WO2017203454A1

WO2017203454A1 - Method for obtaining an extract of azadirachta indica

Info

Publication number: WO2017203454A1
Application number: PCT/IB2017/053073
Authority: WO
Inventors: Fernando OROZCO SÁNCHEZ; Rodrigo Alberto HOYOS SÁNCHEZ; Anny Daniela MARTÍNEZ MIRA; Juan David LÓPEZ TABORDA; Juan Carlos OVIEDO RAMIREZ
Original assignee: Universidad Nacional De Colombia
Priority date: 2016-05-25
Filing date: 2017-05-24
Publication date: 2017-11-30

Abstract

The invention relates to a method for obtaining an extract from a cell culture of Neem (Azadirachta indica). The suspended cell culture is subjected to cellular stress under conditions that promote the production of active metabolites such as azadirachtins and terpenoids, which are subsequently extracted with solvents and/or adsorption resins. The extract obtained, whether alone or incorporated into an agrochemical composition, can be used for pest control in different crops.

Description

PROCESS FOR OBTAINING AN EXTRACT BE Azadirachta indica

FIELD OF THE INVENTION

The present invention is in the field of biotechnology, particularly in processes for obtaining extracts from vegetative cells and their agrochemical compositions for pest control. DESCRIPTION OF THE STATE OF THE TECHNIQUE

Among the most studied agents for the control of insect populations are the limonoids produced by the Neem tree (Azadirachta indica), native to India and Pakistan. These types of compounds are considered environmentally friendly and have an antialimentary effect on many species of insect pests and on some gram-positive bacteria, nematodes, molluscs and harmful fungi, including species of Aspergilius sp. Aflatoxin producers. The production of biocomposites based on limonoids can be carried out by an extraction process from the semilias of the Neem tree (Azadirachta indica), which does not generate heterogeneity in the content of limonoids due to genetic, climatic and geographical variations (Sidhu , Kumar, & Behl, 2003). That is why we have designed processes to obtain a bioreactor scale from in vitro cultures of cells and hairy roots of Neem (Azadirachta indica).

The use of Neem hairy roots to produce limonoids brings with it many difficulties in scaling and industrialization, while the use of suspended cells allows processes to be developed in bioreactors with specific conditions of mixing, aeration, agglomerate formation, and in general, more appropriate conditions for scaling (Prakash & Srivastava, 2007).

To increase the production of the cells in suspension, variables such as the composition of the culture medium, the modification of the nutritional requirements of the cells and the supply of precursors and elicitors have been optimized that improve cell growth and secondary metabolite production (Linden, Haigh, Mirjalili, & Phisaphalong, 2001).

One of the most studied limonoid compounds, azadirachtin, is mainly obtained from the seeds of the Neem tree (Azadirachta indica). Azadirachtin has a high aggregate value and is used commercially as an agent for the control of broad-spectrum insect populations. Since its discovery, research has focused on its activity and processes to increase its production (Srivastava & Srivastava, 2013) (Singh & Chaturvedi, 2013). However, azadirachtin is not the only limonoid identified in Neem, other related limonoids such as nimbin. Salannina, isonimbinolide, among others, also show significant activity against several insect species (Simmonds, Jarvis, Johnson, Jones, & Morgan, 2004). Document IN00148DE2010A describes a procedure for transforming Neem cells and forming hairy roots. The document discloses the growth media and the technical characteristics for the production of azadirachtin. Document CN103493844A illustrates a procedure for the production of Neem mutant cells, the means for the culture of calluses and cells in suspension and the production of azadirachtin, as well as the details of lyophilization and extraction with solvents at laboratory level for analysis. of azadirachtin.

Although several processes for obtaining biocidal compounds from the Neem tree are disclosed in the state of the art, it is necessary to develop processes that optimize the production of extracts and active metabolites that are easily scalable at the industrial level and that do not generate destructive practices such as tree felling and deforestation. It is also necessary to develop formulations from these extracts for use in agriculture as controlling agents for insects and other pests.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a process for obtaining an extract from a culture of Neem cells (Azadirachta indica) in suspension and agrochemical compositions comprising said extract. The process involves the culture of the cells and their subsequent extraction with solvents and / or adsorption resins. The extract obtained comprises active compounds such as azadiractins and Terpenoids, the four can be used to control pests in various crops.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Process diagram for the production and separation of limonoids from Neem cells (based on water vaporization and ethanol extraction).

FIG. 2, Diagram of the process for the production and separation of limonoids from Neem cells (based on adsorption resins and extraction with ethanol).

FIG, 3. Graph of the kinetics of Neem cell production using the MS medium and the cell propagation medium (modified MS) in a stirred tank bioreactor. FiG, 4. Graphs of the kinetics of viable biomass production (live cells}, non-viable biomass (dead cells), limonoids (extract) and sugar consumption in a Neem cell culture in a stirred tank bioreactor. (4A ) Stage 1: culture in the cell propagation medium - modified MS. (4B) Stage 2: culture in the production medium.

FIG. 5. Graph of Concentration vs. Time of the methanolic extract obtained in the Neem cell culture in a stirred tank bioreactor with the limonoid production medium (clear points). Black dots represent the extract in a reference MS medium.

FSG 6. Bioactivity test of the extracts obtained in the Neem cell cultures in the Erienmeyer flask and in a stirred tank bioreactor. (6A) Photograph of the affected leaves without the extract. (6B) Graph of the percentage of anti-food index.

FSG, 7. Graph of the concentration of the ethanol extract obtained from Neem cell cultures using the extraction process 1 (water vaporization + ethanolic extraction) and the extraction process 2 (XAD resins + ethanolic extraction). FIG. 8, Photograph of the trial evaluating the effectiveness of Composition A under laboratory conditions. Choice test on corn discs with larvae of Spodoptera frugiperda or corn worm cogollero. FSG 9. Graph of the effectiveness of Composition A on larvae of Spodoptera frugiperda at the laboratory level. A) Percentage of leaf area affected. B) Amphibious effect.

Treatments with equal tetras do not show significant differences according to the Físher LSD test. The anti-food effect of the chemical insecticide is equal zero (0) because the larvae die inside the Petri dish, without feeding on the control treatment (leaf disc without insecticide).

FIG. 10, Graph of the percentage of affected leaf area of Composition A at field level. The percentages of affected leaf area of the treatments with equal letters do not show significant differences according to Fisher's LSD test.

FIG. 11. Photograph of the degree of involvement of the corn leaves by Spodoptera frugiperda in a bioassay in the field with Composition A. a) Commercial chemical insecticide, b) Water, c) Base emulsion. d) Extract, e) Composition A.

DETAILED DESCRIPTION OF THE INVENTION

The process involves, as an initial stage, the in-vitous culture of Neem leaf or seed cells (Azadirachta indica) in a suitable supplanted culture medium with specific growth conditions, so that the cells grow in shape dedifferentiated and calluses form after several weeks, which for the purposes of the process of the invention is called the propagation stage. The cells obtained can be broken down by agitation to obtain a suspension cell culture, the viability of which can be verified using an exclusion dye (e.g. Evans Blue).

The suspension cell culture is subjected to cellular stress under conditions that favor the production of active metabolites, and subsequently these metabolites are extracted by processes involving centrifugation, lysis, biomass filtration and solvent extraction. As a result, an extract of Neem (Azadirachta indica) is obtained, which can be used in agrochemical formulations as controlling agents for populations of insect pests.

The culture step can be carried out in an appropriate container and employ a culture medium with salts of Murashigue and Skook, MS (commercial composition), supplemented with sucrose, indo acid! butyric acid (IBA) and benzyl amine purine (BAP), and provide adequate conditions of agitation, pH, temperature and absence of light. In a preferred embodiment of the process of the invention, the culture stage is carried out in a bioreactor with suppressed MS medium, stirring between 100 and 150 rpm, pH between 5.0 and 6.0, temperature between 20 ° C and 35 ° C with absence of light.

In the propagation stage, the cells are exposed to a second culture medium with salts of Murashigue and Skook, MS, characterized by having MSx1.5 suppressed with sucrose, indolbutyric acid. benzilarninopurine and sodium acetate. The conditions of pH, temperature, dissolved oxygen, agitation and absence of light are adapted to favor cell propagation. In a preferred embodiment, the propagation stage is carried out in a bioreactor with a supplemented MSx1.5 medium, pH between 5.0 and 7.0, in the dark, stirring between 100 and 120 rpm and with oxygen control between 20 and 40%. The cultures of suspended plant cells obtained in the propagation stage have a water content between 90 and 95%. Alternatively and to decrease the costs associated with water vaporization, the culture medium with cells of the propagation stage can be concentrated by centrifugation and the cells subjected to cell lysis by mechanical disruption.

The dry biomass obtained is subjected to cell lysis with a cell disruption system and subsequently the extraction is carried out with an organic solvent selected from methane !, ethanol, isopropanol, chloroform and mixtures thereof, depending on the polarity of the extract that is desired obtain. Prior to the production and extraction of the bioactive compounds with organic solvents, the cells can be centrifuged and then the biomass can be lyophilized under vacuum conditions and at temperatures below 40'C.

The cell debris is filtered and the liquid is passed through a polymeric adsorption resin, preferably an Amberlite XAD-7HP resin. Subsequently and to separate the active metabolites adsorbed by the resin, an extraction is carried out with organic solvents selected from methane !, ethanol, isopropanol, chloroform and mixtures thereof, with ethanol being the most preferred.

The cell debris or filter cake resulting from the previous step can also be subjected to solvent extraction, preferably with the same solvent, to ensure a total extraction of the produced active metaboits. The two ethanol streams resulting from this stage of the process are concentrated at low pressure and temperatures between 30 and 40 ° C to obtain the concentrated extract containing the active metabolites.

After the extraction and concentration of the meíabolitos, these can be identified by means of techniques such as chromatography. It is possible to detect the presence of fatty acids, phytosterols, triterpenes, azadirazines, limonoids and other terpenoids in the extract resulting from the process. Table 1 presents the fractions obtained from the extract and Sos possible chemical compounds present in each fraction. As it is lowered in the Table, the polarity of the compounds present in each fraction increases, starting with low polar compounds (soluble in 95: 5 v / v hexane / ethyl acetate), such as fatty acids, and ending with polar compounds (soluble in methanol) that could include highly oxygenated terpenoids.

Table 1. Separated chemical fractions of the extract obtained. from Neem cell cultures

Nuclear magnetic resonance imaging (NMR) also identified the presence of stigmasterol and α-sitosterol. The HPLC analyzes allowed confirming the presence of azadirachtin in the extract, although it is not its main compound. By means of HPLC coupled to mass spectrometry (HPLC-MS), different molecular ions were identified in the extract, which allow presuming that there are terpenoids and limonoids presented in Table 2. Table 2. Compounds present in Aleen cell cultures? according to the masses of the ions obtained by HPLC-MS.

ND Not available. Aza, azadirachtin isomers. C1, C2, compounds 1 and 2 observed in the Aza spectrum. Exp, experimental Vaior. (*) Mass of the compound obtained by the expression "mass of the compound = mass of the ion + 17".

With the extract obtained according to the process of the invention, agrochemical compositions can be designed (eg soluble solutions, emulsions, suspensions and granules) by the addition of additives, adjuvants and agrochemically acceptable vehicles, considering for this the physicochemical properties of each of them , such as solubility and polarity. The agrochemical compositions can be applied either alone or together with other biocidal agents for the control of insect populations. The present invention will be presented in detail through the following Examples, which are provided for illustrative purposes only and not for the purpose of limiting its scope.

EXAMPLES

EXAMPLE 1. Establishment of in vitro culture and culture of Neem cells

A medium with salts of Murashigue and Skook, MS (commercial composition), sucrose 30g / L, indole butyric acid (IBA) 4 mg / l, benzyl amino purine (BAP) 1 mg / l, pH 5.8, Phytagel 2g / l, 25ºC, in dark conditions, allows calluses to be obtained in periods between 2 and 4 months. In turn, these calluses can be added to liquid media without the gelling agent, containing the MS salts, sucrose, with the IBA and BAP regulators defined above.

By stirring between 100 and 120 rpm, 28 ° C and darkness, the cells are disintegrated and the suspension cell culture is obtained after several weeks. The viability of the cells can be verified periodically using an exclusion dye such as Evans blue.

EXAMPLE 2. Propagation of Neem cells A medium with 6.6 g / L of salts from Murashigue and Skook, MS, sucrose 45 g / L, indoi butyric acid (IBA) 4 mg / l, benzii amino purine ( BAP) 1 mg / l, pH 5.8, 0.1 g / L sodium acetate, 25 ° C, in the dark, to allow for greater cell growth (FIG. 3). C.S. It corresponds to dry cells. At the level of flasks the cells can be grown at 100-120 rpm in darkness or light. At the agitated tank bioreactor level, these can be grown with speeds of 200 to 450 rpm - depending on the impeller used and with oxygen control between 20 and 40%.

EXAMPLE 3. Production of active metabolites

A bioreactor was conditioned with an internal filter to retain the cells obtained according to Example 2, such that more than 50% of the spent culture medium was removed, thus concentrating the cells therein. Then, a second stage was performed by refilling the reactor with a modified culture medium containing inorganic salts, sucrose 40-60 g / l, salicylic acid 137.3 mg / l, jasmonic acid 2.9 mg / l, chitosan 18 , 5 mg / l, pH 5.8, with a speed of 200 to 450 rpm and with oxygen control between 80 and 90%. Using the conditions indicated in Example 2, the production of bioactive compounds was increased, as shown in FIG. 4 (kinetics in the cell propagation medium and in the production medium) and FIG 5 (complete production kinetics of the extract in the two-stage culture). In addition, the bioactive properties of the extract were preserved when the process was scaled to a stirred tank bioreactor (FIG. 6).

EXAMPLE 4. Extraction and partial purification of the extract

1 Liter of Neem cells suspension, obtained according to Example 2, was taken with a concentration of 12-15 g dry cells / L. These cells were filtered and lyophilized. The dried biomass was mechanically lysed and extracted 3 times with ethanoi. AND! Solvent was evaporated to dryness to obtain the extract of the water + extraction vaporization process.

In parallel, 1 L of the same Neern cell suspension was taken, the biomass was mechanically lysed and then filtered. The filtered liquid is passed through an adsorption bed containing 100 g of Amberlite XAD-7HP resin and then the resin is desorbed with ethanoi. The filtered biomass was desorbed in turn with etanoi. The two ethanoi solutions obtained were evaporated to dryness to obtain the extract with the bioactive compounds of the resin + extraction process. In each case, between 8.3 and 6.8 g of extract were obtained. Although there are no significant differences in the amount obtained between both processes, the resin + extraction process is more efficient because it does not require water vaporization (FIG. 7). EXAMPLE 5. Agrochemical Compositions of Neem Extract

From the Extract obtained according to Example 4, emulsion-type agrochemical compositions were prepared. Table 3 describes composition A, which includes commercial chemical compounds as additives. Table 4 describes Composition B which includes less toxic and environmentally friendly organic compound additives. Table 3. Composition A

To obtain Composition A, the extract, the protectors of the extract (photoprotector, stabilizers) are mixed with the oil, given their hydrophobic character. Once the components have dissolved, the surfactant is added to the oil phase and the mixture is stirred. Slowly, water is added to the previous preparation, constantly stirring until an oil / water type emulsion is obtained. This procedure is known as the phase inversion method (Adamson and Gast, 1997).

For stirring the emulsions, a blender or immersion mixer operated at the minimum speed was used. To obtain Composition B, the extract is mixed with the oil and then the surfactant is added to the oil phase and the mixture is stirred. Separately, the water is mixed with the corresponding photoprotector and stabilizer. Slowly the aqueous phase is incorporated into the oil phase, constantly stirring until an emulsion is obtained. Compositions A and B can be diluted 50 times with water for field applications, depending on the crop and pests to be controlled (Tables 5 and 8),

Table 5. Diluted Composition A

Upon application in the field of compositions A and B, a foliar adherent (eg, INEX-A®) can be added to increase the foliar adhesion of the composition. It is recommended to follow the recommendations for the use of the commercial product.

EXAMPLE 8, Evaluation of the biocidal effect of the Agrochemical Compositions

To evaluate the effect of the compositions obtained according to Example 5, larvae of the second instar of Spodoptera frugiperda were subjected to fasting 6 hours before the experiments. The assembly consisted of a 5 cm diameter plastic Petri dish with an agar-agar film inside. Two corn leaf discs of 1.5 cm in diameter were placed on the agar, of which one was impregnated with the treatment and the other acts as a control. In addition to the discs, a larva of S. frugiperda from the second instar (F! G 8) was deposited on the agar,

The effectiveness of the treatments was evaluated at 98 hours, determining their anti-food index and the percentage of leaf area affected in the corn leaf discs (Capataz-Tafur, Orozco-Sánchez, Vergara-Ruiz, & Hoyos-Sánchez, 2007) . The anti-food index (EAA) was calculated using the formula proposed by Kerney el al, (Keamey, Allan, Hooker, & Mordue (Luntz), 1994)

To determine the area consumed in the leaf disks, photographs of the experiment were taken and analyzed with the program! Mage J 1.44 (FIG. 9).

For the bioassays in the field, a ground was prepared using a chisel plow and rotary harrow to polish it, depending on the characteristics of the soil. For corn crops, a seed of the ICA V109 variety is deposited per site at a distance of 0.80 meters between rows and 0.12 meters between plants.

The reseeding can be carried out one week after the sowing. After eight days of the emergence of the corn, the crop is thinned leaving approximately 4 plants per linear meter. Diluted formulations can be applied weekly, also depending on the environmental conditions and the culture. The affected follicle area, the physiological state of the plants and the amount of corn collected at the end of the harvest can be monitored.

EXAMPLE 7, Bioassays with fas field level formulations

Compositions A and B of Example 5 were evaluated on corn crops planted at the Cotové Agricultural Center of the National University of Colombia, Medellin Headquarters. The area of life of the place is that of the tropical dry forest (bs-T), which is located at an approximate height of 540 m.a.s.l., average annual rainfall of 1031 mm and average temperature between 23 ° C and 33 ° C.

ES land was previously subjected to a chisel plow and rotary harrow to polish it. Sowing was done using a Super Tatu seeder model PS0T2. A seed of the fCA V109 variety was deposited per site at a distance of 0.80 meters between rows and 0.12 meters between plants.

Re-seeding was carried out manually one week after sowing. After eight days of the emergence of corn, the crop was thinned leaving approximately 4 plants per linear meter. Given the continuous rains that took place during the experiment, it was not necessary to install any artificial irrigation system. The land was divided into three blocks of 60 m ² for a total of 180m ² treated and evaluated, although in total 550m ² were sown. In turn, the blocks were divided into 15 spaces of 4 m ² and in each one the application of a treatment was made.

Once a week, 150 mL of diluted compositions A and B were applied. To improve its adhesion to the leaves, the treatments administered are mixed with INEX-A®. The application process began 20 days after the emergence of the plants and lasted 15 days. Spraying was done with a ROYAL CARDEN CONDOR sprinkler with 1.8 L of adjustable nozzle with 200 / mm hole.

For the evaluation at 96 hours after the last application, three leaves of the selected plant were taken at random. In order to eliminate edge effects that could be generated, corresponding to the selected unit planted in the center of the subdivisions of 4m ² floor. In each case a photograph was taken to determine the area consumed by insects (by using the Image J program 1.44) and in this way the percentage of affected leaf area could be calculated (FIG. 10 and FIG. 11).

REFERENCES

[1] M. W. Aktar, D. Sengupta, and A. Chowdhury, "Impact of pesticides use in agriculture: their benefits and hazards.," Interdiscip. Toxic!, Vo !. 2 no. 1, pp. 1-12, 2009.

[2] S. J. Boeke, M. G. Boersma, G. M. Alink, J. J. A. Van Loon, A. Van Huis, M.

Dicke, and I. M. C. M. Rietjens, "Safety evaluation of Neem (Azadirachta indica) derived pesticides.," J. Ethnopharmacol., Vo !. 94, no. 1, pp. 25-41, 2004.

[3] S. Srivastava and A, K. Srivastava, "Production of the biopesticide azadirachtin by hairy root cultivation of Azadirachta indica in liquid-phase bioreactors.," Appl. Biochem Biotechnol., Vol. 171, no. 8, pp. 1351-1381, 2013.

[4] M. Singh and R. Chaíurvedi, "Sustainable production of azadirachtin from differentiated in vitro celi iines of Neem (Azadirachta indica)," AoB Plants, vol. 5 p. plt034. 2013

[5] M. S. J. Simmonds, A. P. Jarvis, S. Johnson, G, R. Jones, and E. D.

Morgan, "Comparison of anti-feedant and insecticidal activify of nimbin and salannin photo-oxidation producís with Neem (Azadirachta indica) limonoids.," Pest Manag. Se / ,, vol. 80, no. 5, pp. 459-484, May 2004.

[8] O. P. Sidhu, V. Kumar, and H. M. Behi, "Variability in Neem (Azadirachta indica) with respect to azadirachtin content.," J. Agric. Food Chem., Vol. 51, no. 4, pp. 910-915, 2003.

[7] G. Prakash and A. K. Srivastava, "Production of biopesticides in an in situ cell retention bioreactor ,," Αρρl. Biochem Biotechnol., Vol. 151, no. 2-3, pp.

307-318, 2008.

[8] R. K. Satdive, D. P. Fu! Zele, and S. Eapen, "Enhanced production of azadirachtin by hairy root cultures of Azadirachta indicates A. Juss by elicitation and media opimimization," J. Biotechnol., Vol. 128, no. 2, pp. 281-289, Feb. 2007,

[9] S. Srivastava and A. K. Srivastava, "Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica.," Appl.

Biochem Biotechnol., Vol, 172, no. 4, pp. 2286-2297, 2014.

[10] S. Jayaram, "Bioreactor And Uses Thereof," IN00148DE2010A, 2010.

[11] G. Prakash and A. K. Srivastava, "Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture," Process Biochem., Vol.

42, no. 1, pp. 93-97, 2007. [12] JC Linden, JR Haigh, N. Mirjalili, and M. Phisaphalong, "Gas Concentration Effects on Secondary Production by Pious Cell Cultures.," Adv. Biochem Eng. Biotechnol., Vol. 72, pp. 27-62, 2001.

[13] F. Orozco-Sánchez, "Effect of oxygen supply on the growth and production of terpenoids with Azadirachta Indica cells in a bioreactor," National Polytechnic Institute, 2009.

[14] P. Dayanandan and J. Ponsamuel, "Ulltrastructure of terpenoid secretory cells of Neem (Azadirachta indica A. Juss)," in Electron Microscopy in Medicine and Biology, no. 18, P, D. Gupta and H. Yamamoto, Eds. New Delhi: Oxford & IBH Publishing Co. Pvt. Ltd., 2000, pp. 179-195.

[15] A. W. Adamson and A. P. Gast, Physica! Chemistry of Suríaces, Sixth Edit.

1997,

[18] J. Capataz-Tafur, F. Orozco-Sánchez, R. Vergara-Ruiz, and R. Hoyos- Sánchez, "Antifeedant effect of cell suspension exíracts of Azadirachta indica on Spodoptera frugiperda JE Smith under laboratory conditions," Rev. Fac. Nac. Agron. Medellin, vol. 80, no. 1, pp. 3703-3715, 2007.

[17] M.-L. Kearney, E. J. Allan, J. E. Hooker, and A. J. Mordue (Luntz), "Antifeedant effects of in vitro culture extracts of the Neem tree, Azadirachta indica against the desert locust (Schistocerca gregaria (Forskál))," Plant Cell. Tissue Organ Cult, vol. 37, no. 1, pp. 87-71, 1994.

Claims

1. A process to obtain an extract from Neem suspension cells (Azadirachta indica) comprising the following steps:

a) culturing and propagating Neem cells (Azadirachta indica) under suitable culture conditions in an MS culture medium supplemented with sucrose, indolbutyric acid and benzylaminopurine;

b) stress the cells obtained in step a) to favor the production of active metabolites;

c) release the active metabolites from cells by extraction; and d) purify the fractions obtained in c) until the extract is obtained.

2. The process according to Claim 1, wherein the suitable culture conditions of step (a) are: pH between 5.0 and 6.0; room temperature; dissolved oxygen, agitation and absence of light.

3. The process according to Claim 1, wherein the propagation conditions of step a) are: MSxl.5 medium supplemented with sucrose, indole butyric acid, benzylaminopurine and sodium acetate; pH between 5.0 and 6.0; room temperature; dissolved oxygen, agitation and absence of light;

4. The process according to Claim 1, wherein the stress conditions of step (b) are: presence of precursor and elicitors, pH between 5.0 and 6.0; room temperature; dissolved oxygen, agitation and absence of light;

5. The process according to Claim 1, wherein the extraction of step (c) is carried out by one or more centrifugation, lysis, biomass filtration and solvent extraction processes.

6. The process according to Claim 1, wherein the solvents used in the extraction of step (c) are selected from the group consisting of methanol, ethanol, isopropanol, chloroform and mixtures thereof.

7. The process according to Claim 1, wherein the purification of step d) is carried out by drying, adsorption and desorption and vaporization.

8. The process according to Claim 7, wherein the purification of step d) is carried out with polymeric resins.

9. An extract of Neem cells (Azadirachta indica) obtained according to the process of Claim 1.

10. The extract according to Claim 9, comprising: fatty acids, phytosterols, triterpenes, azadiractins, limonoids and other terpenoids.

11. The extract according to claim 9, comprising as active metabolites: limocinin, azaridactin, aziracdactolium, salanine.

12. The extract according to Claim 9, with biocidal activity against populations of species of different insects, including Spodoptera frugiperda.

13. An agrochemical composition comprising the extract according to Claim 9, together with adjuvants and an acceptable carrier.

14. The agrochemical composition of Claim 13, in the form of a solution, suspension, emulsion or soluble granulate.

15. The agrochemical composition of Claim 13, comprising:

16. The agrochemical composition of Claim 13, comprising: