In Vitro Azadirachtin Production

Abstract

The secondary metabolite azadirachtin (C35H44O16) is a tetranortriterpenoid obtained from the neem tree (Azadirachta indica). It has long been investigated for its biopesticidal properties. It is a natural insecticide, known to affect feeding, growth, reproduction and metamorphosis of the insect pests. Because of the broad-spectrum control of insects and the relatively low nontarget toxicity it has been widely used in agriculture. To add to its advantage the biopesticidal property of azadirachtin is not only limited to phytophagous insects, but is also known to affect the other pathogenous organisms like nematodes, fungi and micro-organisms. It is a highly oxygenated and complex molecule, which makes its chemical synthesis difficult as well as uneconomical. Studies are still in progress to make its chemical synthesis successful and practically feasible for the mass production of azadirachtin. Currently, azadirachtin is isolated by solvent extraction of the seeds of the Azadirachta indica tree. There are various limitations in extracting azadirachtin from plant sources, majorly due to its limited availability/short shelf-life of seeds, degradation during storage and considerable genotypic/environmental variation in its content from different sources. At present, the demand for azadirachtin is greater than the supply. However, due to variability of azadirachtin available in seeds (0.2–0.6 %) it is difficult to base its mass production on natural sources. A significantly larger amount of material (seeds) would need to be processed to yield a reasonable amount of azadirachtin. Instead, it would be better to rely on a rather stable parent cell line that can be cultivated in vitro (in bioreactors) with a faster doubling time. A biotechnological approach can be very useful for reaching longterm goals. A deeper understanding of different aspects of large-scale azadirachtin production is therefore very important. In recent years, a considerable amount of information has been obtained on the production of azadirachtin by cell and tissue cultures of Azadirachta indica. This approach has an added potential of increasing yield by culture selection and manipulation using elicitors, precursors, permeabilising agents and growth regulators. Azadirachtin is extremely liable to atmospheric degradation in the presence of sunlight. Although few investigations regarding enhancement in its atmospheric stability have been done, more detailed analysis is required to select appropriate stabilisers against the photo-degradation of azadirachtin. A great deal of work with Azadirachta indica has been focused on the extraction and quantification of azadirachtin. Purification of azadirachtin is difficult to accomplish, especially on a preparative scale due to its complexity and similarity in structure of the chemicals found in the seeds, foliage and cell culture. Reverse-phase high-performance liquid chromatography is widely used for the qualitative and quantitative estimation of azadirachtin. Use of other methods like super-critical fluid chromatography (SFC) and liquid chromatography-mass spectrometry combined with flash chromatography, thin-layer chromatography and SFC have also been documented for authentic quantification. Even though azadirachtin has long been recognised as a potent biopesticide, no reports are available to date on the commercial production of azadirachtin by plant cell/tissue culture. This chapter provides a deeper insight with respect to the origin, chemical nature, application, mode of action and prevalent technologies for the production of this high-value bioactive molecule.