published studies describing techniques for producing Neem extracts and fractions and powder azadirachtin, reproducibility of the technique generally depends on seasonal effects, and on the quality of the chosen seed.
There are also several studies which aim at proposing viable compositions for stabilizing formulations made with Neem oil and extracts, and azadirachtin.
Thus, novel alternatives have arisen. U.S. Pat. No. 5,698,423 relates to a procedure for growing plant cells and for producing azadirachtin, and reactors under controllable conditions.
U.S. Pat. No. 6,733,802 describes a formulation for preparing a Neem derived natural insecticide. Said study describes the use of surfactants derived from plants of the Saponaria, Quillaja, Chlorogalum, or Sapindus genera, and of antioxidants such as vitamin C, tocopherol and other derivatives from the Zingiber or Curcuma genera.
U.S. Pat. No. 6,703,034 introduces a formulation for preparing microemulsions with Neem oil using non-ionic surfactants such as, for example, alkylphenol etoxylates.
U.S. Pat. No. 6,635,757 introduces powder insecticide formulations comprised by a Neem extract complex comprising azadirachtin with water dispersible cyclodextrines which are dried using a Lyophilizer or Spray-Drying. In said study, several variations in the composition of azadirachtin, solar protection agents (hydroquinone and anacardic acid), and salicylic acid were performed, resulting in formulations which increase the defense mechanism lifetime of such insecticides in crops.
U.S. Pat. No. 6,667,277 describes chemically modified gums which increase biological efficiency for use in several insecticide, herbicide, or fungicide formulations, having several active ingredients, including solid Neem oil, which are dispersed in aqueous medium with rapid release of biological agents.
U.S. Pat. No. 5,643,351 makes use of polymer melts dispersed in water or organic solvents, encapsulating agricultural ingredients with, for example, Neem products in the form of polymeric films. In said patent, polyethylene glycol and styrene oxide and propylene oxide co-polymers, non-ionic surfactants, liquid emulsifiers, dispersion agents, and ultraviolet protectors were used.
U.S. Pat. No. 7,538,079 describes a process for producing, through Spray-Drying, powder capsules formulated with inorganic salts and beneficial agents such as essential oils for perfumes, aromatherapeutic materials, vitamins, insect repellents, etc., possibly having in their composition detergents, polymers, sequestering agents, and diverse oils such as, for example, citronella oil and Neem oil, applied in therapeutic or stimulating processes.
U.S. Pat. No. 5,009,886 introduces a process for producing dentifrice microparticles or microfibers formulated with Neem branch or root extracts.
U.S. Pat. No. 7,872,067 innovates with an amphiphilic polymer composition applied in order to prepare water insoluble compound compositions dispersed in aqueous medium, which are applied in the protection of crops. Azadirachtin is listed among the many insecticides that may be formulated.
U.S. Pat. No. 7,871,645 provides a method and composition with ion exchange resins loaded with one or more active compounds. There are several pesticides and drugs that may be adsorbed in the resins, including azadirachtin.
Carrier particles for several pesticides having diameters between 500 and 3000 micrometers are introduced in U.S. Pat. No. 7,867,507. The process consists of applying a layer of a liquid comprising solvent, specific fixation agent for the groups, bentonite, carbohydrates, proteins, lipids, synthetic groups over powder carbaryl carrier group previously adsorbed with either insecticides, fertilizers, herbicides, or stimulants, comprising adjuvants or not. Azadirachtin lies among the several listed pesticides which are liable to be applied with carrier particles. Among the advantages, the authors give emphasis to environmental impact reduction.
In a similar study, U.S. Pat. No. 7,754,655 describes preparing agrochemical formulations as microcapsules. Microformulation consists of a dispersion particulate phase of polyurea and/or polyurethane microparticles with penetrating agents and, if suitable, additives, said phase being poured onto a suspension of a solid agrochemical active compound, additives (protective colloid and emulsifiers), and water. Azadirachtin is also listed among compounds.
U.S. Pat. No. 7,655,599 and U.S. Pat. No. 7,655,597 describe agrochemical formulations based on emulsifiers and adjuvants, respectively.
U.S. Pat. No. 7,655,599 describes a suitable formulation for several compounds, such as azadirachtin, based on ethylenediamine alkoxylate emulsifier, which acts either as emulsion stabilizer, crystallization inhibitor, or both. If necessary, penetration promoters, emulsifiers, and δ-butyrolactone are added to the formulation.
U.S. Pat. No. 7,655,597 describes a pesticide (insecticide or herbicide) composition which is formulated with adjuvant or additive co-polymers, where the co-polymer consists of individual proportions of maleic or itaconic groups. Such formulation promotes efficiency gain when compared to the non-formulated active compound, which allows it to be applied for azadirachtin.
Excellent stability for water dispersed granulated solid formulation was achieved according to U.S. Pat. No. 6,596,292. The formulation comprises active compound, including azadirachtin, dispersant, wetting agent, boron, water-soluble carrier, and smectite.
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