**Conflict of interest**

Although the highest concentrations of azadirachtin were obtained with the enzymatic preparations, Cellulase 17600L and Crystalzyme PML-MX, also Crystalzyme Cran and Crystalzyme 100XL could be used to elaborate neem extracts because of their azadirachtin concentrations of 1540 and 1690 ppm, respectively, are similar to those contained in commercial acaricidal products elaborated based on neem, but with the use of solvents [32]. This result indicates an advantage and a possible solution to the indiscriminate use of synthetic pesticides and the organic solvents employed in several extractions of active biomolecules. On the other hand, after 18 h of enzymatic hydrolysis, there are no changes on azadirachtin release, which means it is time required to carry out the obtaining of neem seed extracts. In addition, the conditions identified in this study as necessary to neem seed hydrolysis are not extreme or aggressive and could be easily scalable.

The present study had the objective of generating an alternative solution to the multiple problems generated by the indiscriminate use of synthetic pesticides. The use of enzymes in the production of metabolites with biological interest has been widely studied in the last decade, due to their specificity and their effect on the cellulose substrate hydrolysis, minimizing the times of obtaining and the necessary costs for their implementation. In addition, its application allows reducing the use of solvents, normally employed in the extraction of neem oil. Besides, the conditions required for neem seed hydrolysis under optimal activity of the enzyme preparations can be easily standardized and scaled for its industrial production. In addition, it was found no affectations on azadirachtin concentration when temperatures of 45 and 50°C were used during enzymatic hydrolysis coinciding with that reported in [34]. However, more studies are needed to determine the stability of azadirachtin under different

Although the enzyme-assisted extraction allows the obtaining of neem extracts with higher azadirachtin concentrations than those obtained with conventional methods such as extrusion, cold extrusion, water maceration, and percolation with hexane [32, 33] and in similar concentrations to those obtained by extrusion with cold methanol [32], the amount of neem seeds used in this method was lower, and therefore, the yield was higher than obtained with all these methods. In next studies, components identified as repellents and/or acaricides such as salannin and nimbin should be analyzed. In addition, it is necessary to carry on shortterm and long-term *in vitro* and *in vivo* analysis of enzymatic neem extracts on pest with public health impact, defining the vehicle of application and the lethal concentrations for its

This study was funded by the Dirección General de Educación Superior Universitaria

**4. Conclusions**

implementation.

**Acknowledgements**

(DGESU/SEP) (Project DSA/103.5/14/7147).

temperatures and pH conditions during storage.

120 Soil Contamination and Alternatives for Sustainable Development

All authors who participate in the elaboration of this manuscript have no conflict of interest to declare.
