Abstract
Plant tissue culture has emerged as an important tool to produce bioactive compounds from various plant species, including the sustainable production of limonoids that are receiving considerable attention due to the benefits associated with human health such as anticancer activities. The purpose of the present study was to evaluate the capacity of limonoids aglycone production from callus culture from sweet orange cv. Pera (Citrus sinensis) seeds and identify the compounds produced in this cell line. Callus induction occurred in Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic (2,4-D), malt extract, agar and coconut water. For the analysis and identification of the limonoids, CG-MS-EI ion-positive mode and UPLC-QTOF-ESI were used operating in positive and negative mode. An intense peak corresponding to limonin appeared in the callus extracts at a retention time of 58.1 min. in CG-MS-EI and four major limonoids aglycone by positive ion mode UPLC-QTOF-ESI: limonin, nomilin, deacetylnomilin, and nomilinic acid. The culture medium was efficient at the bioproduction of limonoids aglycone in callus cultures of C. sinensis seeds. Therefore, data obtained from UPLC-QTOF-ESI proved its importance at identifying new compounds that benefit human health, and may assist future work in the identification of known or new limonoids in Citrus species and related genera.
Highlights
The production of original and/or modified bioactive compounds through living organisms is a major goal of biotechnology
The objectives of this study were to evaluate the ability of limonoid aglycone bioproduction from calluses from C. sinensis seeds and identify the compounds present in this cell line through Gas Chromatography coupled to Mass Spectrometry by electronic impact ionization (CG-MS-EI) and UltraEfficient Liquid Chromatography coupled to high-resolution quadrupolar-flight time analyzer (UPLC-QTOFESI) mass spectrometry
We proceeded with C. sinensis callus extraction with acetone and analysis through gas chromatography-mass spectrometry (GC-MS)-EI, which almost only showed the presence of the limonin (1), the major limonoid aglycone found in the genus Citrus (Figure 2), which can be identified by comparison of its mass spectrum with NIST Library spectra and limonin fragmentation mode (Ren et al, 2014)
Summary
The production of original and/or modified bioactive compounds through living organisms is a major goal of biotechnology. Callus production consists of a plant tissue culture technique aimed at genetic enhancement and the production of bioactive compounds from many woody plant species, including the genus Citrus (Fallico, Ballistreri, Arena, Brighina, & Rapisarda, 2017). This technique offers a defined production system and is not subject to climatic and soil influences for being conducted in a controlled environment, which allows it to be applied in biomass production and development of somaclonal variations and exploited through biotechnological production means for new cultivars (Gonçalves & Romano, 2013). Peels and seeds can be used as alternative sources of bioactive compounds such as flavonoids and limonoids (Avula et al, 2016; Zou, Xi, Hu, Nie, & Zhou, 2016; Celano et al, 2018; Russo et al, 2019)
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