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. 2020 Nov 11:8:e10172.
doi: 10.7717/peerj.10172. eCollection 2020.

Chemical analysis of callus extracts from toxic and non-toxic varieties of Jatropha curcas L

Affiliations

Chemical analysis of callus extracts from toxic and non-toxic varieties of Jatropha curcas L

Gerardo Leyva-Padrón et al. PeerJ. .

Abstract

Jatropha curcas L. belongs to Euphorbiaceae family, and it synthesizes flavonoid and diterpene compounds that have showed antioxidant, anti-inflammatory, anticancer, antiviral, antimicrobial, antifungal and insecticide activity. Seeds of this plant accumulate phorbol esters, which are tigliane type diterpenes, reported as toxic and, depending on its concentration, toxic and non-toxic varieties has been identified. The aim of this work was to characterize the chemical profile of the extracts from seeds, leaves and callus of both varieties (toxic and non-toxic) of Jatropha curcas, to verify the presence of important compounds in dedifferentiated cells and consider the possibility of using these cultures for the massive production of metabolites. Callus induction was obtained using NAA (1.5 mg L-1) and BAP (1.5 mg L-1) after 21 d for both varieties. Thin layer chromatography analysis showed differences in compounds accumulation in callus from non-toxic variety throughout the time of culture, diterpenes showed an increase along the time, in contrast with flavonoids which decreased. Based on the results obtained through microQTOF-QII spectrometer it is suggested a higher accumulation of phorbol esters, derived from 12-deoxy-16-hydroxy-phorbol (m/z 365 [M+H]+), in callus of 38 d than those of 14 d culture, from both varieties. Unlike flavonoids accumulation, the MS chromatograms analysis allowed to suggest lower accumulation of flavonoids as the culture time progresses, in callus from both varieties. The presence of six glycosylated flavonoids is also suggested in leaf and callus extracts derived from both varieties (toxic and non-toxic), including: apigenin 6-C-α-L-arabinopyranosyl-8-C-β -D-xylopyranoside (m/z 535 [M+H]+), apigenin 4'-O-rhamnoside (m/z 417 [M+H]+), vitexin (m/z 433 [M+H]+), vitexin 4'-O-glucoside-2″-O-rhamnoside (m/z 741 [M+H]+), vicenin-2 (m/z 595 [M+H]+), and vicenin-2,6″-O-glucoside (m/z 757 [M+H]+).

Keywords: Callus; Glycosilated flavonoids; Jatropha curcas; Phorbol esters; micrOTOF Q-II.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Cell dedifferentiation of petiole explants from both toxic and non-toxic varieties of Jatropha curcas.
(A-D) Explants from non-toxic variety throughout dedifferentiation experiment (0, 7, 14, and 21 d, respectively), (E-H) Explants from toxic variety throughout dedifferentiation experiment (0, 7, 14, and 21 d, respectively). Both induced on MS culture medium added with NAA (1.5 mg.L-1) and BAP (1.5 mg.L-1).
Figure 2
Figure 2. Identification of both diterpenes-type (A), and flavonoids-type (B) compounds in seeds, leaves, and callus of Jatropha curcas, through thin layer chromatography.
Lanes from 2 to 38 correspond to extracts of callus of non-toxic variety throughout 38 d of culture, NTS= Non- toxic variety-seeds, PMA= Phorbol-12-myristate-13-acetate (Sigma) reference standard (Rf 0.42), V= Vitexin (Sigma) reference standard (Rf 0.42), and Q= Quercetin (Sigma) reference standard (Rf 0.37). A) The spots intensity increased throughout to culture time (Rfs 0.71, and 0.27), mobile phase chloroform-methanol (94:6). B) The spots intensity decreased throughout to culture time (Rfs 0.77, and 0.58), mobile phase chloroform-methanol (75:25). Plates were revealed with anisaldehyde.
Figure 3
Figure 3. Spectrophotometrical analysis of phorbol esters in extracts of Jatropha curcas seeds.
MS/MS fragmentation profile of the molecular ion m/z 365 [M+H]+ related to 12-deoxy-16-hydroxy-phorbol, which is the structural core from Jatropha curcas-phorbol esters (referred as Jatropha factors). Predictive structures obtained through CFM-ID platform from each ionized fragment.
Figure 4
Figure 4. Mass spectra from callus extracts of J. curcas showing the relative intensity of the molecular ion m/z 365 [M+H]+ related to the structural core of the Jatropha-phorbol esters.
Callus extracts from toxic variety: (A) 14 d of culture; (B) 38 d of culture; non-toxic variety: (C) 14 d of culture, (D) 38 d of culture. The relative intensity from molecular ion m/z 365 [M+H]+ increased throughout culture time.
Figure 5
Figure 5. Fragmentation profile (MS/MS) of the molecular ion m/z 381 [M+H]+, observed in leaves extracts, and related to fragmentation of two glycosylated apigenin (apigenin (6-C- α-L-arabinopyranosyl-8-C-β-D-xylopyranoside m/z 535 [M+H]+, apigenin 4’-O-rhamnoside m/z 417 [M+H]+)).
(A) Fragmentation profile of the molecular ion m/z 381 [M+H]+; (B) Apigenin 6-C-α-L-arabinopyranosyl-8-C-β-D-xylopyranoside m/z 535 [M+H]+, and Apigenin 4’-O-rhamnoside m/z 417 [M+H]+; (C) structures predicted to each molecular ion (381, 355, 335, and 219 m/z), obtained from CFM-ID platform.
Figure 6
Figure 6. Mass spectra of callus extracts from both toxic and non-toxic varieties of J. curcas at 14 and 38 d culture, showing the relative intensity of the molecular ion m/z 381 [M+H]+ related to the fragmentation profile from two glycosylated apigenin.
(A and C) Extracts of J. curcas callus from J. curcas-toxic variety (14 and 38 d, respectively). (B and D) Extracts of J. curcas callus from non-toxic variety (14 and 38 d, respectively). The relative intensity from molecular ion m/z 381 [M+H]+ diminished throughout culture time.

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